Lipid Droplet-Associated Proteins Perilipin 1 and 2: Molecular Markers of Steatosis and Microvesicular Steatotic Foci in Chronic Hepatitis C.

Chronic infection with hepatitis C (HCV) is a major risk factor in the development of cirrhosis and hepatocellular carcinoma. Lipid metabolism plays a major role in the replication and deposition of HCV at lipid droplets (LDs). We have demonstrated the importance of LD-associated proteins of the perilipin family in steatotic liver diseases. Using a large collection of 231 human liver biopsies with HCV, perilipins 1 and 2 have been localized to LDs of hepatocytes that correlate with the degree of steatosis and specific HCV genotypes, but not significantly with the HCV viral load. Perilipin 1- and 2-positive microvesicular steatotic foci were observed in 36% of HCV liver biopsies, and also in chronic hepatitis B, autoimmune hepatitis and mildly steatotic or normal livers, but less or none were observed in normal livers of younger patients. Microvesicular steatotic foci did not frequently overlap with glycogenotic/clear cell foci as determined by PAS stain in serial sections. Steatotic foci were detected in all liver zones with slight architectural disarrays, as demonstrated by immunohistochemical glutamine synthetase staining of zone three, but without elevated Ki67-proliferation rates. In conclusion, microvesicular steatotic foci are frequently found in chronic viral hepatitis, but the clinical significance of these foci is so far not clear.

Cryopreservation of precision cut tissue slices (PCTS): investigation of morphology and reactivity.

Precision cut tissue slices (PCTS) represent a suitable and convenient tool for pharmacological, toxicological and morphological studies. Cryopreservation would enable to overcome the shortage of liver tissue, in particular in settings using human liver tissue. We investigated the potential of cryopreservation of porcine PCTS as a morphological tool by rapid freezing with 10% and 30% dimethyl sulfoxide as cryopreservation agents and with or without medium using a Brendel/Vitron tissue slicer. Incubation after thawing was done in a static incubation system. Slices were cultured for 3 h, 6 h, 24 h and 48 h and assessed histologically and immunohistologically for proliferation (Ki67) and spontaneous as well as induced apoptotic activity (M30Cytodeath). Vitality was tested using the Tox-8 test. After cryopreservation, morphology of PCTS was well preserved up to 24 h. A reduction of vitality rate took place. Compared to non-frozen PCTS, the rate of spontaneous proliferation of Kupffer cells and apoptosis of hepatocytes were significantly reduced independent of the freezing conditions. The reactivity of PCTS to apoptotic stimuli was significantly reduced in tissue slices after cryopreservation. Apoptotic stimuli could not induce the same amount of cell deaths compared to non-frozen sections. Thus, cryopreservation of PCTS does interfere with pathomechanisms of apoptosis in PCTS.

Acute liver failure is associated with elevated liver stiffness and hepatic stellate cell activation.

UNLABELLED: Acute liver failure (ALF) is associated with massive short-term cell death, whereas chronic liver injury is accompanied by continuous cell death. Hepatic stellate cells (HSCs) contribute to tissue repair and liver fibrosis in chronic liver injury, although their role in ALF remains unexplained. Twenty-nine patients (median age = 43 years, 17 females and 12 males) with ALF according to the Acute Liver Failure Study Group criteria were included. Upon the diagnosis of ALF and after 7 days, we determined liver stiffness (LS) with FibroScan, standard laboratory parameters, and serum levels of matrix metalloproteinase 1 (MMP-1), MMP-2, MMP-9, tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, hyaluronic acid, and markers of overall cell death (M65) and apoptosis (M30). Stellate cell activation and progenitor response were analyzed immunohistochemically in biopsy samples of 12 patients with alpha-smooth muscle actin (alpha-SMA), keratin-17, and keratin-19 staining, respectively. Cell death markers (M30 level = 2243 +/- 559.6 U/L, M65 level = 3732 +/- 839.9 U/L) and fibrosis markers (TIMP-1 level = 629.9 +/- 69.4 U/mL, MMP-2 level = 264 +/- 32.5 U/mL, hyaluronic acid level = 438.5 +/- 69.3 microg/mL) were significantly increased in patients versus healthy controls. This was paralleled by collagen deposition, elevated alpha-SMA expression, and higher LS (25.6 +/- 3.0 kPa). ALF was associated with ductular progenitor proliferation. CONCLUSION: Our results demonstrate HSC activation and a progenitor response in ALF. Positive correlations between LS, the degree of liver cell damage, and the intensity of HSC activation suggest that fibrosis is a response to ALF in an attempt to repair damaged tissue.

