Genome-wide profiling of the expression of serum derived exosomal circRNAs in patients with hepatic alveolar echinococcosis.

The patients with hepatic alveolar echinococcosis is poorly detected due to invasive and slow growth. Thus, early diagnosis of hepatic alveolar echinococcosis is so important for patients. Circular RNAs are crucial types of the non-coding RNA. Recent studies have provided serum-derived exosomal circRNAs as potential biomarkers for detection of various diseases. The clinical importance of exosomal circRNAs in hepatic alveolar echinococcosis have never been explored before. Here, we investigated the serum-derived exosomal circRNAs in the diagnosis of hepatic alveolar echinococcosis. Firstly, High-throughput Sequencing was performed using 9 hepatic alveolar echinococcosis and 9 control samples to detect hepatic alveolar echinococcosis related circRNAs. Afterwards, bioinformatic analyzes were performed to identify differentially expressed circRNAs and pathway analyzes were performed. Finally, validation of the determined circRNAs was performed using RT-PCR. The sequencing data indicated that 59 differentially expressed circRNAs; 31 up-regulated and 28 down-regulated circRNA in hepatic alveolar echinococcosis patients. The top 5 up-regulated and down-regulated circRNAs were selected for validation by RT-qPCR assay. As a result of the verification, circRNAs that were significantly up- and down-regulated showed an expression profile consistent with the results obtained. Importantly, our findings suggested that identified exosomal circRNAs could be a potential biomarker for the detection of hepatic alveolar echinococcosis serum and may help to understand the pathogenesis of hepatic alveolar echinococcosis.

Biochemical Characterization of Echinococcus multilocularis Antigen B3 Reveals Insight into Adaptation and Maintenance of Parasitic Homeostasis at the Host-Parasite Interface.

Alveolar echinococcosis (AE) caused by Echinococcus multilocularis metacestode is frequently associated with deleterious zoonotic helminthiasis. The growth patterns and morphological features of AE, such as invasion of the liver parenchyme and multiplication into multivesiculated masses, are similar to those of malignant tumors. AE has been increasingly detected in several regions of Europe, North America, Central Asia, and northwestern China. An isoform of E. multilocularis antigen B3 (EmAgB3) shows a specific immunoreactivity against patient sera of active-stage AE, suggesting that EmAgB3 might play important roles during adaptation of the parasite to hosts. However, expression patterns and biochemical properties of EmAgB3 remained elusive. The protein profile and nature of component proteins of E. multilocularis hydatid fluid (EmHF) have never been addressed. In this study, we conducted proteome analysis of EmHF of AE cysts harvested from immunocompetent mice. We observed the molecular and biochemical properties of EmAgB3, including differential transcription patterns of paralogous genes, macromolecular protein status by self-assembly, distinct oligomeric states according to individual anatomical compartments of the worm, and hydrophobic ligand-binding protein activity. We also demonstrated tissue expression patterns of EmAgB3 transcript and protein. EmAgB3 might participate in immune response and recruitment of essential host lipids at the host-parasite interface. Our results might contribute to an in depth understanding of the biophysical and biological features of EmAgB3, thus providing insights into the design of novel targets to control AE.

Characterization of the inflammatory cell infiltrate and expression of costimulatory molecules in chronic echinococcus granulosus infection of the human liver.

BACKGROUND: The local immune responses to chronic echinococcal infections in various organs are largely unknown. Since the liver is the most frequently involved organ in such infections in human we aimed to characterize the inflammatory as well as immune cell infiltrate around hydatid cysts in the liver and compared to common inflammatory processes of the liver. METHOD: Surgical samples from the liver of 21 cystic echinococcosis (CE) patients were studied and the distribution of different types of inflammatory and immune cells were determined by immunohistochemistry. Furthermore, expression levels of costimulatory CTLA4, CD28, CD80 and CD86 molecules were measured at RNA level by PCR. Liver biopsy samples from patients with steatohepatitis (SH, n = 11) and chronic hepatitis (CH, n = 11) were used as non-inflammatory and chronic inflammatory controls, respectively. The composition and density of the inflammatory and immune cell infiltrates have been compared by using morphometry. RESULTS: CD3+ T cells predominated the inflammatory infiltrate in all pathological processes, while in CE samples CD20+ B cells, in CH samples CD68+ macrophages were also frequent. Both myeloperoxidase (MPO) + leukocytes and CD68+ macrophages were found to be significantly decreased in CE as compared to either SH or CH samples. Concerning T cell subtypes, only CD8+ T cells were found to be significantly decreased in SH samples. CD1a + dendritic cells were almost completely missing from CE biopsies unlike in any other sample types. There were no differences detected in the mRNA expression of costimulatory molecules except decreased expression of CD28 in CE samples. CONCLUSION: In the hydatid lesions of the liver of chronic echinococcal infections T cell-mediated immunity seems to be impaired as compared to other types of chronic inflammatory processes, suggesting an immunosuppressive role for Echinococcus granulosus, which deserve further attentions.

