de Oca Arjona, Montserrat Montes MD*; Marquez, Mercedes PhD*; Soto, Maria Jose MD, PhD*; Rodriguez-Ramos, Claudio MD, PhD†; Terron, Alberto MD‡; Vergara, Antonio MD§; Arizcorreta, Ana MD, PhD*; Fernandez-Gutierrez, Clotilde MD, PhD‖; Giron-González, Jose Antonio MD, PhD*
Hepatitis C virus (HCV)-related liver disease follows an accelerated course in patients with HIV coinfection.1-6
The intestinal mucosal tissue is an early target organ of HIV.7,8 The disruption of gut epithelial integrity and the subsequent microbial translocation has been linked to systemic immune activation. It has been suggested that the activation of the immune system can play a fundamental role in the accelerated course of liver damage in HCV-HIV patients.9 Once in the circulation, endotoxin promotes the hepatic synthesis of lipopolysaccharide-binding protein (LBP), a plasma protein that enhances the binding of lipopolysaccharide (LPS) to the CD14 monocyte receptor molecule associated with a Toll-like receptor. Endotoxin signalling triggers a cascade that leads to production of proinflammatory cytokines, such as tumour necrosis factor (TNF)-a and interleukin-6.10 Both have been implicated in the increased peripheral vasodilatation observed in advanced phases of liver cirrhosis.11-13
Although it is possible to analyze the intestinal permeability by measuring the serum concentration of LPS, determination of LBP, the serum levels of which are positively correlated with those of LPS, is more reliable because the half-life of LBP is longer. It has been used previously for this reason.10
An increased permeability of the intestinal barrier has been also detected in non-HIV-infected patients with cirrhosis and ascites.14 Furthermore, a significant association has been established between markers of intestinal permeability, such as serum LBP concentration, hemodynamic derangement, incidence of bacterial infections, and death, in non-HIV-infected patients.15,16
The combined effect of increased intestinal permeability in HIV-infected patients with decompensated HCV-related liver disease has not been studied until now. The objective of this work is to analyze the influence of portal hypertension on intestinal permeability in HIV-infected patients with HCV-related cirrhosis, and the prognostic significance of consequent macrophage activation. Therefore, in these patients, we have studied the serum concentration of LBP, the consequences of macrophage activation (soluble CD14 receptor and pro-inflammatory cytokines), the association of these markers with the consequences of peripheral vasodilation (plasma renin activity and aldosterone concentration), and their implication in prognosis.
MATERIALS AND METHODS
Design, Study Population, and Follow-Up
From January 2004 to June 2009, a cohort of 336 HIV/HCV-coinfected patients was prospectively followed up at the 3 participating hospitals. Highly active antiretroviral therapy (HAART) was indicated according to the recommendations of international guidelines. From this cohort, 2 different groups of patients were selected as follows: (1) Patients with HIV infection and HCV-related cirrhosis, without previous or actual decompensation; and (2) Patients with HIV infection and HCV-related cirrhosis, with previous decompensation. These 2 groups of patients, and 2 control groups (HIV-monoinfected individuals and healthy controls), constitute the studied population.
A case-control study was performed to analyze the possible influences of HIV infection and portal hypertension on intestinal permeability. With this objective, patients and controls were classified in the following groups: (1) HIV-infected patients (n = 20), without evidence of liver disease (HbsAg negative, absence of anti-HCV antibodies, normal serum transaminase concentrations); (2) Patients with HIV infection and HCV-related cirrhosis, without previous or actual decompensation (n = 20); (3) Patients with HIV infection and HCV-related cirrhosis, with previous decompensation (n = 50); (4) Healthy controls (n = 20), selected from hospital workers.
Second, a prospective study was performed to analyze the possible influence of intestinal permeability, or the consequent macrophage activation, in those HIV/HCV-coinfected patients with decompensated cirrhosis (n = 50). These patients are those previously analyzed in the case-control study. Criteria for inclusion were as follows: (1) age between 18 and 80 years; (2) histological or clinical diagnosis of cirrhosis; (3) previous or actual cirrhosis decompensation characterized by ascites, gastrointestinal bleeding due to portal hypertension, portal systemic encephalopathy, nonobstructive jaundice or spontaneous bacterial peritonitis; (4) positive serum anti-HIV and anti-HCV antibodies.