Real-time elastography for noninvasive assessment of liver fibrosis in chronic viral hepatitis.

OBJECTIVE: Recently, transient elastography (FibroScan) has been introduced for noninvasive staging of liver fibrosis. Here, we investigated a novel approach for noninvasive assessment of liver fibrosis using sonography-based real-time elastography, which can be performed with conventional ultrasound probes during a routine sonography examination. MATERIALS AND METHODS: Real-time elastography was performed in 79 patients with chronic viral hepatitis and known fibrosis stage and in 20 healthy volunteers. A specially developed program was used for quantification of tissue elasticity. Stepwise logistic regression analysis was performed to define an elasticity score using variables with high reproducibility in a preceding analysis of data from 16 different patients. In addition, aspartate transaminase-to-platelet ratio index (APRI) and routine laboratory values were included in the analysis. RESULTS: The Spearman's correlation coefficient between the elasticity scores obtained using real-time elastography and the histologic fibrosis stage was 0.48, which is highly significant (p < 0.001). The diagnostic accuracy expressed as areas under receiver operating characteristic (ROC) curves were 0.75 for the diagnosis of significant fibrosis (fibrosis stage according to METAVIR scoring system [F] > or = F2), 0.73 for severe fibrosis (F > or = F3), and 0.69 for cirrhosis. For a combined elasticity-laboratory score, the areas under the ROC curves were 0.93, 0.95, and 0.91, respectively. DISCUSSION: Real-time elastography is a new and promising sonography-based noninvasive method for the assessment of liver fibrosis in patients with chronic viral hepatitis.

Expression of epithelial cellular adhesion molecule (Ep-CAM) in chronic (necro-)inflammatory liver diseases and hepatocellular carcinoma.

Epithelial cell adhesion molecule (Ep-CAM) is expressed in a several epithelial tissues and carcinomas, but not on mature hepatocytes. Here, we analysed the expression of Ep-CAM in 230 patients suffering from various liver diseases like chronic hepatitis B and C (HBV and HCV infection), chronic autoimmune hepatitis (AIH), chronic alcoholic liver disease (ALD), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), hereditary hemochromatosis and dysplastic nodules (DNs) as well as hepatocellular carcinomas (HCCs) and cholangiocellular carcinomas (CCCs) by immunohistochemistry. De novo hepatocellular Ep-CAM expression was found in 75.9% of ALD (22/29), 63.6% of HCV (21/33) and 55.6% of each AIH and HBV cases (5/9 and 15/27, respectively). Lower Ep-CAM expression levels were observed for primary sclerosing liver diseases (PBC and PSC) with 25% (3/12) and 7.7% (1/13) of cases. Moreover, only 14.3% of HCCs (9/63) manifested expression, while all CCCs showed strong Ep-CAM expression (5/5). For DNs and hereditary hemochromatosis, Ep-CAM expression was found in 10 and 50% (3/30 and 2/4), respectively. In HBV and HCV, Ep-CAM expression correlated significantly with inflammatory activity as assessed by histological parameters and to the extent of fibrosis. In addition, for HCV also transaminase levels correlated significantly with Ep-CAM expression. Our results indicate that de novo Ep-CAM expression in hepatocytes is frequent in inflammatory liver diseases and is potentially linked to regenerative activity. CCCs and Ep-CAM positive HCCs may represent an attractive target group for Ep-CAM-directed immunotherapies, yet unwanted toxicity may limit the use of such strategies due to Ep-CAM expression in biliary epithelium and several chronic liver diseases such as HBV-and HCV-hepatitis.

