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OBJECTIVE: To investigate intermediate-term clinical results in patients with concomitant anterior cruciate ligament (ACL) reconstruction and chondral defect treated with high-density autologous chondrocyte implantation (HD-ACI) compared to patients without ACL tear but with a chondral lesion and HD-ACI treatment. DESIGN: Forty-eight patients with focal chondral lesions underwent HD-ACI (24 with ACL reconstruction after an ACL injury and 24 with an intact ACL). Follow-up assessments occurred at 6, 12, and 24 months. Patient-reported knee function and symptoms were assessed using the International Knee Documentation Committee (IKDC) questionnaire, pain was measured using the Visual Analog Scale (VAS), and adverse events were monitored. Physical activity was assessed using the Tegner Activity Level Scale, and cartilage healing was evaluated with the Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) score. RESULTS: No significant adverse events occurred during follow-up. Both groups showed significant improvements at 2 years compared to baseline (VAS: 8.0 ± 1.3 to 1.4 ± 2.0 [normal ACL]; 7.4 ± 2.3 to 2.1 ± 2.3 [ACL reconstruction]; IKDC: 39.2 ± 10.6 to 76.1 ± 22.0 [intact ACL]; 35.6 ± 12.1 to 74.6 ± 20.9 [ACL reconstruction]). Patients in both groups exceeded the minimal clinically important difference (MCID) for IKDC scores. The Tegner Activity Level Scale decreased immediately after surgery and increased after 2 years, with 70.6% (normal ACL) and 89.5% (ACL reconstruction) returning to their preinjury activity levels. No significant differences in the MOCART score were observed between the groups. CONCLUSIONS: ACL reconstruction does not appear to reduce the outcomes (at 2 years) of HD-ACI.
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Hyaline cartilage's inability to self-repair can lead to osteoarthritis and joint replacement. Various treatments, including cell therapy, have been developed for cartilage damage. Autologous chondrocyte implantation (ACI) is considered the best option for focal chondral lesions. In this article, we aimed to create a narrative review that highlights the evolution and enhancement of our chondrocyte implantation technique: High-Density-ACI (HD-ACI) Membrane-assisted Autologous Chondrocyte Implantation (MACI) improved ACI using a collagen membrane as a carrier. However, low cell density in MACI resulted in softer regenerated tissue. HD-ACI was developed to improve MACI, implanting 5 million chondrocytes per cm2, providing higher cell density. In animal models, HD-ACI formed hyaline-like cartilage, while other treatments led to fibrocartilage. HD-ACI was further evaluated in patients with knee or ankle defects and expanded to treat hip lesions and bilateral defects. HD-ACI offers a potential solution for cartilage defects, improving outcomes in regenerative medicine and cell therapy. HD-ACI, with its higher cell density, shows promise for treating chondral defects and advancing cartilage repair in regenerative medicine and cell therapy.
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PURPOSE: Two-year follow-up to assess efficacy and safety of high-density autologous chondrocyte implantation (HD-ACI) in patients with cartilage lesions in the ankle. DESIGN: Twenty-four consecutive patients with International Cartilage repair Society (ICRS) grade 3-4 cartilage lesions of the ankle were included. Five million chondrocytes per cm2 of lesion were implanted using a type I/III collagen membrane as a carrier and treatment effectiveness was assessed by evaluating pain with the visual analogue scale (VAS) and American Orthopaedic Foot & Ankle Society (AOFAS) ankle-hindfoot score at baseline, 12-month, and 24-month follow-up, together with dorsal and plantar flexion. Magnetic resonance observation for cartilage repair tissue (MOCART) score was used to evaluate cartilage healing. Histological study was possible in 5 cases. RESULTS: Patients' median age was 31 years (range 18-55 years). Median VAS score was 8 (range 5-10) at baseline, 1.5 (range 0-8) at 12-month follow-up, and 2 (rang e0-5) at 24-month follow-up (P < 0.001). Median AOFAS score was 39.5 (range 29-48) at baseline, 90 (range 38-100) at 12-month follow-up, and 90 (range 40-100) at 24-month follow-up (P < 0.001). Complete dorsal flexion significantly increased at 12 months (16/24, 66.7%) and 24 months (17/24, 70.8%) with regard to baseline (13/24, 54.2%) (P = 0.002). MOCART at 12- and 24-month follow-ups were 73.71 ± 15.99 and 72.33 ± 16.21. Histological study confirmed that neosynthetized tissue was cartilage with hyaline extracellular matrix and numerous viable chondrocytes. CONCLUSION: HD-ACI is a safe and effective technique to treat osteochondral lesions in the talus, providing good clinical and histological results at short- and mid-term follow-ups.