Exclusion criteria were as follows: (1) neoplasias, including liver cancer at entry; (2) clinical, analytical, or histological evidence of active infection by hepatitis B virus (HbsAg negative), alcoholic hepatitis, metabolic or autoimmune liver disease; (3) treatments which could have modified the determination of cytokines (pentoxyfilline, steroidal, or nonsteroidal anti-inflammatory or immunosuppressive drugs); (4) red blood cell or plasma transfusion in the month before inclusion in the study. These were excluded to rule out the possible interference of contamination or reactions between these elements and the inflammatory system. Active infection, including spontaneous bacterial peritonitis, gastrointestinal bleeding, and shock were treated first, and those patients were included only after clinical and hemodynamic stabilization.
Patients were evaluated every 3 months, including assessment of the following: (1) clinical signs and symptoms of HIV disease and opportunistic entities and liver disease; (2) CD4+ cell count and serum HIV load; (3) LBP serum levels; (4) serum concentrations of inflammation-related molecules, namely soluble CD14 (sCD14), soluble TNF-receptor 55 Kda (sTNFR), and interleukin 6 (IL-6); (5) evaluation of the renin-angiotensin-aldosterone axis [plasma renin activity (PRA) and plasma aldosterone concentration (PAC)]. Baseline characteristics are those obtained in the evaluation immediately before decompensation.
The guidelines of the Spanish Association for the Study of the Liver (www.aeeh.org) were used for liver disease management. HAART was continued during and after decompensations, if any.
Diagnostic Criteria and Event Assessment
In patients who acquired HCV through the sharing of needles or via blood transfusion, the duration of HCV infection was estimated as the time elapsed from either the date of the first blood transfusion or the first year of needle-sharing to the date of the inclusion.17
Diagnosis of cirrhosis was established according histological criteria when liver biopsy was performed18 or by the combination of clinical, biochemical, and ultrasound imaging data agreeing with such a diagnosis.19 Patients were grouped according Child-Pugh classification.20 The criteria for the diagnosis of causes of decompensation are those previously established.3
Life Status and Death Determinations
Patients were prospectively monitored until death, liver transplantation, or the end of the observation period (June 30, 2009). Death was classified as liver related, if it was a consequence of portal hypertensive gastrointestinal bleeding, spontaneous bacterial peritonitis, hepatorenal syndrome, portosystemic encephalopathy, hepatocellular carcinoma, or whenever liver failure had a key role in death. AIDS was considered the cause of death when there was a life-threatening disease defining the clinical stage C of the 1993 Centers for Disease Control and Prevention classification. Otherwise, the cause of death was categorized as “other.” Patients who died of conditions not related to liver disease and patients who were lost to follow-up were censored at the time of death or at the time of dropout, respectively. For patients who underwent liver transplantation,21 the date of censoring was that of the surgical procedure.
Plasma HIV RNA load was quantified by polymerase chain reaction assay (Amplicor HIV Monitor test; Hoffman-La Roche, Basel, Switzerland). Plasma HCV-RNA was detected by quantitative polymerase chain reaction (Amplicor HCV Monitor test; Hoffman-La Roche). HCV genotype was identified by line-probe assay (INNOLiPA HCV; Innogenetics, Ghent, Belgium). CD4 cell counts were measured by standard flow cytometry.
Determination of LBP is more reliable than LPS because its half-life is longer. It has been used previously for this reason.10,15,16 Likewise, the sTNFRI, 55 kDa, whose half-life is longer than that of TNF-a, was analyzed; sTNFRI is shed from the cell surface of polymorphonuclear cells and monocytes in response to the same inflammatory agents that are known to induce TNF-a; its concentration is correlated with TNF-a.22
Plasma LBP was measured by immunometric sandwich assay (Immulite LBP; DPC, Los Angeles, CA). Serum levels of sCD14, sTNFRI, and IL-6 were assayed by enzyme-linked immunosorbent assay kits (R &D Systems, Minneapolis, MN). The lower limits of sensitivity (lowest positive standard) were: LBP, 0.2 mg/mL; sCD14, 0.4 ng/mL; TNF-alfa, 0.4 ng/mL; and IL-6, 6 pg/mL.
