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Introduction: Bilateral knee chondral defects can be treated in one or two surgical acts. Objectives: To study clinical outcome of patients with bilateral knee chondral defects treated with High-Density Autologous Chondrocyte Implantation (HD-ACI) during the same surgery. Methods: Study on eight patients (4 women and 4 men) with chondral defects in both knees treated with HD-ACI during the same surgical act. Patients were evaluated 2, 6, 12 and 24 months post-op for treatment safety and efficacy: visual analogic scale (VAS) for pain, International Knee Documentation Committee (IKDC) for subjective patient's perception of both knees and EuroQol five-dimensional five-level questionnaire (EQ-5D-5 L) for quality of life. Integrity of neoformed tissue at 12 and 24-month follow-up was assessed by the magnetic resonance observation of cartilage repair tissue (MOCART) score. Results: VAS decreased from baseline to 24 months from a median of 8 (6 – 9) to a median of 0 (0 – 5) (P < .001). IKDC increased from a median value of 39.0 (17.2 – 48.3) in the basal visit to a median of 83.7 (24.0 – 98.0) in the 24-month visit (P < .001). EQ-5D-5 L decreased at 2 months post-op and increased throughout the follow-up visits thereafter, needing 24 months after surgery to reach a value higher than the basal one. Median MOCART at 12 months was 80.3 (58.0 – 89.7) and 81.3 (54.0 – 89.9) at 24 months. Conclusions: Treatment of both knees with HD-ACI during the same surgical act in patients with bilateral chondral lesions is a safe procedure, providing good clinical results.

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.

DesignIn 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.MethodsArticular 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.ResultsAfter 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.ConclusionCulturing autologous chondrocytes for implantation is not related to cell senescence in terms of telomere length.

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.

Background Futsal started being played in 1930 and the number of futsal players has increased all over the world ever since. Nonetheless, despite the fact that Spain is one of the most relevant national teams worldwide, information on the incidence of injuries and their anthropometric characteristics is sparse in this country. Aim To analyse medical assistance provided to players in their prematch concentration camps with the Spanish national team over five seasons, from 2010–2011 to 2014–2015, and also to collect data regarding anthropometric characteristics. Materials and methods This is a retrospective and detailed study of injuries players suffered over these five seasons. All variables were registered on an Excel spreadsheet and later analysed statistically. Results 411 injuries were studied in total. The dominant somatotype was mesomorph and the injured pivots were both the most endomorphic and the most mesomorphic. The most injured body structure was the hamstring muscles, occurring due to training and intrinsic mechanisms, where fatigue was the most frequent diagnosis. Only a few complementary examinations were carried out and prematch withdrawal was rare. Discussion The skinfold test total sum was lower than that of the Spanish 11-a-side players or than that in the lower category futsal Spanish players. In various research studies analysing exclusively injuries occurring in matches, the most frequent injury is ligament injury by extrinsic mechanism. The body mass index was not a useful parameter when assessing players’ appropriate weight. Most injuries occurred in training sessions, mostly by intrinsic mechanism; the highest percentage of traumatic injuries occurred in official matches.

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.

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 × 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 × 10 4 [range, 7.9 × 10 2 to 1.0 × 10 6 ] versus 2.3 × 10 4 [range, 4.0 × 10 2 to 2.2 × 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.

SUMMARY Background Hepatitis C virus replicates by the synthesis of an antigenomic HCV-RNA. As the end point of anti-viral therapy is to decrease viral replication, the amount of antigenomic HCV-RNA could influence the response. Aim To study if amounts of genomic and antigenomic HCV-RNA in the baseline liver biopsy are predictive factors of response to anti-viral therapy. Methods Eighty-eight patients with chronic HCV infection (anti-HIV-negative) treated with pegyltaed-interferon-α2b plus ribavirin for 12 months were included. Intrahepatic genomic and antigenomic HCV-RNA concentrations were determined by real-time polymerase chain reaction and percentage of infected hepatocytes by in situ hybridization. Results Of the 88 patients, 31% were responders while 69% were not. Median of antigenomic HCV-RNA in liver of responders and non-responders was 120 000 copies/μg RNA (range: 10 000–775 000) vs. 150 000 copies/μg RNA (range: 100–3 200 000; P = 0.38). Median of genomic HCV-RNA in liver of responders was 1 250 000 copies/μg RNA (range: 5000–9 000 000) and in non-responders 3 180 000 copies/μg RNA (range: 4600–18 000 000; P = 0.0191). Predictive factors of response in the logistic regression were: intrahepatic amount of genomic HCV-RNA, percentage of infected hepatocytes and previous therapy. Conclusion Response to 12 months of therapy with pegylated interferon-α2b plus ribavirin depends on the amount of genomic HCV-RNA in the pre-treatment liver biopsy.

