Paolo Gentilini

Infection of peripheral mononuclear blood cells by hepatitis C virus

We investigated the infection of peripheral blood mononuclear cells (PBMNC) by hepatitis C virus (HCV) in 5 patients with HCV-related chronic hepatitis. The presence of HCV-RNA-positive and -negative strands was tested with the polymerase chain reaction (PCR) method. In all subjects, HCV-RNA was shown in PBMNC. In 3 cases, HCV-RNA was shown in the T- and B-cell populations, with viral RNA also present in the monocyte-macrophage fraction of two of these. HCV-RNA-negative stranded molecules, indicative of the viral multiplication, were significantly increased in cells maintained in cultures with PHA/PMA stimulation. The results indicate that HCV infect blood mononuclear cells, thus suggesting that this cellular tropism may play a role in HCV infection.

Fat-storing cells as liver-specific pericytes. Spatial dynamics of agonist-stimulated intracellular calcium transients.

Liver perisinusoidal fat-storing cells (FSC) show morphological and ultrastructural characteristics similar to pericytes regulating local blood flow in other organs. In the present study we have analyzed whether FSC respond to local vasoconstrictors such as thrombin, angiotensin-II, and endothelin-1 with an increase in intracellular free calcium concentration ([Ca2+]i) coupled with effective cell contraction. All agonists tested induced a rapid and dose-dependent increase in [Ca2+]i followed by a sustained phase lasting several minutes in confluent monolayers of Fura-2-loaded human FSC. Pharmacological studies performed using different Ca2+ channel blockers indicated that, at least for thrombin and angiotensin-II, the sustained phase is due to the opening of voltage-sensitive membrane Ca2+ channels. To analyze the temporal and spatial dynamics of Ca2+ release in response to these agonists, we performed experiments on individual Fura-2-loaded human FSC using a dual wavelength, radiometric video imaging system. The rise in [Ca2+]i was exclusively localized to the cytoplasm, particularly in the branching processes. Increases in [Ca2+]i more than four-fold were associated with a simultaneous and transient reduction of cell area indicating reversible cell contraction. Our results indicate that the Ca(2+)-dependent contraction of human FSC in vitro may reflect a potential role in regulating sinusoidal blood flow in vivo.

HNE interacts directly with JNK isoforms in human hepatic stellate cells.

4-Hydroxy-2,3-nonenal (HNE) is an aldehydic end product of lipid peroxidation which has been detected in vivo in clinical and experimental conditions of chronic liver damage. HNE has been shown to stimulate procollagen type I gene expression and synthesis in human hepatic stellate cells (hHSC) which are known to play a key role in liver fibrosis. In this study we investigated the molecular mechanisms underlying HNE actions in cultured hHSC. HNE, at doses compatible with those detected in vivo, lead to an early generation of nuclear HNE-protein adducts of 46, 54, and 66 kD, respectively, as revealed by using a monoclonal antibody specific for HNE-histidine adducts. This observation is related to the lack of crucial HNE-metabolizing enzymatic activities in hHSC. Kinetics of appearance of these nuclear adducts suggested translocation of cytosolic proteins. The p46 and p54 isoforms of c-Jun amino-terminal kinase (JNKs) were identified as HNE targets and were activated by this aldehyde. A biphasic increase in AP-1 DNA binding activity, associated with increased mRNA levels of c-jun, was also observed in response to HNE. HNE did not affect the Ras/ERK pathway, c-fos expression, DNA synthesis, or NF-kappaB binding. This study identifies a novel mechanism linking oxidative stress to nuclear signaling in hHSC. This mechanism is not based on redox sensors and is stimulated by concentrations of HNE compatible with those detected in vivo, and thus may be relevant during chronic liver diseases.

Phosphatidylinositol 3-kinase is required for platelet-derived growth factor's actions on hepatic stellate cells.