Molecular discrimination of Echinococcus granulosus and Echinococcus multilocularis by sequencing and a new PCR-RFLP method with the potential use for other Echinococcus species.

BACKGROUND/AIM: To develop a novel polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol using a new genomic marker sequence and a novel set of restriction enzymes in order to detect and discriminate 2 Echinococcus species, E. granulosus and E. multilocularis, found in formalin-fixed paraffin-embedded (FFPE) human tissues. MATERIALS AND METHODS: DNA was isolated from 11 FFPE human tissue samples positive for cystic echinococcosis or alveolar echinococcosis. A mitochondrial genomic marker region was amplified and sequenced using a novel primer pair and a new PCR-RFLP protocol was developed for the detection and discrimination of E. granulosus and E. multilocularis using a set of restriction enzymes including AccI, MboI, MboII, and TsoI. RESULTS: The selected marker region was amplified using DNA isolated from FFPE human tissue samples positive for cystic echinococcosis or alveolar echinococcosis and the discrimination of E. granulosus and E. multilocularis was accomplished by use of the novel PCR-RFLP method. CONCLUSION: In this PCR-RFLP protocol, use of any single restriction enzyme is enough for the discrimination of E. granulosus and E. multilocularis. The PCR-RFLP protocol can be potentially used for the discrimination of 5 other Echinococcus species: E. oligarthus, E. shiquicus, E. ortleppi, E. canadensis, and E. vogeli.

Transcriptional profiles of cytokine/chemokine factors of immune cell-homing to the parasitic lesions: a comprehensive one-year course study in the liver of E. multilocularis-infected mice.

Pathogenesis of chronically developing alveolar echinococcosis (AE) is characterized by a continuous, granulomatous, periparasitic infiltration of immune cells surrounding the metacestode of Echinococcus multilocularis (E.multilocularis) in the affected liver. A detailed cytokine and chemokine profile analysis of the periparasitic infiltrate in the liver has, however, not yet been carried out in a comprehensive way all along the whole course of infection in E. multilocularis intermediate hosts. We thus assessed the hepatic gene expression profiles of 18 selected cytokine and chemokine genes using qRT-PCR in the periparasitic immune reaction and the subsequent adjacent, not directly affected, liver tissue of mice from day 2 to day 360 post intra-hepatic injection of metacestode. DNA microarray analysis was also used to get a more complete picture of the transcriptional changes occurring in the liver surrounding the parasitic lesions. Profiles of mRNA expression levels in the hepatic parasitic lesions showed that a mixed Th1/Th2 immune response, characterized by the concomitant presence of IL-12α, IFN-γ and IL-4, was established very early in the development of E. multilocularis. Subsequently, the profile extended to a combined tolerogenic profile associating IL-5, IL-10 and TGF-ß. IL-17 was permanently expressed in the liver, mostly in the periparasitic infiltrate; this was confirmed by the increased mRNA expression of both IL-17A and IL-17F from a very early stage, with a subsequent decrease of IL-17A after this first initial rise. All measured chemokines were significantly expressed at a given stage of infection; their expression paralleled that of the corresponding Th1, Th2 or Th17 cytokines. In addition to giving a comprehensive insight in the time course of cytokines and chemokines in E. multilocularis lesion, this study contributes to identify new targets for possible immune therapy to minimize E. multilocularis-related pathology and to complement the only parasitostatic effect of benzimidazoles in AE.

TGF-ß and TGF-ß/Smad signaling in the interactions between Echinococcus multilocularis and its hosts.