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Fracturas Intraarticulares , Astrágalo , Adolescente , Adulto , Tobillo , Articulación del Tobillo/cirugía , Condrocitos , Humanos , Persona de Mediana Edad , Trasplante Autólogo , Adulto JovenAsunto(s)
Cartílago Articular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Glucuronidasa/uso terapéutico , Osteoartritis de la Rodilla/tratamiento farmacológico , Receptor Tipo II de Factor de Crecimiento Transformador beta/uso terapéutico , Animales , Cartílago Articular/patología , Técnicas de Cultivo de Célula , Condrocitos/patología , Humanos , Proteínas Klotho , Osteoartritis de la Rodilla/inducido químicamente , Papaína , Ratas , Proteínas Recombinantes/uso terapéuticoRESUMEN
DESIGN: In the process of cell division, the extremes of the eukaryotic chromosomes are progressively shortening, and this phenomenon is related to cell degeneration and senescence. The treatment of cartilage lesions with autologous chondrocytes implies that cells proliferate in an artificial environment. We have studied the viability of cultured chondrocytes after measurement of their telomere length before implantation. METHODS: Articular cartilage biopsies (B1, B2, and B3) were obtained from 3 patients (2 males and 1 female) with knee cartilage defects, who were going to be treated with chondrocyte implantation. Chondrocytes were cultured in DMEM with autologous serum. After the third passage, an aliquot of 1 million cells was removed to estimate the telomere length and the remaining cells were implanted. Telomere length was measured by quantitative fluorescent in situ hybridization (Q-FISH). Patients' clinical outcome was determined preoperatively, and 12 and 24 months postimplantation with the International Knee Documentation Committee (IKDC) questionnaire. RESULTS: After chondrocyte implantation, IKDC score doubled at 12 and 24 months with regard to the basal value. After 3 passages, chondrocytes were cultured for a mean of 45.67 days, the mean duplication time being 4.53 days and the mean number of cell divisions being 10.04 during the culture period. The 20th percentile of telomere lengths were 6.84, 6.96, and 7.06 kbp and the median telomere lengths 10.30, 10.47, and 10.73 kbp, respectively. No significant correlation was found between IKDC score and telomere length. CONCLUSION: Culturing autologous chondrocytes for implantation is not related to cell senescence in terms of telomere length.