We have recently described the presence of occult hepatitis C virus (HCV) infection (HCV-RNA in liver in the absence of anti-HCV and serum HCV-RNA) in patients with persistently abnormal liver function tests of unknown aetiology. The aim of this study was to compare the characteristics of patients with occult HCV infection vs those of patients with chronic hepatitis C. We compared clinical features of 68 patients with occult HCV infection and 69 untreated chronic HCV patients (anti-HCV and serum HCV-RNA positive), matched for age, gender, duration of abnormal liver function tests and body mass index. Aspartate aminotransferase and alanine aminotransferase were higher (P < 0.001) in chronic HCV, but cholesterol and triglycerides were significantly higher in patients with occult HCV infection (P < 0.001 and P = 0.002). Chronic HCV patients had higher gamma-globulin (P = 0.005), alpha-foetoprotein (P < 0.001) and iron (P < 0.001) levels. Percentage of patients with necroinflammatory activity and fibrosis was higher (P < 0.001) in chronic HCV than in occult HCV infection. Mean percentage of infected hepatocytes was higher (P = 0.001) in chronic HCV (10.1%) than in occult HCV infection (5.3%). This occult HCV infection is a milder disease than chronic HCV, and this could be related to the significantly lower number of infected hepatocytes observed in occult HCV.

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.

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.

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.

Summary Background Occult hepatitis C virus infection is defined by the presence of hepatitis C virus-RNA in liver but with undetectable anti-hepatitis C virus and serum viral RNA. Aim To study the response to anti-viral therapy in occult hepatitis C virus infection to assess the pathogenic effect of occult hepatitis C virus. Methods Ten patients with occult hepatitis C virus infection were treated with pegylated-interferon plus ribavirin for 24 weeks and were followed-up 24 weeks after therapy. All patients had abnormal alanine aminotransferase, hepatitis C virus-RNA positive in peripheral blood mononuclear cells and liver necroinflammation. Results At the end of treatment and follow-up, the percentage of patients with normal alanine aminotransferase was 80% (95% CI: 48–96%) and 60% (95% CI: 31–84%) respectively, and hepatitis C virus-RNA in peripheral blood mononuclear cells was negative in 80% (95% CI: 48–96%) and 70% (95% CI: 40–90%) cases. At the end of follow-up sustained response was observed in 30% (95% CI: 11–61%) of cases. Five patients underwent a second liver biopsy. In all cases, liver hepatitis C virus-RNA persisted, although hepatitis C virus-RNA load was significantly lower (3.2 × 104 ± 5.1 × 104 copies/μg RNA) than in the basal biopsy (2.4 × 105 ± 3.8 × 105 copies/μg RNA); (P = 0.043). Necroinflammation and fibrosis decreased in three cases. Conclusion The biochemical, virological and histological response to therapy achieved in patients with occult hepatitis C virus infection demonstrates the pathologic effects of occult hepatitis C virus.

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.

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.

