Giorgio La Villa

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

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.

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.

High Pretransplant Serum Levels of CXCL10/IP-10 Are Related to Increased Risk of Renal Allograft Failure

In experimental models, the chemokine CXCL10/IP‐10 is required for graft failure owing to both acute and chronic rejection. In the present study, pretransplantation sera from 316 cadaver kidney graft recipients were tested for serum CXCL10 and CCL22/MDC levels by an ELISA assay. Kidney graft recipients with normally functioning grafts showed significantly lower serum CXCL10 levels than patients who experienced graft failure, whereas no differences for serum CCL22 levels were observed. After the assignment of all patients to four groups according to serum CXCL10 levels, the death‐censored survival rates of grafts were 97.5%, 93.6%, 89.7%, 78.7% (p = 0.0006) at 5 years, while no influence was observed on patient survival. Accordingly, patients with the highest CXCL10 levels showed an increased frequency and severity of rejection episodes. Serum C‐reactive protein (CRP) level was also assayed in the same samples. Increase of serum CRP levels represented a predictive parameter for death, but not for graft failure. Multivariate analysis demonstrated that among the analyzed variables, CXCL10 had the highest predictive power of graft loss (RR 2.787). Thus, measurement of pretransplant serum CXCL10 levels might represent a clinically useful parameter to identify subjects who are at high risk of severe rejection and graft failure.

Effect of V1-vasopressin receptor blockade on arterial pressure in conscious rats with cirrhosis and ascites

Angiotensin II blockade with saralasin in human cirrhosis with ascites is associated with a significant reduction in arterial pressure, indicating that endogenous angiotensin II plays an important role in the maintenance of systemic hemodynamics in this condition. The aim of the current study was to investigate whether vasopressin also contributes to the maintenance of arterial pressure in cirrhosis with ascites. The study was performed using three groups of cirrhotic rats with ascites and three groups of control animals. The administration of d(CH2)5Tyr(Me)AVP, a selective antagonist of the vascular effect of vasopressin, to 10 cirrhotic rats induced a significant reduction in mean arterial pressure (from 94 +/- 4 to 85 +/- 4 mm Hg; P less than 0.001) and a significant increase in plasma renin activity (from 24.3 +/- 4.9 to 34.3 +/- 5.9 ng/mL.h; P less than 0.02) and plasma norepinephrine concentration (from 1474 +/- 133 to 2433 +/- 253 pg/mL; P less than 0.01). Similar results were observed following saralasin administration in a second group of 5 cirrhotic rats [mean arterial pressure decreased from 97 +/- 4 to 85 +/- 5 mm Hg (P less than 0.0001); and plasma renin activity and norepinephrine concentration increased from 18.4 +/- 5.8 to 40.3 +/- 5.7 ng/mL.h (P less than 0.02) and from 1383 +/- 70 to 2312 +/- 334 pg/mL (P less than 0.05), respectively]. The simultaneous blockade of angiotensin II and vasopressin in a third group of cirrhotic rats resulted in a significantly greater reduction of mean arterial pressure (from 97 +/- 6 to 74 +/- 6 mm Hg; P less than 0.05). No changes in arterial pressure were observed in the three groups of control rats. These findings indicate that endogenous vasopressin is as important as angiotensin II in the maintenance of arterial pressure in cirrhotic rats with ascites and support the contention that arterial hypotension is the initial event leading to the stimulation of the renin-angiotensin system and vasopressin in this animal model of cirrhosis.

Effects of low-dose captopril on renal hemodynamics and function in patients with cirrhosis of the liver

BACKGROUND In cirrhotic patients with ascites, captopril has deleterious effects on renal function, which have been referred to as captopril-induced arterial hypotension. The effects of this drug on renal function in cirrhosis were evaluated using low-dose captopril, thereby avoiding any change in arterial pressure. METHODS In a randomized, double-blind, placebo controlled, cross-over trial, the effects of 12.5 mg captopril on renal plasma flow, glomerular filtration rate (measured by radioisotopic techniques), and sodium excretion in healthy controls and cirrhotic patients with and without ascites were determined. RESULTS In healthy subjects, captopril only induced a significant, 18% increase in renal plasma flow. In contrast, glomerular filtration rate significantly decreased in patients with (from 108 +/- 7 to 78 +/- 9 mL/min) and without ascites (from 102 +/- 4 to 88 +/- 3 mL/min), whereas renal plasma flow did not change. Urinary sodium excretion also significantly decreased in ascitic patients (from 43.8 +/- 4.4 to 30.6 +/- 3.8 mumol/min). CONCLUSIONS These data suggest that angiotensin II contributes to maintain renal hemodynamics in cirrhosis with and without ascites.

Renal hemodynamic and natriuretic effects of human atrial natriuretic factor infusion in cirrhosis with ascites.