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Enfermedades de los Cartílagos/patología , Cartílago Articular/citología , Condrocitos/patología , Trasplante de Células Madre , Telómero/patología , Adulto , Enfermedades de los Cartílagos/terapia , Cartílago Articular/patología , Células Cultivadas , Femenino , Humanos , Hibridación Fluorescente in Situ , Articulación de la Rodilla/citología , Articulación de la Rodilla/patología , Masculino , Trasplante AutólogoRESUMEN
OBJECTIVE: The aim of this work was to study the short- and mid-term effectiveness and safety of high-density autologous chondrocyte implantation (HD-ACI) in the first 50 patients with knee cartilage damage treated in our unit. DESIGN: Fifty consecutive patients with cartilage lesions (Outerbridge grade III-IV) in the knee treated with HD-ACI were included in this study. Chondrocytes were isolated from a nonbearing cartilage area biopsy and were cultured until 40 to 50 million cells were obtained. Five million chondrocytes per cm2 of a porcine collagen type I/III membrane were implanted covering the defect. Procedure effectiveness was assessed by evaluating pain, swelling, and range of mobility (flexion and extension) at 6-, 12-, and 24-month follow-up. The International Knee Documentation Committee (IKDC) subjective evaluation form was used to evaluate symptoms and functions of the knee. RESULTS: The percentage of patients with pain and swelling decreased progressively in the following visits, with differences being statistically significant ( P < 0.001 and P = 0.040, respectively). IKDC scores improved progressively throughout the 24-month follow-up ( P < 0.001). Thus, the mean IKDC score improvement was 26.3 points (95% confidence interval [CI] = 18.2-34.4 points) at 12 months and 31.0 points (95% CI = 22.9-39 points) at 24 months. No significant differences were found when performing extension ( P = 0.112). Flexion significantly improved by 25.1° at 24-month follow-up ( P = 0.013). CONCLUSIONS: HD-ACI is a safe and effective technique for the treatment of cartilage defects, improving clinical and subjective perception of knee functionality. These preliminary results encourage future studies comparing this technique with traditional ACI.
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Artroplastia Subcondral/métodos , Enfermedades de los Cartílagos/cirugía , Cartílago Articular/cirugía , Condrocitos/trasplante , Adolescente , Adulto , Animales , Femenino , Estudios de Seguimiento , Humanos , Articulación de la Rodilla/cirugía , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Porcinos , Trasplante Autólogo , Resultado del Tratamiento , Adulto JovenRESUMEN
OBJECTIVE: To study if a culture of chondrocytes can be obtained from pathologic hyaline cartilage (PHC) fragments. DESIGN: Twenty-five men and 9 women with osteochondritis dissecans (OCD) in 11 cases, arthrosis in 13 patients, and trauma in the remaining 10 cases were included. The PHC fragments and a small sample of the next healthy cartilage were extracted by arthroscopy. According to the appearance, the PHC samples were divided into fixed (3 cases), flapped (6 patients), or loose bodies (25 cases), depending on the attachment degree of the cartilage to the subchondral bone. Approximately half of each pathologic sample and the whole healthy one were digested to isolate the cells trying to establish the cell culture. RESULTS: We were able to establish a cell culture in 7 out of 34 (20.6%) PHC samples (positive samples), whereas in the remaining 27 (79.4%) no cell growth was observed (negative samples). Most of the negative samples were loose bodies (P = 0.005) taken from patients with OCD or arthrosis (P = 0.001) with an evolution time of more than 1 year (P < 0.001). The best binary logistic regression model (P < 0.001) showed that the only factor affecting the establishment of cell culture was the evolution time (P = 0.044). CONCLUSION: It is possible to culture chondrocytes from osteochondral fragments if they are traumatic, within a year of injury and not from fragments due to arthrosis or OCD.
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BACKGROUND: The factors associated with anterior cruciate ligament (ACL) tears are not completely clear. Some studies have shown that patients with a narrow intercondylar notch have a predisposition for ACL tears. PURPOSE: To determine the relationship between the α angle and intercondylar notch width measurements and ACL tears. STUDY DESIGN: Case-control study; Level of evidence, 3. METHODS: A total of 530 patients (308 with ACL rupture, 222 with healthy ACLs) were included in this study. The α angle and intercondylar width were measured from magnetic resonance images (MRIs). Binary logistic regression analysis was performed to determine the influence of the variables on ACL status (normal or torn). Odds ratios (ORs) and their respective 95% CIs were also calculated. RESULTS: No significant differences in patient age and the affected knee were found between patients with normal or torn ACLs. The mean α angle was higher in patients with a torn ACL than in those with an intact one (57.5° ± 5.5° vs 56.2° ± 4.5°; P = .009). Intercondylar width was significantly lower in patients with a torn ACL than in those with an intact one (18.2 ± 3.1 vs 19.5 ± 3.6 mm; P < .001). A highly significant difference between men and women was found for mean intercondylar notch width (19.3 ± 3.3 vs 17.4 ± 3.1 mm; P < .001). In a logistic regression model, sex, intercondylar width, and α angle were statistically significant when adjusted for age. CONCLUSION: Study results suggest that the ACL tears are associated with a narrow intercondylar notch and a high α angle, and that tears occur more frequently in men than in women. CLINICAL RELEVANCE: The model proposed in this study could be used by the physician in the medical office as a tool to identify the risk factors that may predispose a patient for a potential ACL tear.