Hemodynamic modifications were analyzed in function of median arterial pressure and renin-vasopressin-aldosterone system activation.23 PRA was determined by radioimmunoassay (Clinical Assays, Baxter, Cambridge, MA) of angiotensin I generated after 30 minutes incubation at pH 7.4 and 37°C under conditions to inhibit further conversion of angiotensin I (reference range, 0.4-2.3 ng·mL−1·h−1 [308.8-1775.6 pmol·L−1·h−1]). PAC was measured by radioimmunoassay [ALDOCTK-2-P2714, Sorin Biomedica Diagnostics, Barcelona, Spain; reference range, 3.5-15 ng/dL (0.08-0.42 nmol/L)]. Methods used for these investigations have been described in detail elsewhere.23
Continuous variables are expressed as median (interquartile range) and categorical variables as number (percentage). Comparisons between categorical variables were made by the χ2 test or Fisher test. For continuous variables, analysis of variance was used; when the homogeneity conditions of variance were not fulfilled, Kruskal-Wallis and the Mann-Whitney U tests were used. The Pearson correlation coefficient analyzed the association among quantitative variables.
Survival times were calculated from the date of inclusion to the date of death or when the last information of life status was obtained, and it is expressed as median. Survival estimates at different time points are expressed as the cumulative proportion of survivors at the end of the period. Cox regression analysis was used to identify factors independently associated with liver-related mortality. The variables were entered into a Cox regression model if a difference with a P < 0.1 in the univariate analysis was found. The respective hazard ratios with 95% confidence intervals were obtained.
Continuous variables independently associated with survival were transformed into categorical variables using the value of the median, and survival was analysed by the Kaplan-Meier method. Comparisons between groups were made by the log-rank test. The statistical analysis was carried out using the SPSS 15.0 statistical software package (SPSS Inc, Chicago, IL).
This study was performed according to the Helsinki Declaration. The ethics committee of each participant hospital approved the protocol, and all patients gave their informed consent.
Baseline Characteristics of the Study Population
The characteristics of the patients and healthy controls included in the study are summarized in Table 1. Seventy HIV-infected patients of the different groups were receiving HAART. HAART regimes were based on 2 nucleosides and either protease inhibitors, efavirenz or another nucleoside, without significant differences between HIV-monoinfected and HIV-HCV-coinfected patients, either with or without decompensated cirrhosis.
In the group of HIV-HCV-coinfected patients with decompensated cirrhosis, ascites was the most common liver decompensation [31 (62%) patients] and was complicated with spontaneous bacterial peritonitis in only 1 case. Seven individuals (14%) showed portosystemic encephalopathy as the first hepatic decompensation; 6 patients (12%) presented portal hypertensive gastrointestinal bleeding and 6 (12%) patients nonobstructive jaundice.
Microbial Translocation: Serum Levels of LBP
Serum concentrations of LBP were significantly elevated in HIV-monoinfected patients when compared with healthy controls, and even higher in those with decompensated cirrhosis. However, LBP concentration was similar in HIV-monoinfected patients and in those with compensated cirrhosis (Table 2).
Patients were categorized in function of the serum levels of LBP, the cut-off value being the highest serum level of LBP observed in healthy controls (8.1 mg/mL) (this value also represents the mean LBP concentration plus 3 standard deviations, in healthy controls). Percentages of patients showing LBP concentration higher than the established cut-off value were as follows: 15% (n = 3) of HIV-monoinfected patients; 20% (n = 4) of HIV/HCV patients coinfected with compensated cirrhosis; and 86% (n = 43) of patients with decompensated cirrhosis.
When HIV-infected patients of the different groups were categorized in function of LBP level, it was evident that significantly increased LBP concentrations were detected in those with a CD4+ T-cell count lower than 200 cells per milliliter at HIV diagnosis and in those with currently detectable HIV RNA load. Moreover, when only those patients with liver cirrhosis were considered, increased LBP levels were observed in those with a Child-Pugh score higher than 9 (Child-Pugh stage C) (Table 3).
A pattern similar to that found with LBP concentration was observed when serum levels of sCD14, sTNFRI, and IL-6 were analyzed (Table 2).
