肝片形吸虫对肝纤维化的影响及治疗

作为一种可同时感染家畜和人类的寄生虫,肝片形吸虫对人类和家畜的身体机能造成了不可修复的损伤,在世界范围内造成了巨大的经济损失,尤其是感染早期对肝脏造成的肝纤维化症状不明显,不易被发现。本文主要综述了肝片形吸虫、肝纤维化、三氯苯哒唑的相互关系,并从Th1、Th2类细胞因子、T细胞、血红素氧合酶-1(heme oxygenase 1, HO-1)、巨噬细胞等几个方面探讨了肝片形吸虫对大鼠肝纤维化的影响,包括细胞间的相互作用以及它们所诱导的细胞因子(如:IL-4、IL-10、IFN-γ、TNF-α等)对肝纤维化的促进或抑制作用。最后,还综述了当前唯一可以治疗人类和家畜寄生虫感染的药物三氯苯哒唑对肝片形吸虫所致肝纤维化的治疗作用。     

Local hepatic immune response in rats during primary infection with Fasciola hepatica

The distribution of lymphocyte subpopulations (TCD4+, TCD8+, TCD43+ and Ig+ cells), macrophages and eosinophils were analysed in the inflammatory infiltrates associated with hepatic lesions and in hepatic lymph nodes (HLN) from rats experimentally infected with F. hepatica and necropsied 1, 2, 3, 4, 6 and 8 week post infection (WPI). We also investigated the fixation of immunoglobulin isotypes on migrating flukes in the liver. As early as 1WPI, portal tract areas surrounding migratory tunnels were infiltrated with immune and inflammatory cells. The dominant cells were eosinophils and to lesser extent, macrophages and lymphocytes (TCD4+, TCD8+ and B). Most of the inflammatory and immune cells reached the posterior part of flukes, whereas in front of the parasites these cells were fewer in number. Except for eosinophils, no immune cells penetrated through granuloma consisting of hepatic necrotic cells. As early as 1WPI, IgM could be detected in the liver, and to a lesser extent IgA, IgG2a and IgG2b. At 2WPI, IgE and IgG1 began being detected. IgG2c was detectable at 3WPI. In HLN, we observed numerous microscopic follicles in the cortical zone with proliferation of germinal centres and medullary cords. The protective role of infiltrating cell populations and immunoglobulin isotypes and possible mechanisms of immune evasion by the parasite are discussed.     

The behavior of juvenile Fasciola hepatica

The behavioral repertoire of the infective stage of Fasciola hepatica was qualitatively characterized. During activation, a primary activity was the emptying of the ceca by peristaltic-like contractions. Emergence behavior comprised coordinated patterns of body movement and sucker activity specifically directed at disruption of the ventral plug. The stimulus specificity of the emergence response for glycine-conjugated cholic acid and the log dose-effect relationship of this response with glycocholic acid suggested a receptor-mediated sensory recognition. Extracts from the duodenum (departure organ) and the liver (arrival site) significantly affected the rate of locomotion and the orientation of the migratory stage. The evidence for orientation in the migrating stages is unequivocal, but the mechanisms by which they orient are unclear.     

Studies on Allergen of Fasciola hepatica

Two allergenic substances (C 5 and P 4), which consisted of ribonucleic acid as their main component, were isolated from the heated extract of liver fluke (Fasciola hepatica) by means of precipitation by ammonium sulphate and phenol extraction, followed by extraction with potassium acetate and ethanol fractionation.

One of these substances, C 5, which was extracted with phenol was electrophoretically homogeneous, but ultracentrifugally was shown to contain one other substance in a small quantity.

The other substance, P 4, which was isolated from the phenol insoluble fraction was electrophoretically and ultracentrifugally homogeneous.

Both of these were almost the same in biological activity, and their solutions, which were diluted 1 : 100,000 with Ringer’s solution, were still active in the intradermal reaction to the fascioliasis of cattle. Judging from their activity and yield, it seems that all the allergenic substances which are in the heated extract of liver fluke, are represented by the protein allergen reported already, as well as by C 5 and P 4 described here.     

Studies on Allergen of Fasciola hepatica

Allergenic substance obtained from liver flukes, P4, is composed of ribonucleic acid and peptide, but it was negative in ninhydrin reaction. The preparation was dinitrophenylated without any loss of its biological activity. When the dinitrophenylated material was subjected to digestion by ribonuclease and other enzymes, adenosine-3′-phosphate combined with DNP-peptide was obtained. The DNP-peptide, after hydrolysed with dilute alkali, produced a peptide whose amino terminal was histidine.     

Peptide synthesis by recombinant Fasciola hepatica cathepsin L1

Synthesis of the tripeptide Z-Phe-Arg-SerNH2 has been accomplished by a recombinant cysteine protease, cathepsin L1 from liver fluke (Fasciola hepatica), using Z-Phe-Arg-OMe as acyl acceptor and SerNH2 as nucleophile in 0.1 M ammonium acetate pH 9.0-12.5% v/v acetonitrile at 37 degrees C. LC-MS detection indicated tripeptide formation after 10 min, continuing up to 5.5 h. The ester Z-Phe-Arg-OMe was detected throughout the experiment but the hydrolysis product Z-Phe-Arg-OH appeared early and in quite large amounts. We believe that this is the first application of a parasite protease in enzymatic peptide synthesis.     