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BACKGROUND: We hypothesized that implanting cells in a chondral defect at a density more similar to that of the intact cartilage could induce them to synthesize matrix with the features more similar to that of the uninjured one. METHODS: We compared the implantation of different doses of chondrocytes: 1 million (n = 5), 5 million (n = 5), or 5 million mesenchymal cells (n = 5) in the femoral condyle of 15 sheep. Tissue generated by microfracture at the trochlea, and normal cartilage from a nearby region, processed as the tissues resulting from the implantation, were used as references. Histological and molecular (expression of type I and II collagens and aggrecan) studies were performed. RESULTS: The features of the cartilage generated by implantation of mesenchymal cells and elicited by microfractures were similar and typical of a poor repair of the articular cartilage (presence of fibrocartilage, high expression of type I collagen and a low mRNA levels of type II collagen and aggrecan). Nevertheless, in the samples obtained from tissues generated by implantation of chondrocytes, hyaline-like cartilage, cell organization, low expression rates of type I collagen and high levels of mRNA corresponding to type II collagen and aggrecan were observed. These histological features, show less variability and are more similar to those of the normal cartilage used as control in the case of 5 million cells implantation than when 1 million cells were used. CONCLUSIONS: The implantation of autologous chondrocytes in type I/III collagen membranes at high density could be a promising tool to repair articular cartilage.
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Occult hepatitis C virus (HCV) infection of patients with abnormal liver function tests of unknown origin who are anti-HCV and serum HCV RNA negative but who have HCV RNA in the liver has been described. As HCV replicates in the liver cells of these patients, it could be that the amount of circulating viral particles is under the detection limit of the most sensitive techniques. To prove this hypothesis, serum samples from 106 patients with occult HCV infection were analyzed. Two milliliters of serum was ultracentrifuged over a 10% sucrose cushion for 17 h at 100,000 x g(av), where av means average, and HCV RNA detection was performed by strand-specific real-time PCR. Out of the 106 patients, 62 (58.5%) had detectable serum HCV RNA levels after ultracentrifugation, with a median load of 70.5 copies/ml (range, 18 to 192). Iodixanol density gradient studies revealed that HCV RNA was positive at densities of 1.03 to 1.04 and from 1.08 to 1.19 g/ml, which were very similar to those found in the sera of patients with classical chronic HCV infection. Antigenomic HCV RNA was found in the livers of 56 of 62 (90.3%) patients with detectable serum HCV RNA levels after ultracentrifugation, compared to 27 of 44 (61.4%) negative patients (P < 0.001). No differences in the median loads of antigenomic HCV RNA between patients with an those without serum HCV RNA (4.5 x 10(4) [range, 7.9 x 10(2) to 1.0 x 10(6)] versus 2.3 x 10(4) [range, 4.0 x 10(2) to 2.2 x 10(5)]) were found. Alanine aminotransferase and gamma-glutamyl transpeptidase levels, liver necroinflammatory activity, and fibrosis did not differ between both groups. In conclusion, HCV RNA can be detected in the sera of patients with occult HCV infection after circulating viral particles are concentrated by ultracentrifugation.
