Intermediate-Term Clinical Outcomes of High-Density Autologous Chondrocyte Implantation in Patients with Concomitant Anterior Cruciate Ligament Reconstruction and Focal Chondral Lesions.

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.

Study of Telomere Length in Preimplanted Cultured Chondrocytes.

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.

Cartilage Defect Treatment Using High-Density Autologous Chondrocyte Implantation: Two-Year Follow-up.

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.

Hepatitis C virus replicates in the liver of patients who have a sustained response to antiviral treatment.

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.

Virus-specific T-cell responses associated with hepatitis C virus (HCV) persistence in the liver after apparent recovery from HCV infection.

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.

Detection of hepatitis C virus (HCV) RNA in the liver of healthy, anti-HCV antibody-positive, serum HCV RNA-negative patients with normal alanine aminotransferase levels.

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.

Percentage of hepatitis C virus-infected hepatocytes is a better predictor of response than serum viremia levels.

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.

Hepatitis B infection of the liver in chronic hepatitis C without detectable hepatitis B virus DNA in serum.

Hepatitis B virus (HBV) DNA may persist in the liver in the absence of serum HBV-DNA after a self-limited acute hepatitis B. This may also occur in patients with chronic hepatitis C virus (HCV) infection but its prevalence and its impact on liver histology is unknown. HBV-DNA was tested by polymerase chain reaction (PCR) and by in situ hybridisation in liver biopsies from 98 patients with chronic hepatitis C who were hepatitis B surface antigen negative and serum HBV-DNA negative by PCR. HBV-DNA resulted positive in the liver of 37/98 (37.7%) patients without serum HBV-DNA. To test whether these patients had serum HBV-DNA levels under the detection limit of the PCR assay used in this study (50 copies/ml), PCR products in which HBV-DNA was undetectable after visualization of agarose gels were analysed by dot-blot hybridisation. With this method, HBV-DNA was positive in serum of 12/37 patients with liver HBV-DNA. Thus, 25/98 (25.5%) patients have HBV-DNA detectable only in liver. This was confirmed by in situ hybridisation, the percentage of infected hepatocytes ranging from 0.1% to 12%. In patients in whom the HCV infection was shorter than 20 years, HBV infected patients had higher (P = 0.01) fibrosis score (1.64 +/- 1.21) than HBV negative cases (0.53 +/- 0.66). In conclusion, a significant proportion of patients with chronic HCV infection have HBV-DNA in the liver in the absence of viral DNA in serum. The impact of this finding on liver histology deserves further research.

Occult hepatitis C virus infection in patients in whom the etiology of persistently abnormal results of liver-function tests is unknown.

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.

Distribution of hepatitis B virus in the liver of chronic hepatitis C patients with occult hepatitis B virus infection.

Although occult hepatitis B virus (HBV) infection (HBV-DNA in serum in the absence of hepatitis B surface antigen [HBsAg]) is common in chronic hepatitis C, its characteristics are not well known. In this work, the presence of HBV-DNA (by polymerase chain reaction; PCR) and its distribution (by in situ hybridization) in liver biopsies and peripheral blood mononuclear cells (PBMCs) from 32 patients with chronic hepatitis C and occult HBV infection and in 20 HBsAg chronic carriers were determined. The results showed that serum HBV-DNA levels were statistically lower (P = 0.001) in patients with occult HBV infection than in HBsAg chronic carriers. The HBV infection pattern in liver cells was identical between patients with occult HBV infection and those with chronic hepatitis B. However, the mean percentage of HBV-infected hepatocytes was significantly lower (P = 0.001) in patients with occult HBV infection (5 +/- 4.44%) than in HBsAg chronic carriers (17.99 +/- 11.58%). All patients with chronic hepatitis B have HBV-DNA in their PBMCs while this occurred in 50% of the cases with occult HBV infection. In conclusion, patients with occult HBV infection have a low number of HBV-infected hepatocytes and this fact could explain the lack of HBsAg detection and low viremia levels found in these cases.

Hepatitis C virus replicates in sweat glands and is released into sweat in patients with chronic hepatitis C.

Hepatitis C virus (HCV) replicates in salivary glands of chronic hepatitis C patients and is released into the saliva, suggesting that HCV may replicate in other exocrine glands. The presence of positive and negative HCV RNA strands was demonstrated by in situ hybridization, and of HCV core protein by immunohistochemistry, in sweat glands and keratinocytes in healthy skin biopsies from 15 patients with chronic hepatitis C and 10 anti-HCV negative patients with chronic liver disease. Positive and negative HCV RNA strands were detected in 9.6 +/- 5.2% and 4.2 +/- 3.8%, respectively, of the epithelial cells of eccrine sweat glands. Core protein was detected in 6.0 +/- 3.93% of these cells. HCV RNA resistant to RNase digestion (encapsidated HCV RNA) was detected in 10/10 sweat samples from HCV-infected patients. Positive and negative HCV RNA strands were detected in 6.7 +/- 2.97% and 3.0 +/- 3.08% of the keratinocytes, respectively. HCV core protein was found in 4.5 +/- 2.76% of these cells. No HCV RNA or HCV core protein was detected in the skin biopsies from the 10 anti-HCV negative patients. In conclusion, HCV replicates in eccrine sweat glands cells and keratinocytes in healthy skin and is released into the sweat.

Detection of hepatitis C virus RNA and core protein in keratinocytes from patients with cutaneous lichen planus and chronic hepatitis C.

Cutaneous lichen planus has been associated in patients with chronic hepatitis C virus infection. It is still unknown whether hepatitis C virus infects keratinocytes of lichen planus lesions. In this report we have analyzed the presence of genomic and anti-genomic hepatitis C virus RNA in skin biopsies from 26 patients with chronic hepatitis C and healthy skin and from 24 patients with cutaneous lichen planus (five with and 19 without hepatitis C virus infection) by in situ hybridization. Hepatitis C virus RNA was detected in the keratinocytes of 69% of the patients with healthy skin and chronic hepatitis C, in 100% of the patients with lichen planus and hepatitis C virus infection, and in none of lichen planus patients without hepatitis C virus infection. The percentage of keratinocytes showing genomic or anti-genomic hepatitis C virus RNA was statistically lower (p < 0.01 in all cases) in patients with healthy skin (mean +/- SD: 5.7 +/- 3.5% and 2.7 +/- 3.1% of keratinocytes with genomic or anti-genomic hepatitis C virus RNA, respectively) than in those with lichen planus lesions (31.7 +/- 7.9% and 18.8 +/- 7.4%, mean +/- SD) or the unaffected adjacent skin (24.8 +/- 6.9% and 14.3 +/- 3.8%, mean +/- SD). In conclusion, we have demonstrated that hepatitis C virus infects keratinocytes from patients with lichen planus and hepatitis C virus infection.

TT virus replicates in stimulated but not in nonstimulated peripheral blood mononuclear cells.

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.