Evolución clínica y presencia de edema óseo subcondral a los dos años de tratamiento con implante de condrocitos autólogos de alta de densidad en la rodilla / Clinical outcome and subchondral bone edema presence at two-year follow-up after high density autologous chondrocyte implantation treatment in the knee

Antecedentes y objetivo: Recientemente se ha descrito una nueva modalidad de la técnica de implante de condrocitos autólogos sobre membrana de colágeno i/iii llamada HD-ACI (High Density Autologous Chondrocyte Implantation) que está basada en el aumento de la densidad celular. El objetivo de este trabajo fue estudiar la evolución clínica y la incidencia de la aparición de edema óseo en pacientes con lesiones de cartílago en la rodilla tratados con HD-ACI al año y a los 2 años de la intervención. Métodos: Se trata de un estudio retrospectivo en 40 pacientes con lesiones condrales grado iii-iv. Todos los pacientes fueron tratados con HD-ACI con una dosis celular de 5×106 condrocitos/cm2 de lesión. La percepción subjetiva de la mejora de los síntomas/funcionalidad se valoró mediante la escala del Comité Internacional de Documentación de la Rodilla (IKDC, International Knee Documentation Committee). La presencia de edema óseo se evaluó a los 6, 12 y 24 meses de seguimiento por resonancia magnética. Comité Internacional de Documentación de la Rodilla (IKDC) Resultados: Los valores de IKDC mostraron una mejoría significativa a los 12 y 24 meses (p<0,001). La diferencia media de IKDC entre la visita basal y los 12 meses fue de 26,3 puntos y de 31,6 puntos a los 24 meses. El 27,5% de los pacientes presentaron edema óseo subcondral a los 2 años de seguimiento. Conclusiones: HD-ACI es un tratamiento efectivo y seguro que mejora el dolor, la percepción clínica y la funcionalidad de la articulación. No se ha encontrado correlación entre la presencia de edema óseo y la evolución clínica de los pacientes

Background: Recently, a new approach of autologous chondrocyte implantation technique (using as biomaterial a collagen type i/iii membrane) based on increasing cell density called HD-ACI (High Density Autologous Chondrocyte Implantation) has been described. The objective of this paper was to study the clinical outcome and incidence of subchondral bone oedema in patients with cartilage lesions in the knee treated with HD-ACI at 1-2 years of follow-up. Methods: This is a retrospective study performed with forty patients with chondral injuries grade iii-iv. All patients were treated with HD-ACI with a cellular dose of 5×106 chondrocytes /cm2 of lesion. The subjective perception of improvement of symptoms and functionality was measured with the IKDC score (International Knee Documentation Committee). The presence of bone oedema was assessed at 6, 12 and 24 months of follow-up by magnetic resonance imaging. Results: IKDC values showed a significant improvement at 12 and 24 months (P<.001). The mean difference of IKDC between the baseline visit and 12 months was 26.3 points, and 31.6 points at 24 months. Twenty-seven point five percent of the patients presented subchondral bone oedema at 2 years of follow-up. Conclusions: HD-ACI is an effective and safe treatment that improves pain, clinical perception and functionality of the joint. No correlation was found between the presence of bone oedema and the patients' clinical outcome

Clinical outcome and subchondral bone edema presence at two-year follow-up after high density autologous chondrocyte implantation treatment in the knee. / Evolución clínica y presencia de edema óseo subcondral a los dos años de tratamiento con implante de condrocitos autólogos de alta de densidad en la rodilla.

BACKGROUND: Recently, a new approach of autologous chondrocyte implantation technique (using as biomaterial a collagen type i/iii membrane) based on increasing cell density called HD-ACI (High Density Autologous Chondrocyte Implantation) has been described. The objective of this paper was to study the clinical outcome and incidence of subchondral bone oedema in patients with cartilage lesions in the knee treated with HD-ACI at 1-2 years of follow-up. METHODS: This is a retrospective study performed with forty patients with chondral injuries grade iii-iv. All patients were treated with HD-ACI with a cellular dose of 5×106 chondrocytes /cm2 of lesion. The subjective perception of improvement of symptoms and functionality was measured with the IKDC score (International Knee Documentation Committee). The presence of bone oedema was assessed at 6, 12 and 24 months of follow-up by magnetic resonance imaging. RESULTS: IKDC values showed a significant improvement at 12 and 24 months (P<.001). The mean difference of IKDC between the baseline visit and 12 months was 26.3 points, and 31.6 points at 24 months. Twenty-seven point five percent of the patients presented subchondral bone oedema at 2 years of follow-up. CONCLUSIONS: HD-ACI is an effective and safe treatment that improves pain, clinical perception and functionality of the joint. No correlation was found between the presence of bone oedema and the patients' clinical outcome.

