Involvement of TIGIT in Natural Killer Cell Exhaustion and Immune Escape in Patients and Mouse Model With Liver Echinococcus multilocularis Infection.

BACKGROUND AND AIMS: Alveolar echinococcosis (AE) is a lethal helminthic liver disease caused by persistent infection with Echinococcus multilocularis. Although more attention has been paid to the immunotolerance of T cells caused by E. multilocularis infection, the role of natural killer (NK) cell, a critical player in liver immunity, is seldom studied. APPROACH AND RESULTS: Here, we observed that NK cells from the blood and closed liver tissue (CLT) of AE patients expressed a higher level of inhibitory receptor TIGIT and were functionally exhausted with a lower expression of granzyme B, perforin, interferon-gamma (IFN-γ), and TNF-α. Addition of anti-TIGIT (T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain) monoclonal antibody into AE patients' peripheral blood mononuclear cell culture significantly enhanced the synthesis of IFN-γ and TNF-α by NK cells, indicating the reversion of exhausted NK cells by TIGIT blockade. In the mouse model of E. multilocularis infection, liver and splenic TIGIT+ NK cells progressively increased dependent of infection dosage and timing and were less activated and less degranulated with lower cytokine secretion. Furthermore, TIGIT deficiency or blockade in vivo inhibited liver metacestode growth, reduced liver injury, and increased the level of IFN-γ produced by liver NK cells. Interestingly, NK cells from mice with persistent chronic infection expressed a higher level of TIGIT compared to self-healing mice. To look further into the mechanisms, more regulatory CD56bright and murine CD49a+ NK cells with higher TIGIT expression existed in livers of AE patients and mice infected with E. multilocularis, respectively. They coexpressed higher surface programmed death ligand 1 and secreted more IL-10, two strong inducers to mediate the functional exhaustion of NK cells. CONCLUSIONS: Our results indicate that inhibitory receptor TIGIT is involved in NK cell exhaustion and immune escape from E. multilocularis infection.

Multiple myeloma with Echinococcus granulosus infection diagnosed by detection of oligoclonal bands: A case report.

RATIONALE: Isoelectric focusing electrophoresis (IFE) is currently recognized as the gold standard for detecting oligoclonal bands (OCBs) in cerebrospinal fluid (CSF). To the best of our knowledge, however, no study has reported on type III OCBs using IFE. In this paper, we report on a rare case of multiple myeloma (MM) with Echinococcus granulosus infection diagnosed by IFE. PATIENT CONCERNS: A 71-year-old man complained of weakness of the right lower extremity accompanied with fever (temperature range 37.8°C-38.2°C) for more than 6 months. DIAGNOSES: MM with E granulosus infection. INTERVENTIONS: The IFE results identified a unique monoclonal band, indicating that the patient may have MM in conjunction with a distinct pathogen infection. He received anthelmintic treatment and bortezomib-thalidomide-dexamethasone therapy. OUTCOMES: The patient was followed up for 15 months. During that time, his temperature returned to normal, his Medical Research Council Grading of Muscle Power scale became 5, and his vital signs stabilized. LESSONS: Detection of OCB type III indicated that the patient was diagnosed with MM accompanied by E granulosus infection. Thus, IFE of CSF may be an auxiliary diagnostic method for MM in the future.

Establishing and evaluation of a polymerase chain reaction for the detection of Echinococcus multilocularis in human tissue.

BACKGROUND: Alveolar echinococcosis (AE) is caused by metacestode larva of the tapeworm Echinococcus multilocularis. AE diagnostics currently rely on imaging techniques supported by serology, but unequivocal detection of AE is difficult. Although polymerase chain reaction (PCR)-based methods to detect tapeworm DNA in biopsies have been suggested for several species, no validated protocol adhering to accepted guidelines has so far been presented for AE diagnostics. We herein established a PCR protocol for metacestode biopsies and technically evaluated the method using isolated parasite DNA and cells, biopsies of clinically relevant material, and formalin fixed paraffin-embedded (FFPE) human tissue blocks. We compared the results with an immunochemical (IHC) approach using the monoclonal antibody Em2G11 specific for the antigen Em2 of E. mulitlocularis. METHODOLOGY/PRINCIPAL FINDINGS: Based on tapeworm 12S rDNA sequences we established and validated a PCR protocol for robust detection of as little as 50 parasite cells per specimen and report 127 cases of positive identification of Echinococcus species in samples from humans and animals. For further validation, we analyzed 45 liver, heart, brain, and soft tissue samples as well as cytological probes of aspirates of FFPE-material from 18 patients with clinically confirmed AE. Of each patient we analyzed (i) fully viable lesions with laminated layer; (ii) tissue with mAbEm2G11-positive small particles of E. multilocularis (spems); (iii) mAbEm2G11-negative tissue adjacent to the main lesion; and (iv) lymph node tissue with mAbEm2G11-positive spems. To identify the areas for the PCR-based approach, we performed IHC-staining with the monoclonal antibody Em2G11. Micro-dissected tissue of these areas was then used for PCR-analysis. 9 of 15 analyzed samples with viable E. multilocularis lesions with laminated layer were positive by PCR. Of this group, all samples preserved for less than 6 years (6/6) were tested positive. 11 of 15 samples of spems and 7 of 9 samples of the control group mAbEm2G11-negative tissue were negative by PCR. We further show that all probes from lymph nodes with spems are PCR negative. CONCLUSIONS/SIGNIFICANCE: We present a sensitive PCR method for the detection of E. multilocularis in human tissue, particularly in fresh biopsy material and tissue blocks stored for less than 5 years. While the diagnostic sensitivity of material containing only spems was higher using IHC, PCR detection was possible in IHC negative liver tissue and in patients with negative serology. Our results support the view that spems do not contain parasitic DNA or viable cells of the parasite. spems thus most probably do not directly contribute to metastasis formation during AE.