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Hepacivirus/aislamiento & purificación , Hepatitis C/diagnóstico , ARN Viral/sangre , Secuencia de Bases , Cartilla de ADN , Femenino , Hepacivirus/genética , Hepatitis C/sangre , Hepatitis C/virología , Humanos , Hibridación in Situ , Masculino , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , UltracentrifugaciónRESUMEN
Occult hepatitis B virus (HBV) and occult hepatitis C virus (HCV) infection are two recently described different forms of HBV and HCV infections. This work compares the clinical, virologic, and histologic characteristics of patients with occult dual infection to those of patients with single occult HBV or HCV infection. Seventy-six patients with abnormal liver function tests of unknown etiology (serum HBsAg, anti-HCV, HBV-DNA, and HCV-RNA negative) were included in the study. Viral genomes were tested in liver by real-time PCR and confirmed by in situ hybridization. Of the 76 patients, 17 had occult HBV infection (intrahepatic HBV-DNA positive, HCV-RNA negative), 35 had occult HCV infection (intrahepatic HCV-RNA positive, HBV-DNA negative) and 24 occult dual infection (intrahepatic HCV-RNA and HBV-DNA). No differences among the three groups were found regarding clinical and epidemiologic data. The median load of intrahepatic genomic and antigenomic HCV-RNA strands was similar between single occult HCV infection and occult HBV and HCV dual infection. The percentage of HCV-infected hepatocytes did not differ between these groups. In occult single HBV infection, intrahepatic levels of HBV-DNA and percentage of HBV-infected hepatocytes were similar to the group of patients with occult dual infection. Finally, no differences were found in histological liver damage among the three groups. In conclusion, liver disease in patients with occult dual infection was not more severe than in patients with single occult HBV or occult HCV infection. Moreover, in occult dual infection there is no a reciprocal inhibition of the viral genomes.
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Hepatitis B/patología , Hepatitis B/virología , Hepatitis C/patología , Hepatitis C/virología , Adulto , ADN Viral/análisis , ADN Viral/sangre , Femenino , Hepacivirus , Hepatitis B/complicaciones , Hepatitis B/fisiopatología , Antígenos de Superficie de la Hepatitis B/sangre , Virus de la Hepatitis B , Hepatitis C/complicaciones , Hepatitis C/fisiopatología , Anticuerpos contra la Hepatitis C/sangre , Hepatocitos/virología , Humanos , Hibridación in Situ , Hígado/virología , Pruebas de Función Hepática , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , ARN Viral/análisis , ARN Viral/sangre , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Positive-strand hepatitis C virus (HCV) RNA has been detected in the livers of patients who have achieved a sustained biochemical and virological response to antiviral therapy (hereafter, referred to as sustained responders), but negative-strand HCV RNA was undetectable in the hepatic tissue of these patients. We studied the presence of both positive- and negative-strand HCV RNA in the livers of 20 sustained responders with chronic hepatitis C whose response persisted for a mean (+/- standard deviation [SD]) of 47.4+/-32.8 months after treatment. METHODS: HCV RNA was tested by strand-specific, real-time reverse-transcriptase polymerase chain reaction and by in situ hybridization in posttreatment liver biopsy samples (obtained a mean [+/- SD] 35.4+/-35.0 months after therapy) and in patients' peripheral blood mononuclear cells. RESULTS: Positive-strand HCV RNA was found in 19 (95%) of 20 liver biopsy specimens, and negative-strand HCV RNA was found in 15 (79%) of the 19 samples that had positive-strand HCV RNA. These results were confirmed by in situ hybridization. Regarding peripheral blood mononuclear cells, 13 (65%) of 20 samples had positive-strand HCV RNA, and negative-strand HCV RNA was detected in 12 (92%) of the 13 samples with positive-strand HCV RNA. Liver necroinflammation was still present in the posttreatment liver biopsy specimens of 15 patients, and fibrosis was present in 7, although liver damage improved in all but 2 patients. CONCLUSIONS: HCV persisted and replicated in the livers and peripheral blood mononuclear cells of most sustained responders. Thus, these patients did not experience HCV infection clearance, despite apparent clinical disease resolution.