Estudio piloto para la reconstrucción del ligamento cruzado anterior (LCA) con cultivos de fibroblastos autólogos sobre membranas de colágeno I/III / Pilot study on the anterior cruciate ligament (ACL) reconstruction with autologous fibroblasts culture on collagen I/III membranes

Objetivo: Utilizar cultivos de fibroblastos sobre membranas de colágeno I/III para tratar la rotura del ligamento cruzado anterior (LCA). Pacientes y métodos: Estudio prospectivo con 100 biopsias de LCA de pacientes con rotura de LCA con las que se realizaron cultivos primarios de fibroblastos. Tras alcanzar 20-30 millones, se transfirieron a membranas de colágeno I/III, donde se determinó su disposición mediante tinción con hematoxilina-eosina y la expresión de los genes colágeno de tipo I (Col-I), colágeno de tipo III (Col-III), tenascina-C y Sox-9 mediante PCR en tiempo real. Resultados: A los 30 días de cultivo existía una correlación negativa entre la edad de los pacientes y la velocidad de crecimiento de los fibroblastos (p=0,003). La histología reveló que las células se disponían entre la malla formada por las fibras de las membranas, expresando Col-I, Col-III, Sox-9 y tenascina-C, características del ligamento. Conclusión: Es posible el establecimiento de cultivos primarios de fibroblastos derivados de LCA tanto in vitro como sobre membranas de colágeno I/III sin que pierdan sus características primarias de células de ligamento (AU)

Objective: To study the possibility on the use of the in vitro cultured on I/III collagen membranes for the treatment of the broken anterior cruciate ligament (ACL). Patients and methodology: Prospective study performed with 100 ACL biopsies from patients with broken ACL, in which primary cultures of fibroblasts were established. When a number of 20-30 million was reached, they were transferred to I/III collagen membranes, where the architecture of the cells was examined by hematoxilin-eosin stained and the expression of the collagen type I (Col-I), collagen type III (Col-III), tenascin-C and Sox-9 genes by real time PCR. Results: A negative correlation between the age of the patients and the growth rate of the fibroblasts after 30 days of setting-up the culture (p=0.003) was observed. The histology revealed that the cells were disposed within the membrane fibers net, where the cells expressed the characteristic proteins of the ligament: Col-I, Col-III, Sox-9 and tenascin-C. Conclusion: It is possible to establish the primary culture of the ACL-derived fibroblasts, both in vitro and onto collagen I/III porcine membranes without losing their primary features of the ligament cells (AU)

The role of genomic and antigenomic HCV-RNA strands as predictive factors of response to pegylated interferon plus ribavirin therapy.

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-alpha2b 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/microg RNA (range: 10,000-775,000) vs. 150,000 copies/microg RNA (range: 100-3,200,000; P = 0.38). Median of genomic HCV-RNA in liver of responders was 1,250,000 copies/microg RNA (range: 5000-9,000,000) and in non-responders 3,180,000 copies/microg 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-alpha2b plus ribavirin depends on the amount of genomic HCV-RNA in the pre-treatment liver biopsy.

Comparative study between occult hepatitis C virus infection and chronic hepatitis C.

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.

Effect of anti-viral therapy for occult hepatitis C virus infection.

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 x 10(4) +/- 5.1 x 10(4) copies/microg RNA) than in the basal biopsy (2.4 x 10(5) +/- 3.8 x 10(5) copies/microg 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) and hepatitis B virus (HBV) can coinfect the same hepatocyte in the liver of patients with chronic HCV and occult HBV infection.

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.

Hepatitis C virus replicates in peripheral blood mononuclear cells of patients with occult hepatitis C virus infection.