BACKGROUND & AIMS Platelet-derived growth factor (PDGF) is the most potent mitogen for hepatic stellate cells (HSCs) in vitro. The aim of this study was to investigate the role of phosphatidylinositol 3-kinase (PI 3-K) activation in mediating the biological effects of PDGF on cultured HSCs and its involvement in vivo. METHODS HSCs were isolated from normal human livers. PI 3-K was assayed on phosphotyrosine or PDGF-receptor immunoprecipitates by in vitro kinase assay. RESULTS Incubation of HSCs with PDGF caused a time-dependent increase in PI 3-K activity. Immunoprecipitation of PDGF-alpha and -beta receptors showed that both subunits associate with active PI 3-K in PDGF-stimulated HSCs. Wortmannin, a specific PI 3-K inhibitor, dose-dependently blocked PI 3-K activity induced by PDGF and inhibited DNA synthesis. PDGF (homodimer)-BB also stimulated HSC chemotaxis, which was inhibited by pretreatment with wortmannin. To explore the potential role of PI 3-K in vivo, liver homogenates from rats treated with CCl4 and from control rats were immunoprecipitated with anti-PDGF-beta-receptor antibodies. Liver injury was associated with increased PDGF-beta-receptor autophosphorylation, and greater PI 3-K activity associated with the receptor itself. CONCLUSIONS This study shows that in cultured HSCs, PI 3-K activation is necessary for both mitogenesis and chemotaxis induced by PDGF and that this pathway is up-regulated during liver injury in vivo.

Prevalence of bcl-2 Rearrangement in Patients with Hepatitis C Virus–Related Mixed Cryoglobulinemia with or without B-Cell Lymphomas