We investigated the effect of a continuous infusion (50 micrograms as an initial bolus followed by a maintenance infusion at a rate of 0.1 micrograms/min.kg body wt for 45 min) of synthetic human atrial natriuretic factor (hANF) on renal hemodynamics and the renin-angiotensin-aldosterone system in 15 cirrhotic patients with ascites. Basal hANF levels were higher in cirrhotic patients when compared with normal values. Human atrial natriuretic factor infusion induced a significant decrease in mean blood pressure (from 77.8 +/- 1.1 to 68.6 +/- 1.5 mmHg, p less than 0.001) and a significant increase in heart rate (from 76.4 +/- 2.7 to 89.8 +/- 2.4 beats/min, p less than 0.001) in the patients studied. A remarkable increase in natriuresis (i.e., greater than or equal to 200 muEq/min) was observed in 5 patients (responders), whereas the infusion did not modify sodium excretion (i.e., less than or equal to 20 muEq/min) in 6 patients (nonresponders) and induced an intermediate response in 4 patients. Human atrial natriuretic factor-induced natriuresis was related to changes in renal hemodynamics that occurred during hANF infusion. In responders, the extent of the natriuretic response paralleled the increase of effective renal plasma flow and glomerular filtration rate; in non-responders the absent natriuretic response was associated with an evident reduction of these parameters. The reduction of blood pressure was similar in responders and nonresponders, but in the latter group it was followed by a marked increase of plasma renin activity and heart rate. It is likely that in nonresponders the natriuretic effect of hANF was blunted by the hemodynamic and hormonal changes triggered by the concomitant hANF-induced hypotension. This probably occurs in the presence of a greater reduction of effective arterial blood volume, as suggested by the higher baseline levels of plasma renin activity and the inability to increase free water excretion after a water load observed in nonresponders.

Blockade of the hydroosmotic effect of vasopressin normalizes water excretion in cirrhotic rats.

Water retention in cirrhosis has classically been considered to be due to a low distal fluid delivery secondary to increased proximal sodium reabsorption. However, recent studies showing high plasma vasopressin levels in patients and rats with cirrhosis, ascites, and dilutional hyponatremia suggest that a nonosmotic vasopressin hypersecretion could be an alternative mechanism. To investigate the role of vasopressin in water retention in cirrhosis, the renal ability to excrete a water load (50 ml/kg body wt), as estimated by the minimum urinary osmolality and the percentage of the water load excreted during 3 h, was assessed in 10 control rats and in 20 cirrhotic rats with ascites and impaired water excretion and high urinary excretion of vasopressin. Twenty-four hours later, the same procedure was repeated in cirrhotic rats 20 min after the subcutaneous injection (30 micrograms/kg body wt) of d(CH2)5Tyr(Et) VAVP, an antagonist of the hydroosmotic effects of vasopressin (10 rats), or the vehicle (10 rats). Treatment with the vasopressin antagonist normalized water excretion in 9 of the 10 rats. No significant changes in renal water metabolism were observed in the group of rats given the vehicle. These results indicate that vasopressin hypersecretion is the predominant mechanism of the impairment in water excretion in rats with experimental cirrhosis and ascites.

Low-Dose C-Type Natriuretic Peptide Does Not Affect Cardiac and Renal Function in Humans

In experimental animals, C-type natriuretic peptide (CNP) has vasodilating, hypotensive, and natriuretic activities. The role of circulating CNP in the overall regulation of cardiac and renal function in humans is less defined, in both health and disease. We measured cardiac volumes, diastolic and systolic functions, systemic (Doppler echocardiography) and renal hemodynamics, intrarenal sodium handling (lithium clearance method), plasma and urinary cGMP, plasma renin concentration, and plasma aldosterone level in six healthy volunteers (mean age, 33+/-3 years) receiving CNP (2 and 4 pmol/kg per minute for 1 hour each) in a single-blind, placebo-controlled, random-order, crossover study. During CNP infusion, plasma CNP increased from 1.17+/-0.23 to 41.52+/-4.61 pmol/L (ie, 4- to 10-fold higher levels than those observed in disease states) without affecting plasma and urinary cGMP, cardiac volumes, dynamics of left and right heart filling, cardiac output, arterial pressure, renal hemodynamics, intrarenal sodium handling, sodium excretion, or plasma levels of renin and aldosterone. The finding that increments in plasma CNP within the pathophysiological range have no effects on systemic hemodynamics, renal function, or the renin-angiotensin system do not support the hypothesis that CNP may act as a circulating hormone in humans.

Effects of exercise on natriuretic peptides and cardiac function in man

We evaluated cardiac function and the plasma levels of atrial (ANP) and brain (BNP) natriuretic peptides during bicycle (B) and hand-grip (HG) exercises in eight healthy males. Each test was preceded by a control protocol in resting conditions. Left ventricular (LV) function (echocardiography) was evaluated during both exercises. Atrial function was assessed only during HG. Plasma ANP significantly increased during B (+236%) and HG (+77%), while there was a significant trend towards higher plasma BNP levels during B (+41%) and HG (+30%) than during the corresponding control tests. Plasma ANP correlated with heart rate in both tests, with left atrial volume, pulmonary vein flow systolic fraction and mitral flow E/A ratio in HG; BNP in both test correlated with LV dimensions and function. These data suggest that during exercise the cardiac release of ANP and BNP is differently regulated and related to changes in left atrial and LV function, respectively.