Detection of Lymnaea columella infection by Fasciola hepatica through Multiplex-PCR

From complete mitochondrial DNA sequence of Fasciola hepatica available in Genbank, specific primers were designed for a conserved and repetitive region of this trematode. A pair of primers was used for diagnosis of infected Lymnaea columella by F. hepatica during the pre-patent period simultaneously with another pair of primers which amplified the internal transcribed spacer (ITS) region of rDNA from L. columella in a single Multiplex-PCR. The amplification generated a ladder band profile specific for F. hepatica. This profile was observed in positive molluscs at different times of infection, including adult worms from the trematode. The Multiplex-PCR technique showed to be a fast and safe tool for fascioliasis diagnosis, enabling the detection of F. hepatica miracidia in L. columella during the pre-patent period and identification of transmission areas.     

Disruption of spermatogenesis in Fasciola hepatica by some anthelmintics.

Eight-weeks old F. hepatica were recovered from rabbits and rats that had been treated with known fasciolicides and anthelmintics between 4 and 40 h previously. Examination of the surviving flukes showed that spermatogenesis was significantly disrupted by most of the fasciolicides but by none of the other anthelmintics. It is suggested that disruption of spermatogenesis is particularly associated with compounds showing activity against mature F. hepatica.     

Proteomic analysis of Fasciola hepatica excretory-secretory products

This paper describes a global investigation of the components of Fasciola hepatica excretory-secretory (ES) products by a proteomic approach. Despite the absence of a F. hepatica genome sequencing project we have shown that it was possible to identify 29 of the 60 prominent proteins found using two-dimensional gel electrophoresis. As well as cathepsin L proteases, a number of enzymes implicated in parasite protection from the host immune system were also found to be present in relatively large abundance. These included superoxide dismutase, thioredoxin peroxidase, glutathione S-transferases and fatty acid binding proteins, all of which may play a part in the detoxification of reactive oxygen intermediates. Interestingly, ovine superoxide dismutase was the only protein from the host identified on the gel. We suggest that the relative abundance and protective nature of the components of the ES products of this organism play an important role in its survival within the host. The precise identification, to individual NCBI database entries, of a number of glutathione S-transferases and cathepsin Ls from F. hepatica, by peptide mass fingerprinting, was hampered by multi-database submissions of the two protein superfamilies from this organism.     

Epidemiological observations on iridovirosis of Lymnaea truncatula, host mollusca of Fasciola hepatica]

Epidemiological studies were carried out in 11 populations of Lymnaea truncatula in 1983-1984 and 1987-1989 to determine the prevalence of snail infection by an iridovirus. The virus was found in the different populations and samples, with a frequency ranging from 1.6 to 87%. The virosis would be endemic.     

Enzyme-linked immunosorbent assay (ELISA) using recombinant Fasciola cathepsin L1 for the diagnosis of human fasciolosis caused by Fasciola hepatica/gigantica hybrid type

Recombinant Fasciola cathepsin L-1 (rCatL1) was evaluated in enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of human fasciolosis in Japan. Quality characteristics of the test were accessed by receiver operating characteristic (ROC) analysis, with sera from fasciolosis patients (n = 10), patients with no evidence of parasitic infections (n = 29), and patients with other helminth infections (n = 119). Both the sensitivity and specificity of the test achieved 100% with the control samples. To test the performance of the assay in an authentic situation, 311 serum samples, which had been sent to our laboratory for the diagnosis of parasitic infections from January 2018 to February 2019, were re-assessed using the rCatL1 ELISA. In this case, the sensitivity of the rCatL1 ELISA was 100%, giving positive results to all fasciolosis sera (n = 7), and the specificity was 99.0%, in which three of the 304 non-fasciolosis samples were judged positive. Careful re-examination of the laboratory data and medical imaging of these three patients revealed that one of the patients, who had been diagnosed as having larva migrans syndrome, was judged to be infected with Fasciola, in addition to ascarid nematodes. Thus the true specificity of the assay in the authentic reached 99.3% (302/304). As the rCatL1 ELISA exhibited a highly significant positive likelihood ratio (152.0) and negative likelihood ratio (0.0), calculated from the 311 sample data, this rCatL1 ELISA can be used for routine screening and definitive diagnosis test for fasciolosis in reference laboratories.     

Antigenicity of a proteolytic enzyme of Fasciola hepatica

The possibility was examined of using a haemoglobinase released during in vitro incubation of adult Fasciola hepatica for immunodiagnosis of fascioliasis. By SDS gel electrophoresis the enzyme appeared as two closely migrating bands with a molecular weight of approximately 27,000 daltons. After Western blotting the bands reacted with serum from rats infected with F. hepatica and mice infected with Schistosoma mansoni. The enzyme was therefore not a good antigen for immunodiagnosis.