Context Rearrangement of bcl-2 has an antiapoptotic effect and has been implicated as a potential cause of benign lymphoproliferation (causing mixed cryoglobulinemia) and B-cell lymphoma. Mixed cryoglobulinemia is strongly associated with hepatitis C virus (HCV) infection. Contribution In patients with HCV-associated chronic liver disease, bcl-2 rearrangement occurred significantly more often in patients with chronic HCV infection and mixed cryoglobulinemia than in HCV-infected patients without mixed cryoglobulinemia; it also occurred in three of four patients with B-cell lymphoma. Transient suppression of HCV in two patients was associated with remission of clinical manifestations of mixed cryoglobulinemia. Implications Viral induction of gene sequence translocations may help explain some benign and malignant lymphoproliferative disorders. The Editors Mixed cryoglobulinemia is a distinct syndrome clinically characterized by purpura; weakness; arthralgia; and such conditions as membranoproliferative glomerulonephritis, peripheral neuropathy, skin ulcers, and diffuse vasculitis (1, 2). Cryoprecipitable immune complexes, specifically mixed (IgG-IgM) cryoglobulins, are the serologic hallmark of the disease. Immunoglobulin Gs are the autoantigens, and IgMs with rheumatoid factor activity are the autoantibodies. Mixed cryoglobulinemia is classified as type II or type III according to the presence of polyclonal or monoclonal IgMs (3, 4). Because expansion of rheumatoid factorproducing B cells is the underlying disorder of mixed cryoglobulinemia, this condition is considered a benign B-cell lymphoproliferative disease. Type II and III mixed cryoglobulinemia are similar in terms of organ involvement and clinical course, except that type II disease may evolve into cancer. Type II mixed cryoglobulinemia is often observed in conjunction with bone marrow findings consistent with indolent B-cell lymphoma (5-9) and evolves to frank B-cell malignancy in about 10% of cases (10). A strong association between mixed cryoglobulinemia and infection with hepatitis C virus (HCV), a hepatotropic and lymphotropic virus (10, 11), has been shown. A pathogenetic role of chronic infection with HCV in mixed cryoglobulinemia has been suggested. The mechanisms involved in benign lymphoproliferation of mixed cryoglobulinemia and its evolution to lymphoma remain unknown. However, rearrangement of the antiapoptotic B-cell lymphoma/leukemia 2 (bcl-2) genethe t(14;18) translocationis suggested to play a role in the pathogenesis of HCV-associated mixed cryoglobulinemia (12, 13). The t(14;18) translocation, the most frequent genetic aberration in human lymphoma (14, 15), may be favored by sustained, strong antigenic stimulation (16-18). As a result of bcl-2 rearrangement, the bcl-2 gene on chromosome 18q21 is coupled with the immunoglobulin heavy chain gene (IgH) on chromosome 14q32 by a process frequently involving IgH joining segments (JH) (Figure 1, top). At the junction of the two genes, insertions of variable lengths (N segments) due to random addition of nongermline nucleotides result in a DNA pattern that is clone specific (19, 20). As a consequence of this rearrangement, bcl-2 is activated and B cells bearing the t(14;18) translocation express inappropriately elevated levels of the Bcl-2 protein. Figure 1. Schematic representation of the t(14; 18) translocation and its effects on B cells. Top. bcl-2 Bottom. Bcl-2 is a member of a larger family. Family members can interact with each other in a complex manner; some act to promote and others to inhibit apoptosis (14). The Bcl-2 protein protects cells from apoptosis, whereas its homologue, Bax, kills cells (21). Thus, the ratio of Bcl-2 to Bax is a determinant of susceptibility to apoptosis (14) (Figure 1, bottom). Strong expression of Bcl-2 protein has been observed in lymphoid infiltrates in liver and bone marrow specimens of patients with mixed cryoglobulinemia (22). In a previous study, the prevalence of bcl-2 rearrangement in peripheral blood mononuclear cells was significantly higher in patients with chronic HCV infection than in healthy persons or those without HCV infection but with chronic liver diseases or systemic autoimmune disorders (13). Of note, the prevalence of bcl-2 rearrangement was particularly high in patients with HCV-associated type II mixed cryoglobulinemia. We sought to evaluate the prevalence of bcl-2 rearrangement in peripheral blood cells of patients with mixed cryoglobulinemia, to confirm that results are patient specific by sequencing studies, to analyze Bcl-2 expression and the ratio of Bcl-2 to Bax in these patients, and to observe the effect of antiviral therapy. Methods Patients We enrolled 37 patients (12 men and 25 women; mean age SD, 64 9 years) with HCV infection and mixed cryoglobulinemia who were consecutively referred to the outpatient clinic of the Department of Internal Medicine, University of Florence School of Medicine, a tertiary hepatology center, and the rheumatologic section of the Department of Internal Medicine, University of Pisa School of Medicine, from January 1999 to May 2000. These patients were compared with 101 consecutively recruited patients (62 men and 39 women; mean age, 51 11 years) who had HCV-related chronic liver diseases but not mixed cryoglobulinemia or another lymphoproliferative disease. Hepatitis C virus infection was established by detection of circulating anti-HCV antibodies (EIA-2 and RIBA-2, Ortho Diagnostic Systems, Raritan, New Jersey) and HCV RNA (nested polymerase chain reaction [PCR] for HCV) (10, 13, 23). Essential mixed cryoglobulinemia was diagnosed according to published criteria (10, 13). Serum cryoglobulins, complement fraction levels, rheumatoid factor, and autoantibodies were routinely measured and characterized in all patients as described elsewhere (10, 13, 23). Diagnosis of liver disease was based on results of liver biopsy. Lymphomas were diagnosed by an independent pathologist and classified according to the revised European-American classification of lymphoid neoplasms (24). No patient tested positive for hepatitis B surface antigen, IgM anti-HBc, hepatitis B virus DNA, IgM anti-delta, antiEpsteinBarr virus, anti-cytomegalovirus, antiherpes simplex virus, or anti-HIV. No patient had a history of alcohol abuse or previous antiviral or immunosuppressive treatment. All patients gave informed consent to participate in the study, which was performed in accordance with the principles of the Declaration of Helsinki, and the study was approved by the local ethics committee. Detection of the t(14; 18) Translocation The t(14; 18) translocation in peripheral blood mononuclear cells was detected on total DNA by using nested PCR (major breakpoint region), as described elsewhere (13). Nested PCR is a variant of PCR; after an initial series of amplification cycles, templates are again amplified by using a second set of primers internal to the first ones. The resulting reaction is very specific and sensitive owing to specific binding to the target sequences of four instead of two specific primers. The limit of sensitivity was one rearranged cell in 105 to 106 normal cells. Amplification products were analyzed by both ethidium bromide staining and hybridization with a specific digoxigenin-labeled probe (Southern blot analysis). Each sample was analyzed at least twice, and all samples that tested negative on PCR were analyzed at least four times. Different cell samples that were obtained at the same time (synchronous) or at different times (metachronous) were also analyzed when possible. Approximately 2.5 105 mononuclear cells were tested in each reaction, corresponding to about 1 g of DNA. Positive and negative control samples were included in each experiment (13). To avoid false-positive results caused by carryover of PCR product, precautions were taken, as described elsewhere (10, 13). To ensure DNA amplificability, PCR was also performed by using primers for the human HLA gene (exon 2 of HLA-DRB gene), as previously reported (13). Finally, bcl-2/JH junction sequence was determined in part by cycle sequencing and solid-phase sequencing techniques (13, 25) and in part by automated sequencing (Abi Prism, Perkin Elmer, Norwalk, Connecticut). Measurement of Bcl-2 and Bax Proteins Bcl-2 and Bax proteins were measured as described elsewhere (13) on freshly isolated peripheral blood mononuclear cells and, when possible (9 patients), in separated cell subgroups (T cells, B cells, and monocytes and macrophages). Bcl-2 was detected by using monoclonal mouse anti-human Bcl-2 (Santa Cruz Biotechnology, Inc., Santa Cruz, California), and Bax was detected by using polyclonal rabbit anti-human Bax (Upstate Biotechnology, Inc., Lake Placid, New York). The CD2+ T cells, CD19+ B cells, and CD14+monocytes and macrophages from peripheral blood were separated by immunomagnetic isolation using Dynabeads M450 Pan-T, M-450 Pan-B, and M-450 CD14+, respectively (Dynal A.S., Oslo, Norway), according to the manufacturers instructions. Statistical Analysis Data are expressed as the mean SD. Data were analyzed by performing the Fisher exact test, using True Epistat 4.0 statistical software (Epistat Service, Richardson, Texas). A P value less than 0.05 was considered significant. Role of the Funding Sources The funding sources had no role in the analysis, reporting, or interpretation of the data or in the decision to submit the report for publication. Results The Table shows the clinical, epidemiologic, and pathologic characteristics of patients with HCV-related mixed cryoglobulinemia. The mean duration of mixed cryoglobulinemia syndrome was 9.2 5.2 years. Most of these patients (91%) had chronic liver diseases. Liver biopsy showed chronic hepatitis in 27 patients (72.9%) and cirrhosis in 7 patients (18.9%); of the latter patients, 1 also had superimposed hepatocellular carcinoma. Liver biopsy was not performed in the remaining 3 patients becaus