Cryoglobulin-associated uptake of hepatitis C virus into human hepatocytes.

AIMS: The mechanisms of binding and uptake of hepatitis C-virus (HCV) are critical determinants of the infection-reinfection cycle but due to ongoing absence of a robust cell culture system, these mechanisms are still largely hypothetical. Cryoglobulins are atypical immunoglobulins, present in 40% of HCV patients. The aim of this study was to determine the role of these HCV-containing cryoglobulins as carrier molecules for viral uptake into primary human hepatocytes. METHODOLOGY: Cryoglobulins were precipitated from serum of chronically HCV-infected patients, labeled with biotin and incubated with freshly prepared hepatocytes from human liver tissue. Binding and endocytosis of HCV-cryoglobulins were studied by specific assays, ligand blot analysis and electron microscopy on hepatocellar plasma membranes. RESULTS: Biotinylated HCV-cryoglobulins specifically bound to hepatocytes and inhibitors of homotypic endosomal fusion reduced their uptake and intracellular trafficking, Ligand-blot and electron microscopy analysis revealed adhesion to hepatocellular plasma membranes. Inoculation of human hepatocytes with HCV-cryoglobulins but not serum from the same patients induced HCV infection in vitro. CONCLUSIONS: HCV may enter hepatocytes in conjunction with cryoglobulins via immunoglobulin or related receptors. We hypothesize, that this mechanism plays a role in chronic hepatitis to support the infection-reinfection cycle of the virus.

Precision cut tissue slices of the liver as morphological tool for investigation of apoptosis.

BACKGROUND: Apoptosis, programmed cell death, is involved in a broad range of pathological processes. Dysregulation of apoptosis plays a key role in the pathogenesis of hepatitis, toxic liver disease and also liver tumor development. For the study of apoptosis in liver diseases, different in vivo models and different in vitro approaches have been developed. They include cell culture models based on hepatocellular carcinoma cell lines or isolated primary hepatocytes. MATERIALS AND METHODS: We have established precision cut tissue slices (PCTS) of the liver as a morphological tool for the study of apoptosis. From porcine livers, PCTS were prepared and incubated in a static system with different types and amounts of media. Viability, morphology, spontaneous apoptosis and proliferation were investigated. Apoptosis was induced with actinomycin D and tumor necrosis factor (TNF) alpha. RESULTS: Morphology and viability was well preserved for at least 24 h. After 48 h, deterioration with single and group cell autolysis was seen. There was a low rate of spontaneous apoptosis and proliferation. Using a combination of TNF alpha and actinomycin D, a significant amount of apoptosis occurred. CONCLUSION: PCTS can be used to directly analyse apoptosis at the tissue level in a qualitative and quantitative manner.

Comparison of transcription mediated amplification (TMA) and reverse transcription polymerase chain reaction (RT-PCR) for detection of hepatitis C virus RNA in liver tissue.

BACKGROUND: Transcription mediated amplification (TMA) is known to be one of the most sensitive detection assays for hepatitis C virus (HCV) RNA in serum but has not yet been evaluated in liver tissue. It is unknown whether the higher sensitivity of TMA in comparison with polymerase chain reaction (PCR)-based assays is related to a higher efficiency of the extraction and/or amplification step. OBJECTIVES: The sensitivity of a TMA-based assay (Versant HCV RNA Qualitative assay, Bayer Diagnostics) and a standard RT-PCR-based assay (Cobas Amplicor HCV 2.0, Roche Diagnostics) was compared in formalin-fixed paraffin-embedded liver biopsy specimens of patients with chronic hepatitis C. STUDY DESIGN: After deparaffinization of 7.5 microm liver sections HCV RNA was extracted by standard phenol/chloroform. HCV RNA dilution panels were transferred in parallel to cDNA synthesis and amplification steps of PCR and TMA. Furthermore, TMA amplification from stepwise diluted HCV sera was performed following RNA extraction by either microcentrifuge colums (QIAmp Viral RNA spin Kit, Qiagen, Hilden, Germany) or magnetic microparticles (VERSANT HCV RNA Qualitative assay). RESULTS: The total number of HCV RNA positive liver specimens detected by TMA was higher compared with those detected by RT-PCR (P=0.032). The total number of TMA positive serum samples was higher when HCV RNA was extracted using magnetic microparticles in comparison with multicentrifuge column extraction (P=0.019). CONCLUSION: Our results suggest that both the extraction and amplification step of the TMA-based assay contribute to the higher sensitivity compared with standard RT-PCR.

