Evaluation of the cytokine mannose-binding lectin as a mediator of periportal fi brosis progression in patients with schistosomiasis

Introduction: We hypothesized higher mannose-binding lectin level and classic factors (i.e., age, sex, alcohol consumption, exposure, and specifi c treatment) are associated with the severity of periportal fi brosis in schistosomiasis. Methods: This crosssectional study involved 79 patients infected with Schistosoma mansoni with severe or mild/moderate periportal fi brosis. Serum concentrations of mannose-binding lectin were obtained by enzyme-linked immunosorbent assay (ELISA). Results: Higher serum level of mannose-binding lectin was signifi cantly associated with advanced periportal fi brosis. Conclusions: Mannosebinding lectin may contribute to liver pathology in schistosomiasis and may represent a risk factor for advanced periportal fi brosis in the Brazilian population studied.

Schistosomiasis is a chronic parasitic infection caused by trematode Schistosoma spp.and is a serious public health problem in endemic countries, incurring substantial social and economic burdens.It affects more than 230 million people worldwide and is one of the neglected tropical diseases targeted for elimination by the World Health Organization (1) .Approximately 6 million people are infected with Schistosoma mansoni in Brazil, mainly in the northeast region.The disease causes immense morbidity, especially in the State of Pernambuco, where schistosomiasis is continuously expanding to touristic coastal areas of the state (2) .
Schistosomiasis causes periportal fi brosis (PPF) and portal hypertension in approximately 6% of infected subjects, usually with the preservation of hepatic function (3) .Some patients with schistosomiasis have a poorly regulated immune response to parasite egg antigens and consequently develop extensive hepatic PPF and subsequent hepatosplenic disease (3) .The evolution of PPF may be infl uenced by several exposure-related factors such as local environment and behaviors (e.g., alcohol consumption, exposure frequency, specifi c early treatment, age, and sex) as well as immunogenetic factors that lead to the exacerbation of the host's immune response (4) .Hepatic fibrosis depends on the actions of cytokines including mannose-binding lectin (MBL), which has a central regulatory role (5) .The association between the immune response of MBL and the development of hepatic fi brosis in other liver diseases has been evaluated (5) .The importance of MBL in the innate immune system is related to its multimeric structure as well as its functions as an opsonin and adaptor for the activation of mannose-binding lectin/mannose-binding protein-associated serine protease (MBL/MASPs).Moreover, the MBL serum level and enzyme activity of MBL/MASP-1 complex are elevated in hepatitis C virus (HCV) patients with severe hepatic fi brosis (5) .However, the contribution of MBL in patients with varying severity of S. mansoni remains poorly understood (6) .Considering the central role of MBL in the development of severe hepatic fi brosis in other liver diseases, we determined whether serum levels of MBL and classical factors (i.e., age, sex, alcohol, exposure, and treatment) are associated with PPF severity in schistosomiasis in the population of endemic areas of Northeastern Brazil.All patients infected with S. mansoni examined during the study period were included.Patients with a history of other associated liver diseases including liver cirrhosis, fatty liver disease, or hepatitis B or C, or other clinical forms of diagnosed schistosomiasis were excluded.Hepatitis B and C were excluded on the basis of hepatitis B surface antigen (HBsAg), hepatitis B core antibody (anti-HBc), and hepatitis C virus antibody (anti HCV) measurements (7) .Thus, 79 individuals were included and divided into two groups: the severe group consisted 39 patients with severe and the hepatosplenic form of the disease, and the mild group consisted of 40 patients with no or mild-to-moderate fi brosis and the hepatointestinal form of the disease.All patients had a history of contact with contaminated water and/or had positive stool test results for S. mansoni or prior treatment for schistosomiasis.
The sociodemographic and clinical variables related to the risk factors for developing PPF included sex, age, alcohol consumption, time and site of last contact with contaminated water, and specifi c treatment.Data were collected by using a precoded structured questionnaire that was applied to individuals by a single investigator.
The diagnosis of the clinical form of the disease was determined on the basis of the patient's medical history and clinical examination.In addition, upper-abdominal ultrasonography was performed by a single experienced operator using an Acuson X 150 ® (Siemens, Munich, Germany) with a 3.5-mHz convex transducer in order to confi rm the diagnosis and exclude other liver diseases.PPF pattern was evaluated according to Niamey's classifi cation (8) as follows: A, absence of fi brosis; B, dubious; C, peripheral; D, central; E, advanced; and F, very advanced fi brosis.
Serum MBL levels were measured using the commercial Human MBL Quantikine ® ELISA Kit (R&D Systems, Minneapolis, MN, USA) according to manufacturer's instructions.The results are expressed in pg/mL based on standard curves (limit of detection: 0.029ng/mL).The Cutoff of 881ng/mL was derived from the average serum levels of MBL in the severe group.