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.

Replicación del virus de la hepatitis C en lesiones cutáneas de liquen plano / Hepatitis C virus replication in lichen planus lesions

El liquen plano (LP) es una enfermedad dermatológica cuya etiología es desconocida. En la última década se ha constatado en estudios epidemiológicos una asociación estadísticamente significativa entre dicha enfermedad y la infección crónica por virus de la hepatitis C (VHC). El objetivo de este estudio es investigar si el VHC está presente en las lesiones de LP para así obtener datos que apoyen un papel etiopatogénico del VHC en la inducción de lesiones de LP. Con este fin se ha analizado mediante hibridación in situ la presencia de VHC-RNA genómico y antigenómico en biopsias cutáneas procedentes de 32 pacientes con hepatitis crónica C (26 pacientes en los que la biopsia procedía de piel sana de tronco y 6 pacientes en los que era de piel sana facial), 24 pacientes con LP (5 con y 19 sin infección por VHC) y 6 pacientes sanos.Se detectó VHC-RNA en los queratinocitos del 69,2 por ciento y del 100 por ciento de los pacientes con hepatitis crónica C y piel sana, de tronco y facial respectivamente; en el 100 por ciento de los pacientes con hepatitis crónica C y LP y en ninguno de los pacientes sin hepatitis crónica C pero con LP. El porcentaje de queratinocitos que mostraban VHC-RNA genómico o antigenómico fue estadísticamente menor (p < 0,01) en la piel sana de tronco (5,7 ñ 3,5 por ciento y 2,7 ñ 3,1 por ciento de los queratinocitos con VHC-RNA genómico o antigenómico, respectivamente) que en las lesiones de LP (31,7 ñ 7,9 por ciento y 18,8 ñ 7,4 por ciento) o la piel adyacente no afecta (24,8 ñ 6,9 por ciento y 14,3 ñ 3,8 por ciento). Como conclusión, en este estudio se ha demostrado que el VHC infecta y replica en los queratinocitos de la piel de pacientes con hepatitis crónica C, independientemente de que presenten o no lesiones de LP. El hecho de que el porcentaje de queratinocitos infectados por VHC fuese significativamente mayor en las lesiones de LP que en las pieles dermatológicamente sanas, sugiere que la infección de los queratinocitos por VHC podría ser el agente etiopatogénico del desarrollo de las lesiones de LP cutáneo en estos pacientes, debiéndose aclarar esta cuestión en trabajos futuros (AU)

Detección del virus C de la hepatitis en lesiones cutáneas de liquen plano / Detection of hepatitis C virus in lichen planus lesions

Objetivos: Estudios epidemiológicos han demostrado una correlación entre el liquen plano y la hepatitis crónica por virus C. Para el conocimiento del papel que pueda desempeñar el VCH en el desarrollo de las lesiones de liquen plano, es necesario obtener evidencias morfológicas de la presencia del VCH en esta afección cutánea. Pacientes y métodos: Se ha analizado por hibridación in situ la presencia de VCH-RNA en biopsias de piel de 23 pacientes divididos en tres grupos: 1) once pacientes con liquen plano (cinco con VCH-RNA en suero); 2) seis pacientes sin liquen plano y con VCH-RNA en suero, y 3) seis sin liquen plano ni hepatopatía por VCH. Resultados: El VCH-RNA se detectaba en los queratinocitos de la piel de los pacientes con RNA del virus en suero independientemente de si tenían liquen plano o no y en ninguno de los casos VCH negativos. El porcentaje de células infectadas oscilaba desde un 13,1 per cent a un 44,26 per cent. Conclusión: Se ha demostrado que el VCH infecta los queratinocitos de la piel de los pacientes con hepatitis crónica C tanto en lesiones de liquen plano como de piel sana. Se necesitan investigaciones en esta línea para esclarecer las consecuencias patológicas de este hallazgo (AU)

Expression of factor VII in the liver of patients with liver disease: correlations with the disease severity and impairment in the hemostasis.