Development of skin tumors in mice transgenic for early genes of human papillomavirus type 8.

The cutaneous human papillomavirus (HPV) 8 is clearly involved in skin cancer development in epidermodysplasia verruciformis patients and its early genes E2, E6, and E7 have been implicated in cell transformation in vitro. To examine the functions of these genes in vivo we integrated the complete early region of HPV8 into the genome of DBA/Bl6 mice. To target their expression to the basal layer of the squamous epithelia the transgenes were put under the control of the keratin-14 promoter. Transgenic mice were back-crossed for up to six generations into both FVB/N and Bl6 mouse strains. Whereas none of the HPV8 transgene-negative littermates developed lesions in the skin or any other organ, 91% of HPV8-transgenic mice developed single or multifocal benign tumors, characterized by papillomatosis, acanthosis, hyperkeratosis, and varying degrees of epidermal dysplasia. Squamous cell carcinomas developed in 6% of the transgenic FVB/N mice. Real-time reverse transcription-PCR showed highest expression levels for HPV8-E2, followed by E7 and E6. There was no consistent difference in relative viral RNA levels between healthy or dysplastic skin and malignant skin tumors. Whereas UV-induced mutations in the tumor suppressor gene p53 are frequently detected in human skin carcinomas, mutations in p53 were not observed either in the benign or malignant mouse tumors. Nonmelanoma skin cancer developed in HPV8-transgenic mice without any treatment with physical or chemical carcinogens. This is the first experimental proof of the carcinogenic potential of an epidermodysplasia verruciformis-associated HPV-type in vivo.

Differential efficacy of corticosteroids and interferon in a patient with chronic hepatitis C-autoimmune hepatitis overlap syndrome.

The chronic hepatitis C-autoimmune hepatitis (AIH) overlap syndrome has been described in the literature, but to date appropriate therapy remains controversial. We report on a 28-year-old woman with hepatitis C-AIH overlap syndrome. The patient was infected with HCV genotype 1b and had laboratory and immunologic findings of AIH type 2 such as increased Igs and a high titer of antibodies against liver-kidney microsomes. Initial liver biopsy specimen demonstrated end-stage liver fibrosis due to chronic hepatitis. After long-lasting corticosteroid treatment, only partial remission was achieved. In contrast, short-term antiviral therapy with interferon-alpha2b in combination with ribavirin was followed by complete biochemical and virologic remission. However, 15 months later, a relapse of AIH was observed. After restarting corticosteroid treatment, transaminase levels completely normalized. Surprisingly, in this patient with overlap syndrome, short-term interferon therapy induced complete remission of chronic HCV infection and regression of severe liver fibrosis.

Florid ischemic cholangitis due to leucocytoclastic vasculitis.

Ischemia-induced biliary tract lesions, called ischemic cholangitis, often lead to strictures of biliary ducts and cholestasis. Causes of ischemic changes of the biliary tract can be found in the arterial blood supply or in the peribiliary capillary plexus. Known examples are thrombosis after transplantation, intraoperative ligation, or the application of chemotherapeutic drugs. Rarely, such changes are due to inflammation of the blood vessels, such as occurs in polyarteritis nodosa or giant cell arteritis. We present a report of a 49-year old man with leucocytoclastic vasculitis after viral infection, influenza vaccination, and antibiotic treatment, leading to florid ischemic cholangitis. We conclude that hypersensitivity vasculitis must be included in the differential diagnosis of cholestasis and cholangitis.

