RESUMEN
Hepatitis C virus (HCV) is one of the most important causative agents of hepatitis worldwide. The current study aimed to evaluate the silencing effect of the small interference RNA (siRNA) molecules designed against the core region of HCV genotype 4 (HCV-4) and the CD81 gene, which is the cellular receptor for HCV in the human hepatocytes. RT-PCR was used to measure the changes in both the viral HCV core and the cellular CD81 genes induced by the specific siRNA molecules. Additionally, the fluctuations in either the viral or the cellular proteins of the target regions were tested by flow cytometry and immunofluorescence. The results showed the effectiveness of the used siRNA molecules against the target genes in either RNA or protein levels. The effect of 100 nM of siCD81 and 40 nM of siCore was more evident at 24 and 48 h post-transfection. The combination of the two siRNA molecules resulted in an extra inhibitory effect of the HCV core at both the RNA (85.6%) and protein (98.5%) levels. The current study suggested that targeting of the CD81 cellular receptor and/or the viral HCV core region by the small interference molecules might be a suitable choice in the suppression of HCV-4 replication. This might assist the development of new antiviral medications and provides a new alternative strategy for the targeting and treatment of HCV genotype 4.
Asunto(s)
Hepacivirus/efectos de los fármacos , Hepatitis C/terapia , Fragmentos de Péptidos/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Tetraspanina 28/metabolismo , Proteínas del Núcleo Viral/metabolismo , Línea Celular , Hepacivirus/genética , Hepatocitos/efectos de los fármacos , Hepatocitos/virología , Humanos , Replicación Viral/efectos de los fármacosRESUMEN
OBJECTIVE: To explore the effects of two different methodologies of RNA interference, namely small interfering RNA, and vector-based short hairpin RNA, on the expression levels of hepatitis C virus core RNA and protein of Saudi genotype 4 isolates. STUDY DESIGN: An experimental study. PLACE AND DURATION OF STUDY: Laboratories of the College of Medicine Research Center, King Saud University, Saudi Arabia, from January to December 2018. METHODOLOGY: Hepatitis C virus core small interfering RNA molecule and short hairpin RNA vector were designed against core region. Viral RNA expression was tested by RT-PCR; whereas, core protein was tested by flow cytometry and immunofluorescence. Results were statistically analysed by Chi-square analysis to calculate the p-value. RESULTS: Both molecules caused a reduction in core RNA and protein expression in infected cells. The effect of 100-pmol of small interfering RNA was more evident. For the vector-based short hairpin RNA, inhibition of core RNA expression was quite evident after 96 hours (p = 0.007). The results of flow cytometry and immunofluorescence showed a decline in core protein expression. The most dramatic effect was observed with 100-pmol small interfering RNA treatment of cells for 24 and 48 hours, which resulted in 63.5% and 91.1% core RNA expression reduction, respectively. CONCLUSION: RNA interference of hepatitis C virus core gene efficiently stopped viral replication and offer a promising therapeutic alternative against virus infection.
Asunto(s)
Hepacivirus/crecimiento & desarrollo , Interferencia de ARN , ARN Interferente Pequeño , ARN Viral , Proteínas del Núcleo Viral/metabolismo , Replicación Viral , Técnicas de Cultivo de Célula , Células Hep G2 , Humanos , Arabia SauditaRESUMEN
OBJECTIVES: The "nAG" protein is the key protein mediating the regeneration of amputated limbs in salamanders. The senior author (MMA) developed the original hypothesis that since "nAG" is a "regenerative" protein, it must be also an "antifibrotic' protein. The antifibrotic properties were later confirmed in a rabbit skin hypertrophic scar model as well as in a rat spinal cord injury model. The aim of this study is to evaluate the potential therapeutic properties of the nAG protein in a rat liver fibrosis model. METHODOLOGY: Liver fibrosis was induced using intraperitoneal injections of carbon tetrachloride (CCL4). A total of 45 rats were divided equally into 3 groups: Group I (the control group) received normal saline injections for 8 weeks, Group II received CCL4 for 8 weeks, and Group III received CCL4 and nAG for 8 weeks. At the end of the experiment, the serum levels of 6 proteins (hyaluronic acid, PDGF-AB, TIMP-1, laminin, procollagen III N-terminal peptide, and collagen IV-alpha 1 chain) were measured. Liver biopsies were also taken and the stages of live fibrosis were assessed histologically. RESULTS: The CCL4 treatment resulted in a significant increase in the serum levels of all 6 measured proteins. The nAG treatment significantly reduced these high levels. The degree of liver fibrosis was also significantly reduced in the CCL4/nAG group compared to the CCL4 group. CONCLUSIONS: nAG treatment was able to significantly reduce the serum levels of several protein markers of liver fibrosis and also significantly reduced the histological degree of liver fibrosis.