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Hepacivirus/fisiología , Hígado/virología , ARN Viral/análisis , Replicación Viral/fisiología , Adulto , Antivirales/farmacología , Femenino , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Hepatitis C , Humanos , Hígado/efectos de los fármacos , Masculino , Persona de Mediana Edad , ARN Viral/sangre , Carga ViralRESUMEN
Hepatitis C virus (HCV) RNA persistence in the liver has been described even after apparent resolution of HCV infection. Because T-cell reactivity plays a role in recovery from HCV infection, virus-specific T-cell responses were investigated in apparently recovered individuals in whom hepatic HCV RNA persistence was documented: 15 sustained virological responders to interferon (IFN)-treatment and 9 asymptomatic aviremic anti-HCV carriers. HCV-specific CD4(+) T-cell proliferative responses were detected significantly more often in apparently recovered individuals (sustained virological responders: 60%; asymptomatic anti-HCV carriers: 66%) compared with 50 chronic hepatitis C patients (28%; P < 0.05). However, T-cell frequencies and numbers tended to decline over time and the number of HCV proteins targeted by CD4(+) T-cell proliferative responses was limited. Interestingly, liver viral load correlated inversely with virus-specific immune responses. Thus, CD4(+) T-cell responders showed significantly lower hepatic HCV RNA levels (P < 0.05). HCV-specific IFN-gamma-secreting CD4(+) T-cells were not detected in all the apparently recovered patients although they were found significantly more often compared with chronic hepatitis C patients (P < 0.05). Also, HCV NS3-specific CD8(+) T-cells were detected in 11 HLA-A2-positive apparently recovered individuals (8 sustained virological responders and 3 asymptomatic anti-HCV carriers); T-cell frequencies tended to be greater in those patients who had lower hepatic viral levels. In conclusion, HCV-specific T-cells are detectable in apparently recovered individuals in whom HCV RNA can persist in the liver indicating that HCV replication may be prolonged in the face of an insufficient or inadequate virus-specific CD4(+) and CD8(+) T-cell response.
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Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Hepacivirus/aislamiento & purificación , Hepatitis C/inmunología , Hígado/virología , Subgrupos de Linfocitos T/inmunología , Adulto , Anciano , Biomarcadores , Biopsia , Portador Sano , Células Cultivadas , Femenino , Antígeno HLA-A2 , Hepacivirus/genética , Hepacivirus/inmunología , Hepatitis C/tratamiento farmacológico , Hepatitis C/patología , Humanos , Interferón gamma/análisis , Leucocitos Mononucleares , Hígado/patología , Activación de Linfocitos , Masculino , Persona de Mediana Edad , ARN Viral/genética , Especificidad del Receptor de Antígeno de Linfocitos T , Proteínas del Núcleo Viral/inmunología , Proteínas no Estructurales Virales/inmunología , Replicación Viral/inmunologíaRESUMEN
BACKGROUND: It is unknown whether hepatitis C virus (HCV) is present in the liver of anti-HCV antibody-positive patients with persistently normal alanine aminotransferase (ALT) levels and undetectable serum HCV RNA levels. METHODS: We determined the presence of genomic and antigenomic HCV RNA strands in liver biopsy specimens and peripheral blood mononuclear cell (PBMC) samples obtained from 12 anti-HCV antibody-positive patients who had normal ALT levels and who had been serum HCV RNA negative for at least 12 months, according to the results of quantitative, strand-specific, real-time reverse-transcription-polymerase chain reaction and, also, in situ hybridization of liver cells. Intrahepatic HCV RNA was cloned and sequenced. RESULTS: All patients remained anti-HCV antibody positive and serum HCV RNA negative, and all had normal ALT values during follow-up (mean duration +/- SD, 29.2 +/- 19.8 months). Genomic HCV RNA was detected in liver biopsy specimens obtained from 10 (83%) of 12 patients, and the antigenomic strand was detected in 10 (100%) of 10 liver biopsy specimens in which genomic HCV RNA was detected. Results were confirmed by in situ hybridization. Intrahepatic HCV was of genotype 1b, and HCV sequencing demonstrated no cross-contamination among samples. Genomic HCV RNA was found in 6 (50%) of 12 PBMC samples, and antigenomic HCV RNA was also detected in 5 (83%) of these 6 PBMC samples. CONCLUSION: HCV may persist and replicate in the liver and PBMCs of healthy, anti-HCV antibody-positive, serum HCV RNA-negative patients who have persistently normal ALT levels. These patients should be followed up, because they have an ongoing viral infection.