Successful adjuvant tamoxifen therapy for estrogen receptor-positive metastasizing sweat gland adenocarcinoma: need for a clinical trial?

We report on successful adjuvant tamoxifen therapy for a metastasizing sweat gland adenocarcinoma of the scalp in a 64-year-old woman. Before the antihormonal therapy, the patient had undergone repeated surgery for ipsilateral intraparotid, soft tissue, and lymph node metastases and had had disease-free intervals of less than 5 months. As the immunohistochemical analysis of the tumor tissue revealed a 100% nuclear reactivity to estrogen and progesterone receptors, we started empirical tamoxifen citrate therapy, which dramatically changed the course of the disease. The patient has been in complete remission for 3 years. This is the third report in the literature of substantial therapeutic benefit of antiestrogen therapy in metastasizing eccrine gland adenocarcinoma with positive hormone receptor immunohistochemistry. We suggest examining the hormone receptor expression in these neoplasms regularly. A prospective study should be commenced to assess the benefit of adjuvant antihormonal therapy in eccrine gland adenocarcinomas.

A hemodynamic response to intravenous adenovirus vector particles is caused by systemic Kupffer cell-mediated activation of endothelial cells.

Intravascular injection of adenoviral vectors may result in a toxic and potentially lethal reaction, the mechanism of which is poorly understood. We noted that mice demonstrated a transient change in behavior that was characterized by inactivity and lethargy within minutes after intravenous injection of relatively low doses of adenoviral vectors (including high-capacity gutless vectors). Moreover, immediately after vector injection a significant drop in blood pressure was measured that most probably was caused by the systemic activation of endothelial cells as monitored by detection of phosphorylated Akt/PKB kinase, activated endothelial nitric oxide synthase (eNOS), and nitrotyrosine. The activation of the endothelium was the result of the interaction of viral particles with Kupffer cells, which are resident macrophages of the liver representing the first line of defense of the innate immune system. Surprisingly, the uptake of vector particles by Kupffer cells not only resulted in their strong activation, but also in their nearly complete disappearance from the liver. Our results suggest that the toxicity of intravenously injected adenoviral vectors may be directly linked to the activation and destruction of Kupffer cells.

Human papillomavirus-positive tonsillar carcinomas: a different tumor entity?

Human papillomavirus (HPV) infections are thought to be one of the causal factors in the development of head and neck squamous cell carcinomas (HNSCC), particularly in tumors arising from the Waldeyer's tonsillar ring. We screened 98 carefully stratified HNSCC and different control tissues for the presence of HPV DNA by nested polymerase chain reaction (PCR) specific for genital- and Epidermodysplasia verruciformis (EV)-associated HPVs and by HPV16-specific single step PCR. Typing was performed by direct sequencing and/or sequencing of cloned amplimers. On average HNSCC showed rather low HPV DNA prevalences; 18% of the oral cavity cancers, 8% of nasopharyngeal cancers, 25% of hypopharyngeal cancers and 7% of laryngeal cancers were HPV DNA positive. In contrast, HPV sequences could be detected in 45% of the oropharyngeal cancers, particularly tonsillar carcinomas (58%). Tonsillar carcinomas were significantly more likely to be HPV positive than tumors from any other site ( P<0.001). All tonsillar cancers contained oncogenic HPV types, predominantly HPV16 (13 of 14; 93%). Unaffected tonsils were available from two of these patients, but both tested negative for HPV DNA. Furthermore, no HPV DNA could be found in tonsillar biopsy specimens from control groups. Localization and load of HPV DNA was determined in HPV16-positive tonsillar carcinomas, their metastases and in unaffected mucosa using laser-assisted microdissection and subsequent real time fluorescence PCR. We demonstrated that the HPV genome is located in the cancer cells, whereas the infection of normal mucosa is a rare event. Quantification of HPV16 DNA in samples of seven patients yielded viral loads from 6 to 153 HPV DNA copies per beta-globin gene copy and the load values in both locations were roughly comparable. These loads are comparable with data shown for other HPV-associated lesions. Statistical evaluation of data related to clinicopathological parameters showed a significant correlation of the HPV positivity of tonsillar carcinomas with tumor grading ( P=0.008) and alcohol consumption ( P=0.029). Taken together our findings show a preferential association of HPV DNA with tonsillar carcinomas. Furthermore our results argue for HPV-positive tonsillar carcinomas representing a separate tumor entity, which is less dependent on conventional HNSCC risk factors.