When the overall HIV-infected patients were considered, the concentrations of sCD14, sTNFRI, and IL-6 were significantly higher in those with increased LBP (>8.1 mg/mL) than in those with normal LBP [sCD14, 9411 (5360-10714) vs. 5582 (3660-9739) ng/mL, P = 0.004; sTNFRI, 527 (282-1121) vs. 261 (201-354) pg/mL, P < 0.001; IL6, 18 (9-41) vs. 7 (3-15) pg/mL, P = 0.003]. Moreover, significant correlations were observed between LBP concentration and that of sCD14 (r = 0.260, P = 0.027), sTNFRI (r = 0.481, P < 0.001), and IL-6 (r = 0.443, P < 0.001).
Measurement of mean blood pressure and analysis of the renin-angiotensin-aldosterone axis was performed in cirrhotic patients. Whereas mean blood pressure was similar in compensated and decompensated cirrhotic patients [81 (73-92) versus 84 (69-97) mmHg, P = 0.855], both PRA and PAC were significantly higher in patients with decompensated compared with those with compensated cirrhosis [PRA, 1.8 (0.3-6.6) vs. 0.8 (0.1-2.1) ng/ml.h, P < 0.001; PAC, 28.9 (13.6-70.3) vs. 15.0 (10.3-26.9) ng/dL, P < 0.001].
A correlation analysis was performed to analyze the possible association between proinflammatory cytokines and hemodynamic modifications. Concentrations of sTNFRI and of IL-6, but not those of LBP and sCD14, were significantly correlated with PRA (sTNFRI, r = 0.307, P = 0.010; IL-6, r = 0.414, P = 0.005) and PAC (sTNFRI, r = 0.392, P = 0.012; IL-6, r = 0.382, P = 0.010). Likewise, Child-Pugh score was positively and significantly correlated with concentrations of both hormones (PRA, r = 302, P = 0.041; PAC, r = 0.496, P = 0.005).
Because the detectability of HIV load (either by resistance to prescribed antiretrovirals or because the patient has not complied) influences the serum level of LBP, macrophage-derived molecules and hemodynamic parameters were analyzed in patients, distributed in function of the presence or absence of undetectability (independently of the presence or absence of previous decompensations) (see Table, Supplemental Digital Content 1, http://links.lww.com/QAI/A137). Significantly lower levels of macrophage-derived molecules and PRA and PAC values were detected in patients with undetectable HIV load.
Follow-Up and Analysis of Mortality
Having observed the existence of a more profound inflammatory state in those HIV-HCV patients with decompensated cirrhosis, the possible influence of proinflammatory cytokines on the mortality of these patients was studied.
HIV-HCV-coinfected patients with decompensated cirrhosis were followed-up for a median period of 429 (126-1075) days. HAART was indicated for all patients, although a maintained compliance was obtained in only 90% (n = 45) of them. Twenty-four patients (48%) died during the follow-up period. In 22 (44%) individuals, the cause of death was liver-related. Hepatic encephalopathy was the leading cause of mortality, accounting for 11 deaths (22%). Other causes of death were portal hypertensive gastrointestinal bleeding, 4 cases (8%); hepatorenal failure, 4 cases (8%); spontaneous bacterial peritonitis, 2 cases (4%); hepatocellular carcinoma, 1 case (1.9%). Sepsis by infections not related with liver cirrhosis was the cause of death of 2 patients (4%). In addition, 2 patients (4%) received a liver transplant. There were 2 patients lost in the follow-up and they were considered as dead.
The cumulative probability of survival at 1 and 2 years was 64% and 44%, respectively. The differential characteristics between patients who died and those who survived are presented in Table 4. In the multivariate analysis, the factors that predicted survival were Child-Pugh index, a CD4 T-cell count lower than 200 cells per cubic millimeter, plasma aldosterone, and serum IL-6 concentrations (Table 4, Fig. 1).
The present work has analyzed the intestinal permeability in HIV-infected patients with and without HCV-related liver cirrhosis. In agreement with previous studies,8,24 HIV-infected patients had greater intestinal permeability compared with healthy controls. Furthermore, our results support the existence of an evenly increased alteration in the intestinal barrier in those HIV-infected patients in whom decompensated liver cirrhosis is present.