Hepatitis C Virus Genotype Analysis in Patients with Type II Mixed Cryoglobulinemia

Hepatitis C virus (HCV) infection has been related to different autoimmune-lymphoproliferative diseases such as autoimmune hepatitis [1, 2] and mixed cryoglobulinemia [3, 4]. The latter condition is associated with HCV infection in almost 90% of cases and is characterized by symptoms of systemic vasculitis secondary to deposition of coldprecipitable immune complexes. The remote pathogenesis of mixed cryoglobulinemia is considered to be a B-cell lymphoproliferation, which in many patients can be complicated by malignant lymphoma [5, 6]. Because of the variability of the HCV genome, one might speculate that particular viral variants are responsible for mixed cryoglobulinemia. Thus, we assessed the prevalence of different genotypes in HCV-positive cryoglobulinemic patients and in patients with chronic HCV infection who did not have cryoglobulinemia. Methods From March 1994 to September 1994, we recruited (at ambulatory visits) 29 consecutive HCV-positive (anti-HCV antibody-positive and HCV RNA-positive) patients with type II (IgM ) mixed cryoglobulinemia (9 men, 20 women; mean age SD, 60 7.5 years; age range, 46 to 72 years) and 61 patients with chronic HCV infection who did not have mixed cryoglobulinemia (control group). All patients studied were Italian-born, were heterosexual, and had no history of blood transfusion or drug or alcohol abuse. Cryoglobulinemic and control patients were followed at the rheumatology and hepatology units of the University of Pisa and University of Florence. The mean (SD) duration of follow-up was 8.4 5.5 years (range, 1 to 25 years) for cryoglobulinemic patients and 6.8 4.5 years (range, 2 to 14 years) for controls. A diagnosis of mixed cryoglobulinemia was made if a patient had the typical syndrome (purpura, arthralgias, weakness, and circulating mixed cryoglobulins) and if other well-known systemic disorders could be ruled out. Eight cryoglobulinemic patients developed B-cell non-Hodgkin lymphoma 4.3 2.7 years (range, 1.5 to 8 years) after diagnosis. After informed consent was obtained, percutaneous liver and renal biopsies were done as previously described [4, 7]. Cryocrit determinations were done and cryoglobulin composition was evaluated as previously described [4, 8]. Antinuclear, anti-smooth muscle, anti-liver-kidney microsomal 1, anti-soluble liver antigen, and antimitochondrial autoantibodies were assayed by current techniques [9]. A titer greater than 1:40 was considered positive. Anti-extractable nuclear antigen antibody determinations were done using the method of Bunn and colleagues [10]. Serum samples and aliquots of peripheral blood mononuclear cells with the last washing liquid (phosphate-buffered saline) for HCV polymerase chain reaction (PCR) analysis were collected as previously described [11, 12]. In addition, to ascertain the presence of a latent HCV infection, peripheral blood mononuclear cell samples were cultured for 72 hours in the presence of mitogens (phytohemagglutinin-phorbol myristate acetate) as previously described [11, 12]. Different samples were tested by one-tube nested reverse transcriptase PCR with primers derived from the 5 noncoding region [13]. Several precautions were taken to prevent false-positive results [14], including the incorporation of deoxyuridane-triphosphate instead of deoxythymidine-triphosphate during amplification steps followed by incubation of PCR mixtures for 3 minutes at 50 C in the presence of uracil-N-glycosilase (UNG; Perkin Elmer Cetus, Norwalk, Connecticut). In nine unselected patients with mixed cryoglobulinemia, aliquots of peripheral blood mononuclear cells were also available for HCV genotyping. Hepatitis C virus genotyping was done using two different methods, both based on amplification by PCR. The first technique used type-specific primers localized in the core region, as described by Okamoto and colleagues [15], with the difference that PCR was done without mixing genotype-specific antisense primers. Moreover, for the detection of genotype III, we used a new primer that, in a previous study, made it possible to classify most previously unclassified HCV isolates as genotype 2a/III [16]: This primer was CRIIIa antisense 5-TTCCCCAGGAYT TGCCAGTGG-3 (Y equals C or T). The second one employed biotinyled, universal primers localized in the 5 noncoding region of HCV RNA; amplification products were then hybridized to genotype-specific probes (Line Probe Assay, LiPA, Innogenetics, Brussels, Belgium). Statistical analysis was done using the chi-square test and the Fisher exact test whenever the z approximation was inadequate. Results Table 1 shows the values for the main clinicoepidemiologic and laboratory variables in patients with mixed cryoglobulinemia. The following complications of mixed cryoglobulinemia were recorded: peripheral neuropathy in 15 of 29 patients (52%); mild sicca syndrome in 11 of 28 (39%); glomerulonephritis in 4 of 29 (13%); Raynaud phenomenon in 1 of 