Dominance of CD4+ alpha/beta T-cells and inferior role of innate immune reaction in liver metastases.

BACKGROUND: Tumor infiltrating lymphocytes (TIL) are frequently present in human tumors with CD8+(-)T-cells as effector and CD4+ T-cells as helper cells. Despite the well established knowledge about primary tumors, only little is known about metastatic disease, especially for liver metastases. The role of the innate immune system in the tumor defence is still enigmatic. MATERIALS AND METHODS: We performed a subtyping of TIL in 20 liver metastases. Using immunohistochemistry, CD20+, CD3+, CD56+, CD4+ and CD8+ lymphocytes, gamma/delta-T-cells and alpha/beta-T-cells in the tumor, the peritumoral region, portal tracts and lobules were investigated. RESULTS: The immune response was highly accentuated in the surroundings of the metastases with only few lymphocytes in the tumor itself. There was a dominance of CD3+(-)CD4+(-)alpha/beta-T-cells with a lower number of CD8+(-)T-cells. The CD4+/CD8+ ratio was 6:1. CD56+(-)NK/NKT-cells and gamma/delta-T-cells were rare. No differences were found between metastases from different primaries or according to the number or diameter of the metastases. CONCLUSION: TIL are part of an interaction between the metastatic tumor and the liver. Among them CD4+ T-cells seem to have a unique independent function in tumor response. The localization of the immune response in the tumor periphery might be a reason for insufficient tumor defense. A defect in the innate immune system could be a reason for the escape of the metastatic tumor cells from tumor surveillance.

Allelic losses on chromosome 6q25 in Hodgkin and Reed Sternberg cells.

We established a molecular cytogenetic approach to identify consistent genetic aberrations in classical Hodgkin lymphoma. Single laser-micromanipulated Hodgkin and Reed Sternberg (H-RS) cells and the respective germ line tissue were PCR-amplified using highly polymorphic microsatellite probes. Loss of heterozygosity and genomic imbalances of the fluorochrome-labeled microsatellites were determined by fragment length analysis. Eleven cases of in classical Hodgkin lymphoma (cHL) were initially screened with 21 microsatellite markers scattered over the entire genome. Loss of heterozygosity was detected in >40% of informative loci in most cases indicating a deletion of a substantial part of the genome of H-RS cells. Allelic losses and imbalances on chromosome 6q were detected in most of these cases. A deletion mapping of 6q was performed in 16 cases of cHL. This detailed analysis of 6q led to the identification of a 3.3-Mbp region around D6S311 flanked by D6S978 and D6S1564 that was altered in 11 of 14 cases of cHL analyzed. In conclusion, allelotyping of single H-RS cells revealed monoallelic chromosomal deletions and genomic imbalances on 6q that might affect genes critically involved in the pathogenesis of H-RS cells.

Viral escape and T-cell immunity during ganciclovir treatment of cytomegalovirus infection: case report of a pancreatico-renal transplant recipient.