Asunto(s)
Cirrosis Hepática/tratamiento farmacológico , Proteínas Recombinantes/uso terapéutico , Animales , Biomarcadores/sangre , Modelos Animales de Enfermedad , Cirrosis Hepática/sangre , Cirrosis Hepática/patología , Ratas Sprague-Dawley , Proteínas Recombinantes/sangreRESUMEN
OBJECTIVE: To determine the effects of TGFβ1 and an anti-scar protein nAG on normal (un-injured) 3-D astrocyte cultures with special emphasis on astrocyte proliferation, and the expression of GFAP, CSPG4, S100B, and IL-6. STUDY DESIGN: An experimental study. PLACE AND DURATION OF STUDY: King Saud University, Riyadh, Saudi Arabia, from March to September 2017. METHODOLOGY: 3-D astrocyte gels were treated differently to create one control group (control, untreated astrocytes) and five experimental groups: nAg-only (treated astrocytes with 1 nM nAG recombinant protein), TGFβ1-only (treated astrocytes with 10 ng/ml TGFβ1), TGFβ1+low nAg concentration, TGFβ1+intermediate nAg concentration, and TGFβ1+high nAg concentration. Astrocyte proliferation, and the expression of GFAP, CSPG4, S100B, and IL-6 were studied and compared in these experimental groups. RESULTS: There were major differences in the responses of normal astrocytes in vitro versus the responses of astrocytes in vivo in the setting of injury or disease. The effects of TGFβ1 were dominant over the effects of nAG with regard to changes in CSPG4 and S100B by Real-Time PCR. There was a synergistic inhibitory effect of TGFï¢1 and nAG on the expression of CSPG4 by Western blot. CONCLUSION: The different responses of normal astrocytes in vitro versus astrocytes in vivo in the setting of injury or disease. The response at the gene level might not coincide with the response at the protein level.
Asunto(s)
Proteínas Anfibias/farmacología , Astrocitos/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Salamandridae , Factor de Crecimiento Transformador beta1/farmacología , Animales , Astrocitos/metabolismo , Astrocitos/fisiología , Técnicas de Cultivo de Célula , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Interleucina-6/metabolismo , Proteínas de la Membrana/metabolismo , Subunidad beta de la Proteína de Unión al Calcio S100/metabolismoRESUMEN
The source of HCV transmission in Saudi Arabia is unknown. This study aimed to determine HCV genotypes in a representative sample of chronically infected patients in Saudi Arabia. All HCV isolates were genotyped and subtyped by sequencing of the HCV core region and 54 new HCV isolates were identified. Three sets of primers targeting the core region were used for both amplification and sequencing of all isolates resulting in a 326 bp fragment. Most HCV isolates were genotype 4 (85%), whereas only a few isolates were recognized as genotype 1 (15%). With the assistance of Genbank database and BLAST, subtyping results showed that most of genotype 4 isolates were 4d whereas most of genotype 1 isolates were 1b. Nucleotide conservation and variation rates of HCV core sequences showed that 4a and 1b have the highest levels of variation. Phylogenetic analysis of sequences by Maximum Likelihood and Bayesian Coalescent methods was used to explore the source of HCV transmission by investigating the relationship between Saudi Arabia and other countries in the Middle East and Africa. Coalescent analysis showed that transmissions of HCV from Egypt to Saudi Arabia are estimated to have occurred in three major clusters: 4d was introduced into the country before 1900, the major 4a clade's MRCA was introduced between 1900 and 1920, and the remaining lineages were introduced between 1940 and 1960 from Egypt and Middle Africa. Results showed that no lineages seem to have crossed from Egypt to Saudi Arabia in the last 15 years. Finally, sequencing and characterization of new HCV isolates from Saudi Arabia will enrich the HCV database and help further studies related to treatment and management of the virus.