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Alanina Transaminasa/sangre , Hepacivirus/aislamiento & purificación , Anticuerpos contra la Hepatitis C/sangre , Hígado/virología , ARN Viral/análisis , Adulto , Biopsia , Cartilla de ADN/química , Femenino , Genotipo , Hepacivirus/clasificación , Hepacivirus/genética , Hepacivirus/inmunología , Humanos , Hibridación in Situ , Leucocitos Mononucleares/fisiología , Hígado/patología , Masculino , Persona de Mediana Edad , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factores de TiempoRESUMEN
Pegylated alpha-interferon plus ribavirin is the current therapy for chronic hepatitis C virus (HCV) infection. Serum HCV-RNA concentration before treatment has been identified as an independent predictive factor of response. We have compared the percentage of HCV-infected hepatocytes with the concentration of serum HCV-RNA in baseline samples as predictors of response. We included 97 patients with chronic HCV infection (genotype 1), treated with pegylated-interferon-alpha2b plus ribavirin. Of these 97, 38 (39%) were sustained responders and 59 (61%) were not. Statistical differences between responders and nonresponders were found regarding the percentage of infected hepatocytes (6.83+/-4.50% versus 13.44+/-10.05%; P=0.00003) but not in serum HCV-RNA concentration [1.71+/-2.70 (x10(6) IU/L) versus 1.32+/-1.86 (x10(6) IU/L); P=0.40694]. Other factors associated with response were age, gamma-glutamyl transpeptidase level, and absence of previous therapy. Logistic regression demonstrated that percentage of infected hepatocytes (odds ratio, 1.160; 95% confidence interval, 1.065-1.264) and previous therapy (odds ratio, 0.294; 95% confidence interval, 0.109-0.795) were significant predictive factors for response. Therefore, the percentage of infected hepatocytes in liver biopsy before treatment is a better predictive factor of sustained response to 48 weeks of therapy with pegylated alpha-interferon plus ribavirin than serum HCV-RNA concentration in baseline serum sample.
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Hepacivirus/aislamiento & purificación , Hepatitis C/diagnóstico , Hepatitis C/tratamiento farmacológico , Hepatocitos/virología , Viremia/virología , Adulto , Biopsia , Femenino , Hepacivirus/efectos de los fármacos , Hepacivirus/fisiología , Hepatitis C/virología , Humanos , Hibridación in Situ , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Pronóstico , ARN Viral/genética , Curva ROC , Reproducibilidad de los Resultados , Resultado del Tratamiento , Viremia/sangreRESUMEN
This study analyzes if the profibrogenic factors nitric oxide and transforming growth factor-beta1 (TGF-beta1) affect hepatocyte nuclear factor-4alpha (HNF-4alpha) function. For this purpose, HepG2 cells were treated with TGF-beta1 or with a nitric oxide donor to determine mRNA levels of coagulation factor VII and HNF-4alpha. Treatment effect on factor VII gene promoter was assessed by chloramphenicol acetyl-transferase assays in cells transfected with the pFVII-CAT plasmid. HNF-4alpha binding and protein levels were determined by gel shift assays and Western blot. TGF-beta1 and nitric oxide downregulated factor VII mRNA levels by inhibiting its gene promoter activity. This inhibition is caused by a decrease in the DNA binding of HNF-4alpha. TGF-beta1 induces degradation of HNF-4alpha in the proteasome while nitric oxide provokes nitrosylation of cysteine residues in this factor. TGF-beta1 and nitric oxide inhibit HNF-4alpha activity. These findings may explain the loss of liver functions that occurs during fibrosis progression.