Background: Occult hepatitis C virus (HCV) infection is characterised by the presence of HCV-RNA in the liver in the absence of anti-HCV, and serum viral RNA. Up to 70% of these patients also have HCV-RNA in peripheral blood mononuclear cells (PBMC) but it is not known if HCV is replicating in these cells. Aim: We studied possible HCV replication in PBMC of 18 patients with an occult HCV infection who were selected on the basis of HCV-RNA positivity in PBMC. Methods: Detection of HCV-RNA positive and negative strands in PBMC was done by strand specific reverse transcriptase-polymerase chain reaction (RT-PCR) and by in situ hybridisation. Results: The presence of HCV-RNA positive strand in PBMC was confirmed in all patients by strand specific RT-PCR and by in situ hybridisation. Mean percentage of PBMC which had the HCV-RNA positive strand was 3.3% (95% confidence interval (CI) 2.1–4.4) The HCV-RNA negative strand was found in the PBMC of 11/18 (61%) patients by strand specific RT-PCR and confirmed by in situ hybridisation, and the percentage of PBMC harbouring the HCV-RNA negative strand was 3.1% (95% CI 0.8–5.5). There was a significant correlation (p = 0.001, r = 0.84) between the percentage of PBMC with the HCV-RNA positive strand and that of PBMC with the HCV-RNA negative strand. Conclusion: HCV replicates in the PBMC of patients with occult HCV infection and thus, although these patients do not have serum HCV-RNA, they could be potentially infectious.

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.

Epidemiological studies have demonstrated a correlation between oral lichen planus and different liver diseases. The new virus termed TT virus (TTV) is highly prevalent in patients with chronic hepatitis of different etiology and it may be speculated that TT virus may be involved in the pathogenesis of oral lichen planus. This study examined the presence of TT virus DNA in serum by PCR and in oral mucosa biopsies by in situ hybridization from 20 patients with oral lichen planus (13 with chronic hepatitis and seven without liver disease). Serum and oral mucosa biopsies from six patients all with chronic hepatitis with leukoplakia were also studied as controls. TT virus DNA was positive in the serum of 17/20 (85%) of the patients with oral lichen planus and in all the controls. TT virus DNA hybridization signals were detected in mucosa biopsies from all the patients with TT virus DNA in serum but in none of the three cases without this marker. The percentage of positive cells ranged from 1.6-80%. No differences were found in the percentage of positive cells between TT virus positive patients with and without oral lichen planus and there was no relationship between the number of positive cells and the intensity of the inflammatory infiltrate. In conclusion, TT virus infects oral epithelial cells but the results do not support a role for TT virus in causing oral lichen planus.

The main site of TT virus (TTV) replication remains unknown. Therefore, we have studied the presence and titres of TTV DNA in paired serum, liver and PBMC samples from 50 patients with liver disease (32 with chronic hepatitis B or C, seven with cryptogenic hepatitis and 11 with nonviral liver disease) were included. TTV DNA was analysed by polymerase chain reaction (PCR) using primers from the open reading frame 1 (ORF 1) and from the untranslated region (UTR) and titres were semiquantified by PCR using an external standard. TTV DNA was detected in 26% of serum, 24% of liver and 14% of PBMC samples with ORF 1 primers. When UTR primers were used, 70% of serum and liver samples and 64% of PBMC were TTV DNA positive. No differences between TTV positive and negative patients were found regarding epidemiological or biochemical parameters. Trypsin treatment and fluorescent in situ hybridization confirm the intracellular location of TTV in PBMC. The mean of TTV DNA titres was statistically higher in liver than in serum or PBMC. TTV titres in serum correlated with those in PBMC but not with those in liver. In conclusion, although the liver seems to be the main site for TTV replication, this virus is also able to infect PBMC.

A novel hepatitis-associated virus named TT virus (TTV) has been isolated. However, its hepatotropism has not been proven. We have retrospectively analyzed the presence of TTV-DNA by polymerase chain reaction (PCR) and in situ hybridization in liver biopsies from 30 patients with liver disease (15 TTV-DNA-positive and 15 TTV-DNA-negative in serum), and prospectively in serum and liver from eight patients with normal liver histology. TTV-DNA was detected by PCR in the liver from the 15 patients with serum TTV-DNA and in serum and liver of two of the eight patients without liver disease. TTV-DNA titers in liver were 10 times higher than in serum, although no correlation between TTV-DNA titers in serum and liver were observed. In situ hybridization shows positive signals in the hepatocytes of the 17 patients infected by TTV but in none of the TTV-DNA-negative patients by PCR. No morphological changes were observed in the hepatocytes showing hybridization signals. The percentage of positive hepatocytes ranged from 2.1% to 30% and correlated with the TTV-DNA titers in liver (r = 0.54; P = 0.037). In conclusion, our results show that TTV is able to infect liver cells although they do not support a role for TTV in causing liver disease.