28 (4%); and skin ulcers in 3 of 28 (11%). One or more serum autoantibodies were detected in 8 of 28 (29%) patients with mixed cryoglobulinemia and in 19 of 61 (31%) controls. Table 1. Clinico-epidemiologic Data and Laboratory Findings in 29 Hepatitis C Virus RNA-Positive Patients with Mixed Cryoglobulinemia* Hepatitis C virus RNA sequences were shown in uncultured peripheral blood mononuclear cells from 23 of 29 (75%) patients with mixed cryoglobulinemia and in cultured cells from 3 other patients (total, 90%) (Table 1). In the control group, viral sequences were detected in uncultured or mitogenstimulated peripheral blood mononuclear cells from 46 of 61 (75%) and 49 of 61 persons (total, 80%), respectively (Table 1). Among the 29 patients with mixed cryoglobulinemia, serum specimens showed a single infection with type 1a/I in 1 patient (3 %), with type 1b/II in 14 patients (48%), and with type 2a/III in 12 patients (41%). Two patients (6.6%) had mixed infection (1a/I plus 1b/II and 1b/II plus 2a/III, respectively) (Table 1). Among the 61 controls, genotypes 1a/I, 1b/II, 2a/III, 3a/V, and 4a were observed in 7 (11%), 37 (61%), 9 (15%), 4 (7%), and 1 (1%) patient, respectively, whereas mixed infection (1a/I plus 1b/II; 1b/II plus 2a/III; 1b/II plus 3a/V) was observed in 3 (5%) patients. When HCV genotypes detected in peripheral blood mononuclear cells were also considered, type 2a/III was found in 15 of the 29 (52%) patients with mixed cryoglobulinemia and in most autoantibody-positive patients (6 of 8; 75%) (Table 1). The prevalence of 2a/III genotype was significantly higher in patients with mixed cryoglobulinemia (12 of 29; 41%) than in controls (9 of 61; 15%), a difference of 27 percentage points (95% CI, 6.6% to 46.6%; P = 0.009). No other significant differences were observed between the two groups. Sixteen of the 29 patients with mixed cryoglobulinemia had chronic aminotransferase elevations. Analysis of serum samples showed that 12 of these patients (75%) were infected with HCV genotype 1b/II and that 4 (25%) were infected with HCV genotype 2a/III (Table 1). Of the remaining 13 patients who showed no clinical evidence of liver damage, 8 (61%) had infection with genotype 2a/III, 3 (23%) had infection with genotype 1b/II, 1 (7%) had infection with genotype 1a/I, and 1 had coinfection with types 1b/II and 2a/III. Liver biopsy, done in 14 patients, showed chronic hepatitis in 13 patients and liver cirrhosis in 1 patient; 2 patients with chronic hepatitis and 1 patient with cirrhosis had persistently normal aminotransferase levels (Table 1). Among the 61 controls, 27 (44%) had chronic hepatitis, 21 (34%) had liver cirrhosis, and 13 (21%) had hepatocellular carcinoma; none had normal aminotransferase values. Discussion In our study, HCV genotype 2a/III had a significantly higher prevalence in HCV-positive patients with mixed cryoglobulinemia than in patients with chronic hepatitis who did not have cryoglobulinemia. Among cryoglobulinemic patients, this genotype was more frequent in those without a symptomatic liver disease or with circulating autoantibodies. Recently, several reports have suggested different clinical outcomes for the HCV genotypes. Type 1b/II infection, for example, has been associated with a more severe liver disease and a lower response to interferon treatment, whereas type 2a/III infection has been considered relatively benign [17-19]. This hypothesis is consistent with the observation that genotype 2a/III is more prevalent in cryoglobulinemic patients without symptomatic liver disease than in those with chronic hepatitis. On the other hand, the higher prevalence of genotype 2a/III in patients with mixed cryoglobulinemia than in controls, especially in cryoglobulinemic patients with circulating autoantibodies, suggests that type 2a/III might be involved in the pathogenesis of autoimmune-lymphoproliferative disorders. The recent observation that type 2a/III is particularly frequent in Italian patients with anti-liver-kidney microsomal 1 autoantibody-positive type 2 autoimmune hepatitis further supports the possibility of a peculiar pathogenetic role for this genotype [20]. A recent study [8] showed that patients with mixed cryoglobulinemia have a high prevalence (81%) of HCV infection in peripheral blood mononuclear cells, suggesting that HCV lymphotropism may play a key role in determining the lymphoproliferative disorder underlying the disease. Our study confirms these data and also shows the frequent infection of lymphatic cells in HCV-positive patients with chronic hepatitis who do not have cryoglobulinemia. We can thus hypothesize that different viral, genetic, or environmental factors, in addition to the infection of lymphatic cells, may be involved in the pathogenesis of this disorder. The exact role of HCV variants, namely 2a/III, which are possibly related to different host immune reactivity or to a greater lymphotropism, should be clarified through deeper virologic analysis, including examination of lymph-node and bone