Alveolar echinococcosis (AE) is characterized by the development of irreversible fibrosis and of immune tolerance towards Echinococcus multilocularis (E. multilocularis). Very little is known on the presence of transforming growth factor-ß (TGF-ß) and other components of TGF-ß/Smad pathway in the liver, and on their possible influence on fibrosis, over the various stages of infection. Using Western Blot, qRT-PCR and immunohistochemistry, we measured the levels of TGF-ß1, TGF-ß receptors, and down-stream Smads activation, as well as fibrosis marker expression in both a murine AE model from day 2 to 360 post-infection (p.i.) and in AE patients. TGF-ß1, its receptors, and down-stream Smads were markedly expressed in the periparasitic infiltrate and also in the hepatocytes, close to and distant from AE lesions. Fibrosis was significant at 180 days p.i. in the periparasitic infiltrate and was also present in the liver parenchyma, even distant from the lesions. Over the time course after infection TGF-ß1 expression was correlated with CD4/CD8 T-cell ratio long described as a hallmark of AE severity. The time course of the various actors of the TGF-ß/Smad system in the in vivo mouse model as well as down-regulation of Smad7 in liver areas close to the lesions in human cases highly suggest that TGF-ß plays an important role in AE both in immune tolerance against the parasite and in liver fibrosis.

Identification of genetic loci affecting the establishment and development of Echinococcus multilocularis larvae in mice.

Alveolar echinococcosis (AE) is a severe hepatic disorder caused by larval infection by the fox tapeworm Echinococcus multilocularis. The course of parasitic development and host reactions are known to vary significantly among host species, and even among different inbred strains of mice. As reported previously, after oral administration of parasite eggs, DBA/2 (D2) mice showed a higher rate of cyst establishment and more advanced protoscolex development in the liver than C57BL/6 (B6) mice. These findings strongly suggest that the outcome of AE is affected by host genetic factor(s). In the present study, the genetic basis of such strain-specific differences in susceptibility/resistance to AE in murine models was studied by whole-genome scanning for quantitative trait loci (QTLs) using a backcross of (B6×D2)F(1) and D2 mice with varying susceptibility to E. multilocularis infection. For cyst establishment, genome linkage analysis identified one suggestive and one significant QTL on chromosomes (Chrs.) 9 and 6, respectively, whereas for protoscolex development, two suggestive and one highly significant QTLs were detected on Chrs. 6, 17 and 1, respectively. Our QTL analyses using murine AE models revealed that multiple genetic factors regulated host susceptibility/resistance to E. multilocularis infection. Moreover, our findings show that establishment of the parasite cysts in the liver is affected by QTLs that are distinct from those associated with the subsequent protoscolex development of the parasite, indicating that different host factors are involved in the host-parasite interplay at each developmental stage of the larval parasite. Further identification of responsible genes located on the identified QTLs could lead to the development of effective disease prevention and control strategies, including an intensive screening and clinical follow-up of genetically high-risk groups for AE infection.

Primary alveolar echinococcosis: course of larval development and antibody responses in intermediate host rodents with different genetic backgrounds after oral infection with eggs of Echinococcus multilocularis.

We investigated parasite establishment, subsequent larval development and antibody responses in gerbils, cotton rats and 4 inbred mouse strains until 16 weeks post inoculation (p.i.) with 200 eggs of Echinococcus multilocularis. The rate of parasite establishment in the liver determined at 4 weeks p.i. was highest in DBA/2, followed by AKR/N, C57BL/10 and C57BL/6 mice, whereas gerbils harboured few parasite foci. The accurate number of liver lesions in cotton rats could not be determined due to rapid growth and advanced multivesiculation of the parasite observed at 2 weeks p.i. The course of larval development was most advanced in DBA/2 mice with mature protoscolex formation at 16 weeks p.i., followed by AKR/N harbouring metacestodes with sparsely distributed immature protoscoleces. On the other hand, C57BL/6 and C57BL/10 mice had infertile metacestodes without any protoscolex formation. The parasite growth in mice was totally slower than those in gerbils and cotton rats. Specific IgG and IgM responses against 3 types of native crude antigens of larval E. multilocularis were evaluated using somatic extracts of and vesicle fluid of metacestode, and somatic extracts from purified protoscoleces. The 4 mouse strains demonstrated basically similar kinetics with apparent IgG and IgM increases at 9 weeks p.i. and thereafter, except C57BL/10, exhibited higher levels of IgM against crude antigens at some time point of infection. On the other hand, a follow-up determination of specific IgG and IgM levels against recombinant antigens from larval E. multilocularis revealed that each mouse strain showed different antibody-level kinetics. The findings in the present study demonstrate that the course of host-parasite interactions in primary alveolar echinococcosis, caused by larval E. multilocularis, clearly varies among intermediate host rodents with different genetic backgrounds.