Twenty-four patients with chronic hepatitis B virus (HBV), antibody to hepatitis B e antigen (anti-HBe), HBV DNA positivity, and alanine transaminase (ALT) elevation who failed previous interferon alfa (IFN-α) therapy were included in a pilot study of combination therapy with ribavirin and IFN-α. The patients received daily oral ribavirin (1,000-1,200 mg according to body weight) plus 5 million units (MU) IFN-α2b three times a week for 12 months and were followed-up for 12 months. The median viremia level decreased significantly at the end of treatment (1.2 × 103copies/mL) and follow-up (4.0 × 102 copies/mL) compared with the baseline (3.0 × 106 copies/mL; P < .05). After 12 months, 8 of 24 (33%) patients had cleared HBV DNA and 12 (50%) had normal ALT levels. At the end of the study virological and biochemical response was 50% and 21%, respectively. Thus, virological and biochemical response sustained in 5 of 24 (21%) patients retreated with ribavirin and IFN-α; none of them lost hepatitis B surface antigen (HBsAg). Liver histology improved in 2 of 4 sustained responders but in none of the 12 nonresponders with paired biopsies (P = .05). The response was independent of dose and duration of previous treatment, viral load, or the distribution of HBV precore wild-type/mutant variants. However, sustained responders had significantly higher necroinflammation (P = .036) and fibrosis (P = .007) scores. IFN-α–related side effects were mild and reversible on discontinuation. In 4 (17%) patients who suffered nausea and diarrhea the ribavirin dosage was reduced by 50% after 1 month of therapy and finally discontinued in all of them. No patient had liver disease decompensation. In summary, combination therapy with ribavirin and IFN-α may be efficacious to treat viremic anti-HBe–positive patients with chronic hepatitis B who have failed previous IFN therapy.

The existence of four GB C virus/hepatitis G virus (GBV-C/HGV) subtypes has been reported. The subtype was determined in 16 multitransfused GBV-C/HGV infected patients prior to bone marrow transplantation by comparing the 5' untranslated region (5' UTR) sequence with 39 available sequences. Phylogenetic and bootstrap analyses were carried out with PHYLIP package 3.5c. In the samples with undefined subtype, the whole 5' UTR was cloned and sequenced. Comparison of distances showed that the isolates from 12/16 and 4/16 patients belonged theoretically to subtypes 2a and 2b, respectively. The phylogenetic tree and bootstrap analyses confirmed this result in only 11/16 samples. Analysis of the entire 5' UTR from the remaining five samples with undefined GBV-C/HGV subtype revealed genomic variability within the isolates from each patient and between the isolates of different patients. Evolutionary distances, phylogenetic tree, and bootstrap showed that the isolates from these samples were grouped in a separate branch, different from the published subtypes. In conclusion, a novel GBV-C/HGV subtype was found in a group of multitransfused patients with GBV-C/HGV infection.

Serum samples from 20 anti-hepatitis B e antigenpositive patients with and without normal alanine aminotransferase (ALT) levels who had serum hepatitis B virus (HBV) DNA detectable only by polymerase chain reation (PCR) were examined. Viral DNA was amplified by PCR, using primers that encompassed precore and ORF-X regions and sequenced directly, to investigate whether mutations in the nucleotide sequences of X and precore gene regions of HBV-DNA might be responsible for the difference in the activity of disease and in the levels of viral replication. The HBV-DNA concentration in patients with abnormal ALT levels was higher than in those with normal ALT. The amount of HBV-DNA correlated with the ALT levels (P < 0.05). Seventy-two percent of patients had HBV-DNA harboring the 1896 precore stop mutation, and there was a negative correlation between the percentage of precore mutant genotype and the HBVDNA concentration (P < 0.05). Thirty percent of patients had mutations in ORF-X. Patients with ORF-X mutations had lower levels of HBV-DNA than those who had wild-type virus. The presence of mutations in precore and X regions may be related to a low HBVDNA concentration and reduced biochemical activity in patients with anti-HBe. J. Med. Virol. 56:294‐299, 1998. © 1998 Wiley-Liss, Inc.