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Proteínas de Unión al ADN/metabolismo , Óxido Nítrico/metabolismo , Fosfoproteínas/metabolismo , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Línea Celular Tumoral , Cloranfenicol O-Acetiltransferasa/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Cisteína Endopeptidasas/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN/genética , Factor VII/genética , Factor VII/metabolismo , Fibrosis/metabolismo , Fibrosis/patología , Regulación de la Expresión Génica , Factor Nuclear 4 del Hepatocito , Humanos , Hígado/patología , Hígado/fisiología , Complejos Multienzimáticos/metabolismo , Fosfoproteínas/genética , Fosforilación , Regiones Promotoras Genéticas , Complejo de la Endopetidasa Proteasomal , Unión Proteica , ARN Mensajero/metabolismo , S-Nitroso-N-Acetilpenicilamina/metabolismo , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta1RESUMEN
BACKGROUND: There are patients in whom the etiology of long-standing abnormal results of liver-function tests is unknown (ALF-EU) after exclusion of all known causes of liver diseases. We analyzed the presence of hepatitis C virus (HCV) RNA in liver-biopsy specimens from 100 patients who were negative for anti-HCV antibodies and for serum HCV RNA and who had ALF-EU. METHODS: HCV RNA status was tested by reverse-transcription polymerase chain reaction (RT-PCR) and by in situ hybridization, in liver and peripheral-blood mononuclear cells (PBMCs). RESULTS: HCV RNA was detected in liver-biopsy specimens from 57 of 100 patients negative for anti-HCV antibodies and for serum HCV RNA (i.e., who had occult HCV infection). HCV RNA of negative polarity was found in the liver of 48 (84.2%) of these 57 patients with occult HCV infection. Nucleotide-sequence analysis confirmed the specificity of detection of HCV RNA and that patients were infected with the HCV 1b genotype. Of these 57 patients with intrahepatic HCV RNA, 40 (70%) had viral RNA in their PBMCs. With regard to liver histology, patients with occult HCV infection were more likely to have necroinflammatory activity (P=.017) and fibrosis (P=.022) than were patients without intrahepatic HCV RNA. CONCLUSIONS: Patients with ALF-EU may have intrahepatic HCV RNA in the absence of anti-HCV antibodies and of serum HCV RNA.
Asunto(s)
Hepacivirus/aislamiento & purificación , Hepatitis C/diagnóstico , Hepatopatías/etiología , Hígado/enzimología , ARN Viral/análisis , Adulto , Anciano , Alanina Transaminasa/sangre , Biopsia , Estudios de Cohortes , Femenino , Genotipo , Glutamil Aminopeptidasa/sangre , Hepacivirus/genética , Hepacivirus/patogenicidad , Hepatitis C/sangre , Hepatitis C/complicaciones , Anticuerpos contra la Hepatitis C/sangre , Humanos , Hibridación in Situ , Leucocitos Mononucleares/virología , Hígado/virología , Hepatopatías/enzimología , Pruebas de Función Hepática , Masculino , Persona de Mediana Edad , ARN Viral/sangre , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , gamma-Glutamiltransferasa/sangreRESUMEN
TT virus (TTV) is an unenveloped, single-stranded, circular-DNA virus which resembles members of the Circoviridae, that is commonly found in humans and which lacks pathological consequences for the infected host. TTV replication has been demonstrated in bone marrow cells but not in peripheral blood mononuclear cells (PBMC), suggesting that hematopoietic cells must be activated to support TTV replication. To test this hypothesis, PBMC from two naturally TTV-infected individuals and from two healthy TTV-DNA negative donors infected in vitro with a TTV-DNA-positive serum were cultured in the presence (stimulated) or absence (unstimulated) of phytohemagglutinin, lipopolysaccharide, and interleukin-2. TTV-DNA was detected in both stimulated and unstimulated PBMC. However, TTV-DNA replicative intermediates and mRNA were detected only in stimulated PBMC. Furthermore, TTV-DNA and mRNA were detected in PBMC from two TTV negative donors reinfected with supernatants from TTV-infected stimulated cells but not when using culture supernatants from unstimulated cells. These results demonstrate that TTV replicates in PBMC only when stimulated.