Hidatidosis hepática: revisión de tres casos / Hepatic hydatidosis: A review of three cases

La hidatidosis es una parasitosis zoonótica causada por cestodos del género Echinococcus. La única que tiene relevancia clínica en España es la E. granulosus. En la mayoría de los casos se trata de quistes hidatídicos abdominales cuya localización más frecuente es el hígado. La forma de presentación más habitual es el descubrimiento accidental. El diagnóstico se realiza fundamentalmente mediante ecografía, tomografía axial computarizada y resonancia magnética. Presentamos tres casos clínicos de hidatidosis hepática, dos de ellos diagnosticados de manera casual y uno, en una paciente con un síndrome febril. En todos los casos se realizó tratamiento quirúrgico, y únicamente en el último se realizó tratamiento médico con albendazol. La hidatidosis hepática sigue siendo un problema de salud importante en España. Los programas de control no han resultado totalmente eficaces para modificar la distribución global de la enfermedad hidatídica (AU)

Hydatidosis is a zoonotic parasitic infection caused by the cestode Echinococcus. The only clinically relevant one in Spain is E. granulosus. In most of the cases, these are abdominal hydatic cysts, whose most frequent location is in the liver. The most common presentation is discovered causally. The diagnosis is basically made by means of an ultrasound, computed tomography and magnetic resonance imaging. We present three cases of hepatic hydatidosis, two of which were diagnosed casually and one in a patient with febrile syndrome. Surgical treatment was performed in all the cases and only the latter received medical treatment with albendazole. Hepatic hydatidosis continues to be an important health problem in Spain. The control programs have not been effective to modify the global distribution of the hydatid disease (AU)

Descripción de genotipos de Echinococcus granulosus obtenidos de especímenes de hidatidosis humana / Echinococcus granulosus genotypes in samples of human hydatidosis

Introducción: Se ha descrito variabilidad ambiental de Echinococcus granulosus (Eg). Se han reportado 10 genotipos y cierta heterogenicidad intergenotipo en estudios con material proveniente de animales. El objetivo de este estudio es describir los resultados de un protocolo de genotipificación de Eg en muestras de hidatidosis humana. Material y método: Estudio de corte transversal. Se recolectó el líquido hidatídico de una muestra consecutiva de pacientes intervenidos quirúrgicamente por hidatidosis hepática y pulmonar en hospitales de Temuco entre julio de 2004 y septiembre de 2005. Se diseñó un protocolo de extracción de ADN para Eg en muestras homogeneizadas de aspirado de quistes hidatídicos. Se emplearon 2 sistemas de reacción de polimerasa en cadena (PCR): PCREg9 y PCREg16; ambos en concentraciones de MgCl2 al 2mM. Para PCREg9, se utilizaron los primers Eg9F y Eg9R a concentraciones de 0.5 mM, empleándose 35 ciclos con temperatura de hibridación a 60°C. Para PCREg16 se utilizaron los primers Eg16F y Eg16R a concentraciones de 0,5 mM, empleándose 35 ciclos con temperatura de hibridación a 65°C. Los productos de las PCR fueron digeridos con una enzima de restricción (Rsa1) para la discriminación de los genotipos. Resultados: Se analizaron 25 muestras, 4 provenientes de quistes pulmonares y 21 de quistes hepáticos. Se logró amplificación de Eg en 22 de 25 muestras (88 por ciento). La digestión enzimática reveló la presencia de 3 genotipos posibles: en 21 de 22 muestras (95,45 por ciento) se observó un patrón de restricción correspondiente a los genotipos G1 ó G7 y en la muestra restante a los genotipos G4 ó G7. Conclusión: Con los sistemas de PCR empleados se detectó ADN de Eg.