Fifty percent of healthy hepatitis B surface antigen carriers may have histologically proven chronic hepatitis. Our aim was to study the benefit of interferon-alpha in healthy patients. Twenty-nine hepatitis B surface antigen carriers with normal liver enzymes and with serum hepatitis B virus DNA were randomized into two groups: Group I, 14 patients treated with 9 megaunits of interferon alpha-2a thrice weekly for six months, and Group II, 15 control patients. A liver biopsy was obtained from each patient at study initiation. A second biopsy was available in nine treated patients and six controls. During treatment, a significant increase in alanine amino transferase levels was observed in treated patients as compared with the controls (P < 0.05). After treatment, transaminase levels decreased to normal values. No differences between treated and control patients were observed in clearance of hepatitis B virus markers. A significant increase in the total histological activity index between base line and final liver biopsies was observed in treated patients (P < 0.05). It is concluded that interferon alpha treatment may induce a biochemical and histological activation of liver disease. Accordingly, interferon alpha should not be administered to healthy hepatitis B surface antigen carriers, at least with the schedule used in this work.

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.

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.

The aim of this work was to study the presence of the hepatitis GB virus type C (HGBV-C) in liver and serum samples of children with chronic viral hepatitis, the time course of changes in viral RNA, and the possible acquisition routes of infection. Frozen serum and liver samples from 58 children with chronic hepatitis B (n = 33) or C (n = 25) were analyzed using polymerase chain reaction. Twenty-seven children had been included in different interferon trials. Two additional serum samples from the HGBV-C-positive children as well as serum samples from 29 of their relatives were also analyzed. HGBV-C RNA was detected in serum and liver samples from 9 of 58 (15%) of children as well as in serum samples from 3 of 29 of the relatives of the HGBV-C-infected children: the mother and the brother of one child (index case A) and the mother of another child (index case B). The homologies of the HGBV-C RNA sequences were 93% between index case A and his mother, 88% between index case A and his brother, and 94% between index case B and his mother. In the 3 children receiving alpha-interferon, HGBV-C RNA became undetectable during treatment although it reappeared in 2 of them after therapy. In conclusion, we found that 15% of children with chronic viral hepatitis were coinfected with HGBV-C. HGBV-C RNA was simultaneously present in serum and liver samples and tended to remain detectable even after alpha-interferon therapy. Our results suggest that vertical transmission of HGBV-C may occur.

In this work, we have shown that hepatitis C virus (HCV) and hepatitis B virus (HBV) can coexist in the same hepatocyte using double fluorescent in situ hybridization in liver biopsy samples from patients with chronic HCV infection with occult HBV infection. Digital image analysis of hybridization signals showed that the HBV DNA levels in coinfected hepatocytes were lower than those in cells infected only with HBV. This finding supports the hypothesis of inhibition of HBV replication by HCV. Furthermore, HCV RNA levels were lower in coinfected cells than in cells infected only with HCV, suggesting that HBV may also inhibit HCV replication.

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.

Patients with chronic renal failure on hemodialysis have a high risk of infections with viruses such as hepatitis B (HBV), hepatitis C (HCV), GB virus C/hepatitis G (GBV-C/HGV) and TT (TTV) viruses. The prevalence of HBV, HCV, GBV-C/HGV and TTV in patients with chronic renal failure who are on conservative management before entering into a hemodialysis program (predialysis) in comparison with hemodialyzed patients was studied to elucidate whether the high prevalence of these viruses is influenced by that observed in the predialysis stage. The presence of hepatitis B virus surface antigen (HBsAg), HCV RNA, GBV-C/HGV RNA and TTV DNA was analyzed in sera from 80 patients with chronic renal failure (35 on predialysis and 45 on hemodialysis). HBsAg, HCV RNA, GBV-C/HGV RNA and TTV DNA were detected in one (2.8%), six (17.1%), eight (22.5%) and 16 (45.7%) of the 35 patients on predialysis. Two (5.7%) of these patients were coinfected with HCV and GBV-C/HGV, whereas six (17.1%) had GBV-C/HGV and TTV coinfection. In the 45 hemodialyzed patients, HBsAg, HCV RNA, GBV-C/HGV RNA and TTV DNA were detected in one (2.2%), two (4.4%), seven (15.5%) and 26 (57.7%). One (2.2%) patient had HBV and TTV coinfection, two (4.4%) HCV and TTV coinfection whereas four (8.8%) were coinfected with GBV-C/HGV and TTV. No differences regarding age, gender, previous surgery and number of transfusions were found between infected and uninfected patients within and between both groups. In conclusion, the prevalence of the viruses studied in predialysis may influence their prevalence in dialysis units. J. Med. Virol. 63:103–107, 2001. © 2001 Wiley-Liss, Inc.