Asunto(s)
Hepacivirus/genética , Hepatitis C Crónica/epidemiología , Hepatitis C Crónica/virología , Antivirales/uso terapéutico , Secuencia de Bases , Teorema de Bayes , Egipto , Femenino , Genotipo , Geografía , Hepacivirus/aislamiento & purificación , Humanos , Funciones de Verosimilitud , Masculino , Cadenas de Markov , Filogenia , Reacción en Cadena de la Polimerasa , ARN Viral/genética , Arabia SauditaRESUMEN
OBJECTIVE: To evaluate the therapeutic properties of nAG protein during the recovery following acute spinal cord injuries in the rat. STUDY DESIGN: An experimental study. PLACE AND DURATION OF STUDY: King Saud University, Riyadh, Saudi Arabia, from September 2014 to September 2015. METHODOLOGY: Eight rats were studied (4 control rats and 4 experimental rats; and hence 50% were controls and 50% were experimental). All rats were subjected to an acute spinal cord injury using the aneurysmal clip injury model. Immediately after the injury, a single intra-dural injection of either normal saline (in the control group) or the nAG protein (in the experimental group) was done. Assessment of both groups was done over a 6-week period with regard to weight maintenance, motor recovery scores, MRI and histopathology of the injury site. RESULTS: Weight maintenance was seen in the experimental and not in the control rats. Starting at 3 weeks after injury, the motor recovery was significantly (p<0.05) better in the experimental group. MRI assessment at 6 weeks showed better maintenance of cord continuity and less fluid accumulation at the injury site in the nAG-treated group. Just proximal to the injury site, there was less gliosis in the experimental group compared to the control group. At the crush injury site, there was less tissue architecture distortion, less vacuole formation, and less granulation tissue formation in the experimental group. CONCLUSION: The local injection nAG protein enhances neuro-restoration, reduces gliosis, and reduces vacuole/ granulation tissue formation following acute spinal cord crush injury in the rat aneurysmal clip animal model.
Asunto(s)
Lesiones por Aplastamiento/terapia , Factor 15 de Diferenciación de Crecimiento/administración & dosificación , Traumatismos de la Médula Espinal/terapia , Animales , Inyecciones Espinales , Masculino , Ratas , Ratas Sprague-Dawley , Resultado del TratamientoRESUMEN
BACKGROUND/AIMS: The lack of a reliable cell culture system allowing persistent in vitro hepatitis C virus (HCV) propagation is still restraining the search for novel antiviral strategies. HepG2 cells transfection with HCV allows for viral replication. However, the replication is weak presumably because of HepG2 lack of miRNA-122, which is essential for viral replication. Other agents such as polyethylene glycol (PEG) and dimethyl sulfoxide (DMSO) have been shown to increase the efficiency of infection with other viruses. This study included comparison of HCV genotype 4 5'UTR and core RNA levels and HCV core protein expression at different time intervals in the absence or presence of PEG and/or DMSO postinfection. MATERIALS AND METHODS: We used serum with native HCV particles in infecting HepG2 cells in vitro. HCV replication was assessed by reverse transcriptase polymerase chain reaction for detection of HCV RNA and immunofluorescence and flow cytometry for detection of HCV core protein. RESULTS: HCV 5'UTR and core RNA expression was evident at different time intervals after viral infection, especially after cells were treated with PEG. HCV core protein was also evident at different time intervals using both immunofluorescence and flow cytometry. PEG, not DMSO, has increased the HCV core protein expression in the treated cells, similar to its effect on viral RNA expression. CONCLUSIONS: These expression profiles suggest that the current model of cultured HepG2 cells allows the study of HCV genotype 4 replication and different stages of the viral life cycle.
Asunto(s)
Carcinoma Hepatocelular/virología , Hepacivirus/fisiología , Neoplasias Hepáticas/virología , Regiones no Traducidas 5' , Dimetilsulfóxido/farmacología , Genotipo , Células Hep G2 , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Humanos , Polietilenglicoles/farmacología , ARN Viral/genética , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND: nAG and Prod-1 are proteins responsible for the regeneration of completely amputated limbs in salamanders (which are lower vertebrates). The purpose of this study was to introduce an experimental distal phalanx amputation model in mice (which are higher vertebrates) in which nAG and Prod-1 are expressed in the amputation stumps. METHODS: Sixteen mice with amputation of the distal two-thirds of the distal phalanx were used. One hind limb was used and the central three digits were amputated. Injection of nAG and Prod-1 plasmids was performed in the footpad twice weekly in experimental mice (n = 8), and injection of solution only (without the plasmids) was performed twice weekly in control mice (n = 8). RESULTS: nAG and Prod-1 were expressed in experimental stumps only. This expression results in quicker and more mature bone regeneration in experimental animals, and this was shown using histology and immune stains to osteocalcin (an osteoblast marker). Finally, quantitative mRNA showed a 21-fold increase of osteocalcin in experimental stumps compared with control stumps, and this was statistically significant. CONCLUSION: Injection of nAG and Prod-1 into the footpad will result in their expression in the distal amputation stumps, and this will enhance bone regeneration in the model described.