Hidatidosis hepatopulmonar en una preescolar, caso clínico: case report / Hepatopulmonary hydatidosis in a preschool female: case report

Se denomina Hidatidosis a la zoonosis parasitaria que causa la infección de herbívoros o del hombre con el estado larval (hidátide) de parásitos del género Echinococcus. Objetivo: Revisión del tema y presentación del primer caso en Chile estudiado mediante técnicas de biología molecular. Caso Clínico: Preescolar de 3 años 9 meses procedente de Punta Arenas portadora de una hidatidosis múltiple de 6 quistes (4 pulmonares y 2 hepáticos), trasladada a la V Región para su tratamiento. Se realizó 3 cirugías y tratamiento médico asociado (Albendazol en dosis de 15 mg/kg/día vía oral) durante 73 días. Las hidátides extraídas fueron medidas, se efectuó estudio de fertilidad y vitalidad e identificación de cepa de Echinococcus granulosus mediante técnicas de biología molecular. La paciente mostró notable mejoría clínica e imagenológica, y mantuvo serología positiva. El tratamiento fue bien tolerado no presentando reacciones adversas. El tamaño de las hidátides fluctuó entre 6 y 11 cm de diámetro, todas fueron fértiles y vitales en diferente porcentaje y en todas se identificó cepa oveja de E. granulosus. De regreso en Punta Arenas, a los 4 meses se le detecta una nueva hidátide hepática que fue extirpada. El complejo manejo de este caso resultó exitoso.

EmsB, a tandem repeated multi-loci microsatellite, new tool to investigate the genetic diversity of Echinococcus multilocularis.

In order to explore the genetic diversity within Echinococcus multilocularis (E. multilocularis), the cestode responsible for the alveolar echinococcosis (AE) in humans, a microsatellite, composed of (CA) and (GA) repeats and designated EmsB, was isolated and characterized in view of its nature and potential field application. PCR-amplification with specific primers exhibited a high degree of size polymorphism between E. multilocularis and Echinococcus granulosus sheep (G1) and camel (G6) strains. Fluorescent-PCR was subsequently performed on a panel of E. multilocularis isolates to assess intra-species polymorphism level. EmsB provided a multi-peak profile, characterized by tandemly repeated microsatellite sequences in the E. multilocularis genome. This "repetition of repeats" feature provided to EmsB a high discriminatory power in that eight clusters, supported by bootstrap p-values larger than 95%, could be defined among the tested E. multilocularis samples. We were able to differentiate not only the Alaskan from the European samples, but also to detect different European isolate clusters. In total, 25 genotypes were defined within 37 E. multilocularis samples. Despite its complexity, this tandem repeated multi-loci microsatellite possesses the three important features for a molecular marker, i.e. sensitivity, repetitiveness and discriminatory power. It will permit assessing the genetic polymorphism of E. multilocularis and to investigate its spatial distribution in detail.

Survival strategy of Echinococcus multilocularis in the human host.

As exemplified by "aborted" calcified liver lesions commonly found in patients from endemic areas, Echinococcus multilocularis metacestodes develop only in a minority of individuals exposed to infection with the papasite. Clinical research has disclosed some aspects of the survival strategy of E. multilocularis in human hosts. Clinical observations in liver transplantation and AIDS suggest that suppression of cellular/Th1-related immunity increases disease severity. Most of the studies have stressed a role for CD8+ T cells and for Interleukin-10 in the development of tolerance. A spontaneous secretion of IL-10 by the PBMC seems to be the immunological hallmark of patients with progressive forms of alveolar echinococcosis (AE). IL-10-induced inhibition of effector macrophages, but also of antigen-presenting dendritic cells, may be operating and allowing parasite growth and survival. The genetic correlates of susceptibility to infection with E. multilocularis are clearer in humans than in the mouse model. A significant link between MHC polymorphism and clinical presentation of AE has been shown, and the spontaneous secretion of IL-10 in patients with a progressive AE is higher in patients with the HLA DR3+, DQ2+ haplotype. Clustering of cases in certain families, in communities otherwise exposed to similar risk factors, also points to immuno-genetic predisposition factors that may allow the larva to escape host immunity more easily. The first stage of larval development may be crucial in producing "danger signals" stimulating the initial production of cytokines. Therapeutic use of Interferon alpha is an attempt to foil the survival strategy of E. multilocularis.

HLA-DRB1 allele in 35 patients with alveolar echinococcosis in Gansu Province of China.