To study the existence of GB virus C/hepatitis G virus (GBV-C/HGV) variants with different tropism, we have analyzed the heterogeneity and quasispecies composition of GBV-C/HGV isolated from in vitro-infected peripheral blood mononuclear cells (PBMC) and from sera, livers, and PBMC from two chronically infected patients. For this purpose, the GBV-C/HGV 5′ noncoding region (5′NCR) was amplified by reverse transcription-PCR and the amplified products were cloned and sequenced. These analyses showed that the master 5′NCR sequences isolated from the in vitro-infected PBMC and from the PBMC isolated from the patient whose serum was used as the inoculum were identical but different from that of the inoculum. Furthermore, phylogenetic analysis revealed that all PBMC sequences grouped together into a branch which was separate from those of the inoculum. For one of the two chronically infected patients, all the sequences from the PBMC and one from the liver clustered into a single branch while the sequences from the serum and all the other liver sequences grouped together in the other branch. For the other patient, the sequences from the serum and PBMC and three sequences from the liver grouped together into one branch, while the remaining five sequences from the liver were separated in a different cluster. In conclusion, our results support the existence of different GBV-C/HGV variants with different tissue tropism.

The nucleotide sequence of hepatitis GB virus type C (HGBV-C)/hepatitis G virus (HGV) NS3/helicase and 5′-untranslated regions from 23 Spanish patients were analyzed to assign the HGV isolates one of the proposed HGBV-C/HGV genotypes. The analysis of the evolutionary distance frequency showed that the distances among all sequences in NS3/helicase region were distributed around a single peak of 0.20, suggesting that all included sequences belonged to the same HGBV-C/HGV genotype. By contrast, in the 5′-untranslated region, all the distances corresponding to our sequences and those of the HGBV-C/HGV types 2 and 3 were distributed around a major peak of 0.03. The remaining distances corresponding to the HGBV-C/HGV type 1 sequences were distributed around a minor peak of 0.11. The phylogenetic tree and pairwise comparison of evolutionary distances among the 5′-untranslated region of the infected patients and each HGBV-C/HGV genotype demonstrated that our HGBV-C/HGV isolates belonged to subtype 2a (17/23; 78%) and 2b (5/23; 22%). No relation was found between HGBV-C/HGV subtype and hepatitis B or C virus infection.

Summary. We compared the response and the relapse rates of HBeAb-positive patients treated with interferon-alpha for 6 or 12 months. Thirty-eight HBeAb-positive patients which chronic hepatitis B were randomly allocated into two groups: Group I (19 patients receiving 10 MU of recombinant interferon-alpha 2b three times a week for 2 months, followed by 5 MU three times a week for 2 months and then 3 MU three times a week for 2 months); Group II (19 patients receiving 10 MU of recombinant interferon-alpha 2b three times a week for 2 months, followed by 5 MU three times a week for 2 months and then 3 MU three times a week for 8 months). At the end of treatment, alanine aminotransferase normalization was higher but not more significant in Group I than in II (53%vs 26%), while hepatitis B virus DNA clearance was similar in both groups (21% in Group I vs 26% in Group II). However, at 12 months of followup, biochemical relapses occurred only in Group I (60%vs 0% in Groups I and II, respectively). Five complete responded cleared hepatitis B surface antigen at that time. In conclusion, prolonged treatment of HBeAb patients is efficient in reducing the biochemical relapse.