Albumin improves the response to diuretics in patients with cirrhosis and ascites: results of a randomized, controlled trial.

BACKGROUND/AIMS Diuretic treatment of ascites could result in intravascular volume depletion, electrolyte imbalance and renal impairment. We investigated whether intravascular volume expansion with albumin exert beneficial effects in cirrhosis with ascites. METHODS In protocol 1, 126 cirrhotic inpatients in whom ascites was not relieved following bed rest and a low-sodium diet, were randomly assigned to receive diuretics (group A) or diuretics plus albumin, 12.5 g/day (group B). In protocol 2, group A patients continued to receive diuretics and group B diuretics plus albumin (25 g/week) as outpatients and were followed up for 3 years. End points were: disappearance of ascites, duration of hospital stay (protocol 1), recurrence of ascites, hospital readmission and survival (protocol 2). RESULTS The cumulative rate of response to diuretic treatment of ascites was higher (p < 0.05) and hospital stay was shorter (20 +/- 1 versus 24 +/- 2 days, p < 0.05) in group B than in group A patients. After discharge, group B patients had a lower cumulative probability of developing ascites (19%, 56%, 69% versus 30%, 79% and 82% at 12, 24 and 36 months, p < 0.02) and a lower probability of readmission to the hospital (15%, 56%, 69% versus 27%, 74% and 79%, respectively, p < 0.02). Survival was similar in the two groups. CONCLUSIONS Albumin is effective in improving the rate of response and preventing recurrence of ascites in cirrhotic patients with ascites receiving diuretics. However, the cost/benefit ratio was favorable to albumin in protocol 1 but not in protocol 2.