Factor VII (FVII) plasma levels in patients with liver disease may be below the normal range. However, no data are available on FVII expression in liver biopsies from patients with liver diseases other than cirrhosis. We have analyzed the expression of FVII by in situ hybridization in liver biopsies from 50 patients in comparison with the procoagulant activity of FVII, and with the plasma levels as activated FVII (FVIIa) and FVII antigen. The level of FVIIa was significantly lower in stage 4 liver fibrosis patients than in the remaining ones (P < 0.05). The percentage of hepatocytes expressing FVII was significantly lower in stage 4 liver fibrosis patients (4.1+/-1.3%) than in stage 3 (22.7+/-6.1%), stage 2 (31.5+/-6.1%), stage 1 (43.7+/-8.2%) and stage 0 patients (63.8+/-4.4%) (P < 0.001). These percentages correlated inversely in a statistically significant way with the histological activity index and the liver function tests. We have demonstrated that the FVIIa plasma levels in patients with chronic liver disease other than cirrhosis may be below the normal range in the absence of blood coagulation impairment. The percentage of hepatocytes expressing FVII decreases as the severity of liver damage increases.

TT virus detection in oral lichen planus lesions.

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.

Distribution of hepatitis C virus infection in liver biopsies from children and adults with chronic hepatitis C.

Chronic hepatitis C in children is characterized by milder forms of liver damage than those found in adults. Such a difference has been attributed to a low viral load in children that may lead to poor recognition of infected cells by the immune system. One approach that could be used to confirm this hypothesis may be to examine the number of infected hepatocytes in liver biopsies. Paraffin embedded liver biopsies from 21 children and 15 adults with chronic hepatitis C virus (HCV) infection (with a similar duration of the infection) were hybridized in situ and the percentage of infected hepatocytes was correlated with the histological activity index, alanine aminotransferase levels and HCV viraemia levels. Histological activity index and HCV viraemia levels were statistically higher (P < 0.05 and P < 0.01 respectively) in adults than in children, and the percentage of infected hepatocytes was higher in adults (11.0 +/- 19.7%) than in children (4.6 +/- 3.6%), although it did not reach statistical significance. Also, the percentage of infected hepatocytes correlated with HCV-RNA concentration in serum in both children (r = 0.683, P = 0.001) and adults (r = 0.768, P = 0.001). The results show that liver damage in children with chronic hepatitis C is not related to the extent of infection in the liver. This findings support the hypothesis of that liver injury in chronic HCV infection is mediated by the host immune response.

In situ detection of hepatitis C virus RNA in salivary glands.

Chronic hepatitis C virus (HCV) infection has been associated with several extrahepatic manifestations, among these, to diseases with oral manifestations such as Sjögren's syndrome or sialadenitis. HCV-RNA has been detected in saliva and in salivary glands from patients with sialadenitis by polymerase chain reaction. However, morphological evidence of HCV replication in salivary gland cells is needed to support a role for HCV in causing sialadenitis or Sjögren's syndrome. We have used in situ hybridization and immunohistochemistry to analyze the presence of HCV-RNA of sense and antisense polarity and HCV core antigen, respectively, in salivary gland biopsies from 19 patients with chronic sialadenitis or Sjögren's syndrome (eight anti-HCV-positive; 11 anti-HCV-negative). HCV-RNA of both positive and negative polarity as well as HCV core antigen were detected in the epithelial cells of the salivary gland biopsies from all of the anti-HCV-positive patients but in none of the anti-HCV-negative cases. The percentage of HCV-infected cells ranged from 25 to 48.8% in the patients studied. In conclusion, we have shown that HCV infects and replicates in the epithelial cells from salivary glands of patients with Sjögren's syndrome or chronic sialadenitis. However, its implication in the pathogenesis of these diseases deserves future research.

Presence of TTV DNA in serum, liver and peripheral blood mononuclear cells from patients with chronic hepatitis.

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.

Detection of hepatitis C virus replication by In situ hybridization in epithelial cells of anti-hepatitis C virus-positive patients with and without oral lichen planus.