BACKGROUND: Pancreas-kidney transplant recipients are at high risk for cytomegalovirus (CMV) disease despite prophylactic ganciclovir therapy. Because the impact of antiviral therapy on anti-CMV immune reactions is unknown, CMV-specific T-cell subsets in primary and recurrent CMV infection were analyzed in a pancreas-kidney transplant case study. METHODS: Major histocompatibility complex class I tetramers were used to detect peripheral CMV pp65-specific CD8 T cells. Intracellular cytokine staining was used to determine the frequency of CMV-specific CD4 T cells. Conventional virologic parameters and routine laboratory parameters were monitored. For ganciclovir resistance testing, CMV-UL97 genotyping was performed. RESULTS: Despite prophylactic ganciclovir therapy, primary CMV infection induced in vivo expansion of activated CMV-specific CD8 T cells. Interestingly, viral dissemination during recurrent CMV disease was a result of partially ganciclovir-resistant CMV. Recovery after discontinued ganciclovir treatment was associated with the expansion of CMV-specific CD4 T cells. CONCLUSION: Immunologic monitoring may contribute to clinical management of recurrent CMV disease.

Expression of p16 protein identifies a distinct entity of tonsillar carcinomas associated with human papillomavirus.

Recent analyses of head and neck squamous cell carcinomas revealed frequent infections by oncogenic human papillomavirus (HPV) type 16 in tonsillar carcinomas. Concerning involvement of risk factors, clinical course of the disease, and prognosis there are strong indications arguing that the HPV-positive tonsillar carcinomas may represent a separate tumor entity. Looking for a surrogate marker, which in further epidemiological studies could replace the laborious and expensive HPV detection and typing we analyzed p16 protein expression in 34 tonsillar carcinoma for correlation to HPV status and load of viral DNA. p16 has been shown to be of diagnostic value for clinical evaluation of cervical dysplasia. We found 53% of the tested tonsillar carcinomas to be HPV-positive. Fifty-six percent of all tumors tested were immunohistochemically positive for the p16 protein. In 16 of 18 of the HPV-positive carcinomas diffuse p16 expression was observed. In contrast, only one of the HPV-negative carcinomas showed focal p16 staining (P < 0.001). As determined by laser-assisted microdissection and quantitative real-time polymerase chain reaction, p16 expression correlated with the presence of HPV-DNA in the individual tumor specimens. Clinical outcome analysis revealed significant correlation of p16 expression with increased disease-free survival (P = 0.02). These data indicate that p16 is a technically simple immunohistological marker, applicable for routine pathological histology, and its prognostic value for survival is fully equivalent to HPV-DNA detection.

Selective depletion or blockade of Kupffer cells leads to enhanced and prolonged hepatic transgene expression using high-capacity adenoviral vectors.

Tissue macrophages, in particular hepatic Kupffer cells (KCs), contribute to early inflammatory responses following adenoviral vector administration. This study evaluates the effect of selective and transient (3 days) depletion of KCs by a single injection of clodronate liposomes on the in vivo performance of high-capacity adenoviral (HC-Ad) vectors. In KC-depleted C57BL/6 and C3H mice increased and stabilized hAAT levels were observed following intravenous injection of HC-Ad vectors expressing human alpha-1 anti-trypsin (hAAT) either from the hAAT promoter or from the human cytomegalovirus promoter. Comparable increases in hAAT levels were obtained in mice preinjected with a transcriptionally silent HC-Ad vector. Interestingly, in the majority of animals of both strains depletion of KCs was sufficient to prevent the generation of anti-hAAT antibodies, resulting in prolonged transgene expression. Thus, short-term and selective depletion of hepatic macrophages at the same time significantly increased hepatic transgene expression and reduced the humoral immune response to the transgenic protein.

Autoimmune hepatitis and overlap syndromes.

Autoimmune hepatitis (AIH) is an immune-mediated, autodestructive liver disease with hepatocytes as target cells, mostly affecting young women. Primary biliary cirrhosis (PBC) is also regarded as an autoimmune liver disease with bile duct epithelia as the target cells, resulting in a continuous loss of bile ducts. Both diseases may occur simultaneously in their full manifestations in about 10% to 20% of cases, thus constituting an overlap syndrome with PBC directing the course of the disease. AIH may also occur simultaneously with primary sclerosing cholangitis (PSC), with a frequency of between 2% and 8% of patients with PSC. In most cases, AIH precedes manifestation of PSC. In children, the overlap syndrome of AIH and PSC seems to make up an entity of its own: autoimmune sclerosing cholangitis.