Asunto(s)
Muñones de Amputación/patología , Regeneración Ósea/efectos de los fármacos , Citocromo P-450 CYP2B1/farmacología , Proteínas/farmacología , Amputación Quirúrgica/métodos , Animales , Biomarcadores/metabolismo , Biopsia con Aguja , Citocromo P-450 CYP2B1/metabolismo , Modelos Animales de Enfermedad , Inmunohistoquímica , Ratones , Ratones Endogámicos , Proteínas/metabolismo , ARN/metabolismo , Distribución Aleatoria , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sensibilidad y EspecificidadRESUMEN
Generally speaking, the excessive expression of myofibroblasts is associated with excessive collagen production. One exception is seen in patients and animal models of Ehlers-Danlos syndrome type IV in which the COL3A1 gene mutation results in reduced collagen III but with concurrent increased myofibroblast expression. This paradox has not been examined with the use of external drugs/modalities to prevent hypertrophic scars. In this paper, we injected the rabbit ear wound model of hypertrophic scarring with two doses of a protein called nAG, which is known to reduce collagen expression and to suppress hypertrophic scarring in that animal model. The higher nAG dose was associated with significantly less collagen III expression and concurrent higher degree of myofibroblast expression. We concluded that collagen III content of the extracellular matrix may have a direct or an indirect effect on myofibroblast differentiation. However, further research is required to investigate the pathogenesis of this paradoxical phenomenon.
Asunto(s)
Colágeno Tipo III/metabolismo , Miofibroblastos/metabolismo , Enfermedades de la Piel/metabolismo , Heridas y Lesiones/metabolismo , Animales , Diferenciación Celular/fisiología , Cicatriz Hipertrófica/metabolismo , Modelos Animales de Enfermedad , Síndrome de Ehlers-Danlos/metabolismo , Matriz Extracelular/metabolismo , Humanos , ConejosRESUMEN
The current study was designed to determine the Hepatitis C Virus (HCV) genotypes in a representative sample of HCV chronically infected patients in Saudi Arabia. All HCV isolates were genotyped by sequencing of the 5'UTR region and newly identified HCV isolates were identified. Specific universal primers targeting 5'UTR region were used for both amplification and sequencing of all isolates that resulted in 244 bp fragment which represent about 80% of 5'UTR region. Most of HCV isolates in this study were genotype 4 (76.4%) where only few isolates were recognized as genotype 1 (19.6%). All results were compared to HCV reference sequences from LOS ALAMOS HCV database, considering only the complete full genomes for the main phylogenetic analysis. Sequences that showed maximum identity (98% -100%) were selected. Most isolates were identical with HCV genotype 4 references. Some isolates were similar to different subtypes of HCV genotypes 4, 1 and 6. Phylogenetic analysis showed resemblance of most isolates to similar ones from the Far East, North America and Egypt. Using sequence Weblogo, Alignment analysis of isolated HCV genotypes 4 and 1 showed 92% and 95.5% nucleotide conservation, respectively. There was no predominant nucleotide in the varied sites, in both genotypes. All isolated sequences were submitted to GenBank database.
Asunto(s)
Regiones no Traducidas 5' , Genotipo , Hepacivirus/genética , Hepatitis C Crónica/genética , ARN Viral/genética , Análisis de Secuencia de ARN , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Bases de Datos de Ácidos Nucleicos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Filogenia , Arabia Saudita , Alineación de SecuenciaRESUMEN
nAG (newt-Anterrior Gradient) protein is the key mediator of regrowth of amputated limbs in salamanders. In a previous work in our lab, a new nAG gene (suitable for humans) was designed and cloned. The cloned vector was transfected into primary human fibroblasts. The expression of nAG in human primary fibroblasts was found to suppress collagen expression. The current study shows that local injection of recombinant nAG reduces scar hypertrophy in the rabbit ear model. This is associated with lower scar elevation index (SEI), lower levels of collagen I & III, higher levels of MMP1, and a higher degree of scar maturation in experimental wounds compared to controls.
Asunto(s)
Proteínas Anfibias/administración & dosificación , Productos Biológicos/administración & dosificación , Cicatriz Hipertrófica/tratamiento farmacológico , Cicatriz Hipertrófica/patología , Enfermedades del Oído/tratamiento farmacológico , Enfermedades del Oído/patología , Urodelos/metabolismo , Proteínas Anfibias/genética , Animales , Colágeno/metabolismo , Enfermedades del Oído/metabolismo , Inyecciones Intralesiones , Conejos , Proteínas Recombinantes/administración & dosificación , Resultado del Tratamiento , Urodelos/genética , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Unlike humans, salamanders regrow their amputated limbs. Regeneration depends on the presence of regenerating axons which upregulate the expression of newt anterior gradient (nAG) protein. We had the hypothesis that nAG might have an inhibitory effect on collagen production since excessive collagen production results in scarring, which is a major enemy to regeneration. nAG gene was designed, synthesized, and cloned. The cloned vector was then transfected into primary human fibroblasts. The results showed that the expression of nAG protein in primary human fibroblast cells suppresses the expression of collagen I and III, with or without TGF- ß 1 stimulation. This suppression is due to a dual effect of nAG both by decreasing collagen synthesis and by increasing collagen degradation. Furthermore, nAG had an inhibitory effect on proliferation of transfected fibroblasts. It was concluded that nAG suppresses collagen through multiple effects.