Epidemiological studies have demonstrated that there is a correlation between oral lichen planus and chronic hepatitis C virus (HCV) infection. HCV RNA has been recently detected in epithelial cells from oral lichen planus lesions by reverse-transcription polymerase chain reaction (RT-PCR). However, this technique does not discriminate which types of cells are infected by the virus or if the viral RNA is present in the serum that contaminates the biopsy. Morphological evidence of viral replication in cells from these lesions is needed to establish a role for HCV in oral lichen planus. Consequently, we have analyzed the presence of positive and negative HCV-RNA strands in oral mucosa biopsies from 23 patients (14 anti-HCV-positive) diagnosed as having oral lichen planus and from 5 patients with chronic hepatitis C without oral lichen planus. Positive and negative HCV-RNA strands were detected in epithelial cells of the mucosa biopsies from all anti-HCV-positive patients independently of whether or not they had oral lichen planus, but in none of the anti-HCV-negative cases. The percentage of stained cells ranged from 4.4% to 14.3%. These percentages do not correlate with the serum viremia levels or the intensity of the cellular infiltrate in patients with oral lichen planus. In conclusion, we have shown that HCV replicates in epithelial cells of patients with and without oral lichen planus. The pathological consequences of this finding remain to be elucidated.

Detection of TT virus DNA in liver biopsies by in situ hybridization.

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.

Hepatitis C virus RNA in kidney biopsies from infected patients with renal diseases.

Hepatitis C virus (HCV) infection has been associated with several renal pathologies, including membranoproliferative and membranous glomerulonephritis. Although the presence of HCV proteins has been reported, there are no data concerning detection of the viral RNA in renal cells from HCV-infected patients with kidney disease. In this report we analysed, by in situ hybridization, the presence of HCV RNA in renal biopsies from 10 patients who were positive for antibodies to HCV (anti-HCV) and serum HCV RNA positive, and from four patients without HCV infection, with different renal disease. HCV RNA was detected in the renal biopsies from all of the 10 HCV-infected patients. Hybridization signals were detected in the tubular and capillary endothelial cells. No hybridization signals were found in the renal biopsies of the four anti-HCV-negative patients. In conclusion, our results demonstrate that HCV RNA is common in kidney cells of patients with renal diseases who are infected with HCV. The presence of HCV RNA is not necessarily associated with a pathogenetic consequence.

Fluorescent "in situ" hybridization of hepatitis C virus RNA in peripheral blood mononuclear cells from patients with chronic hepatitis C.

Although the liver is the main target for hepatitis C virus (HCV) infection, HCV RNA of positive and negative polarity has also been detected in peripheral blood mononuclear cells (PBMCs) by polymerase chain reaction. However, no data have been published on the relationship between the number of HCV-infected PBMCs and serum viremia levels. To address this issue, PBMC samples from 20 patients with chronic hepatitis C were examined by fluorescent "in situ" hybridization. Serum viremia levels and viral load in infected PBMC were measured using the Amplicor Monitor test. HCV was detected in all PBMC samples corresponding to the HCV-positive patients. Fluorescent signals were found mainly in the cytoplasm of the cell. The percentage of positive cells ranged from 0.08% to 4%, with a statistical correlation with the viral load in PBMC (r = 0.69; p =. 001) but not with the serum viremia levels (r = 0.23). It was demonstrated that HCV infection of PBMCs is a common feature of HCV chronic carriers. The results suggest that HCV infection of PBMCs does not contribute significantly to HCV viremia.

Transplantation of allogeneic CD34-selected peripheral stem cells does not prevent transmission of hepatitis C virus from an infected donor.

There is little information on the clinical course of transplantation from HCV-positive donors. However, it seems that there is no increased risk of acute liver failure after the procedure and that the presence of HCV-RNA in serum is necessary for transmission to take place. We report a case of allogeneic CD34-selected peripheral stem cell transplantation from an HCV-infected donor with viremia with a special clinical and virological course. After the selection procedure and cell washing we could not detect HCV-RNA by PCR in the wash buffer, but HCV-RNA was positive by PCR in the selected cells. Once the patient received the transfusion of the selected product HCV was detected in the PBMCs and at very low concentration in serum. HCV was also demonstrated in the hepatocytes with the in situ hybridization technique. In conclusion, we have shown that CD34+ cell selection from an HCV-positive allogeneic donor does not prevent HCV infection in the recipient. Our results also suggest that HCV replicates in PBMCs in vivo and that these cells release viral particles that can infect the liver.