We observed that LBP concentration, as a marker of intestinal permeability, was associated with a decreased CD4+ T-cell count at HIV diagnosis and with currently detectable HIV RNA. Early HIV infection is consistently associated with a rapid, dramatic, and largely irreversible depletion of mucosal CD4 T cells, followed by activation-induced cell death responsible for the subsequent depletion of peripheral blood CD4+ T cells.25 Thus, it not was surprising that LBP concentrations were higher in those with a lower CD4+ T cell at diagnosis of HIV infection, as this reflected a more prolonged course of HIV infection and higher intestinal lymphocyte depletion.
Specifically, the alteration in the intestinal barrier is not secondary to the absence of HAART therapy. Patients with liver cirrhosis had received this therapy less frequently, and consequently, the percentage of patients with undetectable HIV load was lower in this group, according to previous data.26,27 However, although the proportion of patients with compensated cirrhosis receiving HAART was similar to that of those with decompensated liver disease, only in the former group were LBP levels significantly elevated compared with HIV-monoinfected patients. The absence of HIV replication control, either by nonadherence or by resistance to antiretrovirals, was the parameter clearly associated with increased LBP levels, and not the absence or presence of HAART.
We also noted a significant association between LBP concentration and the Child-Pugh score, an index which includes parameters indicative of liver function (serum concentrations of albumin and bilirrubin, prothrombin activity, encephalopathy), and of portal hypertension (ascites). Although it is generally accepted that the presence of decompensations of cirrhosis is associated with a significant increase in portal pressure,28 no direct measurement of portal pressure, such as the hepatic venous pressure gradient, was performed in the present study. Thus, it can be stated that the increased LBP levels detected in HIV-infected patients with HCV-related decompensated liver cirrhosis can be attributed either to the influence of portal hypertension or/and liver insufficiency.
Microbial translocation has been correlated with markers of systemic immune activation. Endotoxin signalling triggers a cascade that leads to production of proinflammatory cytokines, such as TNF-a and IL 6, and to systemic immune activation.9,10 We found that the serum levels of these cytokines were elevated in HIV-infected patients, with a pattern similar to that observed with LBP as follows: both sTNFRI and IL-6 concentrations were higher in HIV-infected patients when compared with healthy controls and even higher in those patients with decompensated cirrhosis. Moreover, a significant correlation was detected between serum levels of LBP and proinflammatory cytokines.
After proving the existence of a significant inflammatory state in HIV-HCV patients with decompensated cirrhosis, we analyzed the possible influence of proinflammatory cytokines on the mortality of these patients. In the present study, survival was influenced by liver function indices (Child-Pugh score) and by the immunosuppression level, findings previously detected in other series,3,4,6 and by haemodynamic markers and macrophage-derived parameters. The absence of evidence of prognostic capability for LBP levels may be explained because this molecule is not directly involved in changes in liver pathology or in hemodynamic alterations; rather, its effects are mediated by secretion of proinflammatory cytokines.
In advanced phases of the natural history of liver cirrhosis, peripheral vasodilatation with decreased circulatory volume and activation of vasoactive components (renin-angiotensin-aldosterone, norepinephrine, and vasopressin) has been detected.23,29-31 The independent influence of plasma aldosterone concentration on survival found here, indicating the presence of a more profound hemodynamic alteration, supports the view that effective hypovolemia is also important in HCV-related decompensated cirrhosis in HIV-coinfected patients.
Moreover, IL-6 levels have an independent prognostic capability in the multivariate analysis. TNF-a and IL-6, possibly acting through nitric oxide-independent mechanisms, play a role in the hemodynamic alterations and mortality in cirrhosis.11,13,32-34 In fact, our study revealed that serum levels of IL-6 correlate with measures of vasoactive response (plasma renin activity and aldosterone concentration) to vasodilation. In line with this, some studies have reported the role of proinflammatory cytokines in the pathogenesis of hepatic encephalopathy, the main cause of death in HIV/HCV-coinfected patients with liver cirrhosis.35,36
In conclusion, increased intestinal permeability, as measured by serum LBP levels, observed in patients with HIV infection, is significantly higher in patients with decompensated liver cirrhosis. We can state that, in addition to previously described markers (immunodepression, liver function scores), the consequences of hemodynamic alterations (plasma aldosterone concentration) and monocyte-derived inflammatory molecules are prognostic markers in HIV-infected patients with decompensated HCV-related liver cirrhosis.
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