Evidence for a Storage Pool Defect in Platelets From Cirrhotic Patients With Defective Aggregation

The mechanisms underlying the defective platelet function in cirrhotic patients were investigated. Eleven cirrhotic patients with mild disease (group 1), 20 patients with severe cirrhosis (group 2), and 31 controls were studied. Platelet aggregation was significantly reduced in cirrhotics compared with controls. Compared with controls, cirrhotic patients in group 2 showed a significant reduction in the total content of adenosine triphosphate (57.8 +/- 7.8 vs. 26.1 +/- 6.3 mumol/10(11) platelets; P less than 0.05), 5-hydroxytryptamine (285 +/- 26 vs. 104 +/- 38 nmol/10(11) platelets; P less than 0.05), beta-thromboglobulin (2129 +/- 120 vs. 1223 +/- 161 ng/10(8) platelets; P less than 0.01), and platelet factor 4 (1389 +/- 108 vs. 805 +/- 176 ng/10(8) platelets; P less than 0.05). In patients with severe disease, an increase in plasma beta-thromboglobulin-platelet factor 4 ratio, an index of in vivo platelet activation, was observed (controls, 3.50 +/- 0.50; group 1, 4.02 +/- 0.80; and group 2, 6.59 +/- 1.15). Our data indicate the existence of a platelet storage pool defect, which may favor the bleeding tendency of cirrhotic patients.

Altered renal and platelet arachidonic acid metabolism in cirrhosis

Urinary excretion rates of prostaglandin (PG) E2, PGF2 alpha, 6-keto-PGF1 alpha, and thromboxane (TX) B2 were evaluated in three groups of cirrhotic patients [without ascites (group 1, 13 cases), with ascites and normal renal function (group 2, 15 cases), and with ascites and renal failure (group 3, 5 cases)] and in 14 healthy controls. All urinary arachidonate metabolites were significantly increased in group 2 patients. Patients with renal failure showed lower PGE2, PGF2 alpha, and TXB2 values than those from group 2; PGF2 alpha values were also lower than controls. Platelet TXA2 production during whole blood clotting was significantly reduced in all groups of patients. Administration of low-dose aspirin and sulindac, two cyclooxygenase inhibitors selectively sparing renal cyclooxygenase activity, effectively inhibited platelet TXA2 production without affecting urinary TXB2 excretion, thus ruling out platelets as a possible source of urinary TXB2. We conclude that patients with ascites and normal renal function show an overall activation of the renal PG system. Renal production of vasodilating PGE2 and PGI2 may be involved in supporting renal function in these patients. A reduced platelet synthesis of proaggregatory TXA2 also occurs in cirrhotic patients. This may play a role in the bleeding tendency of cirrhosis.

Up-regulated expression of fractalkine and its receptor CX3CR1 during liver injury in humans

BACKGROUND/AIMS Little is known about the role of fractalkine (CX3CL1) in the liver. The aim of this study was to investigate the expression patterns of fractalkine and its receptor CX3CR1 in normal human liver and in conditions of injury. METHODS Distribution and expression of fractalkine and its receptor were investigated using immunohistochemistry, in situ hybridization, flow cytometry and reverse transcriptase-polymerase chain reaction. In vitro experiments were conducted in HepG2 cells. RESULTS Both fractalkine and CX3CR1 were up-regulated during chronic injury, in areas of portal and lobular inflammation. In severe acute hepatitis, fractalkine and CX3CR1 were expressed at high levels not only in areas of inflammation but also in regenerating epithelial cells within bile duct-like structures, which showed co-expression of fractalkine and cytokeratin-7 or CX3CR1. The human hepatocarcinoma cell line HepG2 expressed fractalkine at the gene and protein level, and HepG2-conditioned medium was chemotactic for cells overexpressing CX3CR1. Transcripts for CX3CR1 were detected in HepG2, and exposure of these cells to recombinant fractalkine induced cell migration. CONCLUSIONS This study shows that the fractalkine system is up-regulated during liver damage, and suggests that fractalkine may play a role in the recruitment and adhesion of inflammatory cells and in the biology of liver epithelial cells.