Summary. To assess the effects of prednisone and interferon on the distribution of hepatitis B virus (HBV) precore mutants, nine hepatitis B e antibody (HBeAb)-positive patients with HBV chronic infection were studied. Patients were treated with prednisone (30 mg day-1 for 4 weeks, followed by 20 mg day-1 for 2 weeks and by 10 mg day-1 for 1 week), followed by recombinant interferon-α (15 MU thrice per week) for 6 months, without a clearance period. The HBV precore region was amplified by polymerase chain reaction (PCR) and distribution of the precore mutants was determined by hybridization of PCR products. Moreover, the glucocorticoid-responsive element (GRE) was sequenced to determine whether changes in the sequence were produced at the end of prednisone treatment. During prednisone treatment, changes in alanine transaminase (ALT) were observed in only two patients, in whom ALT decreased to nearly normal values. In three patients ALT normalized at the end of interferon treatment. At baseline, wild-type HBV alone was detected in one patient, while seven patients were infected by a mixture of wild-type and precore mutants, predominantly wild type. At the end of prednisone treatment, two patients were infected by only wild-type HBV. The proportion of precore mutants decreased in three cases, while no changes were observed in three. At the end of interferon treatment, the precore mutant proportion decreased in the three responders, while tending to increase or remain unchanged in the rest. No significant changes in GRE sequence were found as a result of prednisone treatment. Our results would appear to confirm the role of the immune system in the selection of precore mutants.

ResumenObjetivoEvaluar los resultados clínicos en pacientes con lesiones condrales en rodilla y tobillo, a los que se les ha realizado implante de condrocitos autólogos en membrana (MACI). Se han incluido 150 pacientes tratados con MACI (siempre por el mismo equipo quirúrgico) durante los años 2002-2007.MétodosEn todos los casos se tomó una biopsia de cartílago para utilizarla como fuente para el cultivo celular mientras se evaluaba la lesión condral por artroscopia. El implante celular se realizó por artroscopia en 53 casos, mientras que en el resto fue por cirugía abierta. Los pacientes se evaluaron siguiendo un protocolo clínico interno. En 50 pacientes se realizó seguimiento de los resultados durante un periodo medio de 2 años. En 5 pacientes se realizó una biopsia de second look a los 2 años de seguimiento.ResultadosEn el 89% de los pacientes sin cirugías previas y en un 66% de los restantes, los resultados fueron buenos/excelentes en referencia al dolor y la movilidad; la mayoría de ellos pudieron retomar su actividad física previa, incluyendo deporte profesional. El análisis histológico de las 5 biopsias mostró que el tejido neoformado era cartílago tipo hialino, pero con menos celularidad y subjetivamente más blando que el cartílago hialino normal.ConclusiónLa técnica artroscópica del MACI es un procedimiento prometedor para el tratamiento de las lesiones condrales y osteocondrales de rodilla y tobillo, aunque el resultado histológico evidencia que el aumento de células proporcionaría todavía resultados mejores.Nivel de evidenciaNivel III.Relevancia clínicaLa importancia de este estudio radica en que se presenta un gran número de casos de pacientes con lesiones condrales tratados con MACI, muchos de ellos con implante mediante artroscopia. En este estudio se comprueba que el tratamiento con MACI proporciona buenos resultados.AbstractAimThe aim of this work was to evaluate the clinical results of the matrix-induced autologous chondrocyte implantation (MACI) technique on chondral defects of the knee and ankle. A study was conducted on 150 patients treated with MACI (performed by the same team of surgeons in all cases) during 2002-2007.MethodsThe lesion was evaluated by arthroscopy, and a cartilage biopsy was taken. The cellular implant was performed by arthroscopy in 53 patients, and by open surgery in the remaining cases. The results were measured following an in-house clinical protocol. Data corresponding to a mean follow-up period of 24 months were available in 50 out of the 150 implanted patients. Histological studies were performed in a second biopsy taken by arthroscopy in 5 patients 2 years after cell implantation.ResultsIn 89% of patients without previous surgeries, and in 66% of patients with previous surgeries, the results were good or excellent for pain and motion, and most of them were able to return to their previous physical activities, including professional sport. Histological studies performed on a second biopsy showed that the new tissue is hyaline-like cartilage with lower cell numbers and subjectively softer than normal cartilage.ConclusionArthroscopic MACI is a promising procedure to treat chondral lesions in the knee and ankle, although the histological analysis suggests that increasing cellular dosage would improve the results of the technique.Level of evidenceLevel III.Clinical relevanceThe importance of this study is that it presents an increased number of patients with chondral lesion treated with MACI. A high number of these patients were treated with arthroscopic MACI. In the present study it is shown that the treatment gives good results.