The Kruskal-Wallis test was used to compare the serum levels of MBL between groups.Variables showing p < 0.20 in the univariate analysis were entered into non-conditional logistic regression analysis.Odds ratios (ORs) and 95% confi dence intervals (CIs) were calculated using the fibrosis pattern as the dependent variable and the selected factors as independent variables.The level of signifi cance was set at p < 0.05.Epi Info version 3.5.5.(CDC, Atlanta, GA, USA) was used for all statistical analyses.
The associations of sociodemographic and clinical variables with PPF severity are shown in Table 1.The mean ± SD age of the patients was 54 ± 13 years.The patients were predominantly women.Male sex tended to be associated with severe PPF (OR = 2.85; 95% CI: 1.80-8.18;p = 0.050) with borderline signifi cance.Moreover, higher serum MBL level was signifi cantly associated with severe PPF (OR = 2.97; 95% CI: 1.08-8.29;p = 0.032).Median serum MBL level differed signifi cantly between the mild and severe PPF groups (867.62 vs. 874.04ng/mL, respectively p = 0.003).
The fi nal logistic regression model included age and alcohol consumption along with serum MBL level.Only serum MBL level was signifi cantly associated with severe PPF after adjusting for confounding factors (p = 0.012, Table 2).As this association was inverse, serum MBL level may be a protective factor against severe PPF.
The relationship between MBL and hepatic fi brosis in other hepatic diseases remains controversial (5) .The present study evaluated the infl uences of serum MBL level, age, sex, alcohol, exposure, and treatment on PPF severity in schistosomiasis.However, the classic factors studied were not associated with PPF severity.Thus, immunogenetic factors appear to be more important when predicting PPF severity.
PPF is a well-known multifactorial manifestation caused by complex interactions between genetic and environmental factors (9) .However, the classic factors should be considered when evaluating the role of MBL in PPF.
Serum MBL levels were signifi cantly higher in the severe PPF group than the mild PPF group; this result is concordant with recent data indicating increased complement activation and activity of the MBL/MASP-1 complex in HCV-induced hepatic fi brosis (5) (10) (11) , thus corroborating the proinfl ammatory role of MBL in chronic disease.
Hepatitis C virus patients have higher MBL levels than controls and are associated with more severe hepatitis (5) (10) (12) (13) .Brown et al. (5) investigated the infl uence of MBL level in patients with chronic HCV infection, non-HCV liver disease, and healthy controls from the Trent HCV cohort study in the UK (14) (15) ; they analyzed 147 infected patients and 111 healthy controls, and found MBL levels were signifi cantly higher in HCV patients with severe fi brosis than that in patients with mild fi brosis and controls.However, MBL levels did not differ signifi cantly between HCV and non-HCV liver disease patients.
Most studies that compared the distribution of MBL levels with respect to the severity of fi brosis report no signifi cant association (11) .Pedroso et al (12) compared 102 Euro-Brazilian patients with moderate and severe chronic hepatitis C with sex-and age-matched HCV-seronegative healthy controls and found overall circulating levels of MBL did not differ significantly between groups.However, it is important to note that the exclusion of patients with no or little fi brosis from that study may have weakened the association between disease progression and MBL concentration.
El Saadany et al. (13) studied 80 Egyptian patients with chronic HCV infection with different patterns of hepatic fi brosis and 20 control subjects; they found MBL levels tended to be higher in the mild liver fi brosis group than the controls, while MBL levels were signifi cantly higher in the severe fi brosis group than the mild fi brosis and control groups.Thus, the present fi nding suggesting high serum MBL levels play an important role in the severity of PPF corroborates other studies reporting similar fi ndings (5) (12) .Therefore, further larger studies analyzing the association between MBL expression and disease outcomes are required to corroborate the present fi ndings and confi rm the profi brotic activity of MBL.Furthermore, the infl uences of MBL and other classic factors on the severity of immune fi brosis in schistosomiasis warrant a large cohort study with PPF monitoring in order to confi rm this association in different ethnicities as well as clarify the roles of other environmental risk factors and immunogenetic factors in the evolution of this disease.
In conclusion, MBL may contribute to liver pathology in schistosomiasis and may be a risk factor for PPF severity in the Brazilian population.Moreover, it could be used to predict the severity of advanced PPF in schistosomiasis and thus the severity of liver pathology.

Silva PCV et al. -Schistosomiasis
-related fi brosis and MBLOutpatient Clinic of the Hospital das Clínicas/Universidade Federal de Pernambuco (CH/UFPE), a reference center for schistosomiasis treatment.All patients lived in endemic areas of schistosomiasis in the State of Pernambuco.This study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the CH/UFPE (CAAE: 03161512.6.0000.5208).Informed consent was obtained from all patients before blood sampling.

TABLE 1 -Associations of sociodemographic and clinical variables with periportal fi brosis severity in patients with schistosomiasis in Pernambuco, Brazil.
OR: odds ratio; CI: confi dence interval; MBL: mannose-binding lectin.