OBJECTIVE: To investigate the association between histocompatibility leukocyte antigen (HLA)-DRB1 alleles and alveolar echinococcosis (AE). METHODS: Thirty-five patients with AE in high prevalence areas in Gansu Province of China were tested for the HLA-DRB1 gene using the polymerase chain reaction with sequence-specific primer (PCR-SSP) technique. The results were compared with those of 104 healthy individuals. RESULTS: The frequency of the HLA-DRB1 * 040x gene was 26% in the patient group, which was significantly higher than that in the control group (9.62%) with a relative risk (RR) of 4.45 (chi(2) = 13.67, P < 0.01), and an etiological fraction (EF) of 0.20. The frequency of the HLA-DRB1 * 0701 allele was significantly lower in the patient group (2.86%) as compared to the control group (13.94%; chi(2) = 6.67, P < 0.05) with a preventable fraction (PF) of 0.30. The frequencies of other DRB1 alleles were not significantly different. CONCLUSION: Susceptibility to AE is significantly associated with the HLA-DRB1 * 040x. HLA-DRB1 * 0701 gene might confer protection against AE in humans.

Polymorphisms of the TAP1 and TAP2 genes in human alveolar echinococcosis.

We postulated that TAP genes may influence the susceptibility of some individuals to Echinococcus multilocularis infection. Six coding region variants (codons 333 and 637 in TAP1, and 379, 565, 651 and 665 in TAP2) were typed in 94 patients and 100 controls. Thr/Thr homozygosity at TAP2/665 was more prevalent in patients than in controls [64% vs. 45%, respectively; odds ratio (OR) = 2.1 (95% confidence interval (CI) 1.1; 2.7)] and Thr/Ala heterozygozity was less prevalent (32% vs. 50%, respectively) (P = 0.014). Of the 38 patients with progressive lesions, 76% were Thr/Thr, as compared with 55% of patients without progressive lesions and 45% of controls (P = 0.058 and 0.02, respectively), independent of HLA status. To determine whether this association is functionally relevant, functional analyses and/or confirmation in distinct populations of patients with alveolar echinococcosis would be required.

[Association of HLA-DRB1 allele and the susceptibility to alveolar echinococcosis in the west of China].

OBJECTIVE: To investigat the association of HLA-DRB1 alleles and the susceptibility to alveolar echinococcosis (AE). METHODS: Thirty-five patients with AE in the high prevalence areas in the west of China were investigated for HLA-DRB1 gene by PCR/SSP technique. The results were compared with 104 normal healthy peoples. RESULTS: Frequency of HLA-DRB1 * 040x was 26% in patient group, which was significantly higher than in control group (9.62%), with a relative risk (RR) of 4.45 (chi(2) = 13.67, P < 0.01), and with an etiological fraction of 0.20. Frequency of HLA-DRB1 * 0701 allele was decreased in patient group (patient 2.86%, control 13.94%; chi(2) = 6.67, P < 0.05), with preventable fraction of 0.30. While the frequencies of other DRB1 alleles were not significantly different. CONCLUSION: Susceptibility to AE is strongly associated with the HLA-DRB1 * 040x allele. The high prevalence of human AE in this region was associated with genetic factor. HLA-DRB1 * 0701 geners resistant to Echinococcus multilocularis metacestode infection.

Cellular localisations of interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha mRNA in a parasitic granulomatous disease of the liver, alveolar echinococcosis.

Alveolar echinococcosis (AE), an uncommon and very severe parasitic liver disease, can be considered as an "infectious model" of granulomatous disease, where cellular immunity plays a key role in the defence against Echinococcus multilocularis, the larval cestode responsible for the disease. We analysed the localisation of the expression of the pro-inflammatory cytokines IL-1 beta, IL-6 and TNF-alpha mRNA in human AE liver lesions, in the periparasitic granulomas and in the hepatic parenchyma, as well as the phenotypic characteristics of the cells on serial sections. In situ hybridizations, using anti-sense 35S dUTP-labeled IL-1 beta, IL-6 and TNF-alpha riboprobes, were performed on cryostat liver sections; the sense probes were used as negative controls. IL-1 beta, IL-6 and TNF-alpha mRNA were observed in macrophages located at the extreme periphery of the granuloma, between the lymphocytic ring and the liver parenchyma, in patients with active AE. No cytokine mRNA expression was observed in a patient with an abortive case. Only TNF-alpha mRNA was located in the periparasitic area, in cells morphologically identified as macrophages but exhibiting an unusual phenotype (CD 11b-, CD 25+); this particular expression was observed only in those patients with very fertile lesions, associated with centro-granulomatous necrosis. These results show that pro-inflammatory cytokines are consistently produced by macrophages at the periphery of the periparasitic granuloma and can serve as mediators of acute-phase protein secretion and of fibrogenesis in that location.(ABSTRACT TRUNCATED AT 250 WORDS)