Linking murine resistance to secondary cystic echinococcosis with antibody responses targeting Echinococcus granulosus tegumental antigens.

Successful establishment of a parasite infection depends partially on the host intrinsic susceptibility to the pathogen. In cystic echinococcosis (CE), a zoonotic disease caused by the cestode parasite Echinococcus granulosus, the infection outcome in the murine model of secondary CE varies according to the mouse strain used. In this regard, intrinsic differences in susceptibility to the infection were previously reported for Balb/c and C57Bl/6 mice, being C57Bl/6 animals less permissive to secondary CE. Induction of parasite-specific antibodies has been suggested to play relevant roles in such susceptibility/resistance phenomena. Here, we report an in deep comparison of antibody responses induced in both mouse strains. Firstly, only C57Bl/6 mice were shown to induce specific-antibodies with efficient anti-parasite activities during early secondary CE. Then, through ImmunoTEM and Serological Proteome Analysis (SERPA), an evaluation of specific antibody responses targeting parasite tegumental antigens was performed. Both strategies showed that infected C57Bl/6 mice -unlike Balb/c animals- narrowed their IgG recognition repertoire against tegumental antigens, targeting fewer but potentially more relevant parasite components. In this sense, tegumental antigens recognition between Balb/c and C57Bl/6 mice, either by natural and/or induced antibodies, was analyzed through SERPA and MALDI-TOF/TOF studies. A total of 13 differentially recognized proteins (DRPs) uniquely targeted by antibodies from C57Bl/6 mice were successfully identified, wherein a subset of 7 DRPs were only recognized by infection-induced antibodies, suggesting their potential as natural protective antigens. In this regard, immunoinformatic analyses showed that such DRPs exhibited higher numbers of possible T cell epitopes towards the H-2-IAb haplotype, which is present in C57Bl/6 mice but absent in Balb/c animals. In summary, our results showed that the genetic predisposition to generate better T-dependent antibody responses against particular tegumental antigens might be a key factor influencing host susceptibility in the murine model of secondary CE.

Echinococcus multilocularis inoculation induces NK cell functional decrease through high expression of NKG2A in C57BL/6 mice.

BACKGROUND: Alveolar echinococcosis (AE) is caused by the larval stage of Echinococcus multilocularis (E. multilocularis), and considered as public health issue. Parasite-host immune interaction is pivotal during infection. As a subset of innate lymphoid cells, NK cells are known to play an important role during virus, bacteria, intra/extracellular parasitic infections and tumor progression. However, the possible role of NK cells in E. multilocularis infection in both human and murine is little known. Herein, the functional alteration of hepatic NK cells and their related molecules in E. multilocularis infected mice were studied. METHODS: 2000 protoscoleces (PSCs) were injected to C57BL/6 mice via the portal vein to establish secondary E. multilocularis infection. NK cells population and their related molecules (CD69, Ly49D, Ly49G2, Ly49H, Ly49I, NKG2A, NKG2D, granzyme B, IFN-γ, TNF-α) were assessed by using fluorescence-activated cell sorter (FACS) techniques and qRT-PCR. NK cell depletion was performed for further understanding the possible function of NK cells during infection. RESULTS: The total frequencies of NK cells and NK-derived IFN-γ production were significantly reduced at designated time points (2, 4, 12 weeks). The liver resident (CD49a+DX5-) NK cells are decreased at 4 weeks after inoculation and which is significantly lower than in control mice. Moreover, in vivo antibody-mediated NK cell depletion increased parasitic load and decreased peri-parasitic fibrosis. Expression of the inhibitory receptor NKG2A was negatively related to NK- derived IFN-γ secretion. CONCLUSIONS: Our study showed down regulates of NK cells and upper regulates of NKG2A expression on NK cells during E. multilocularis infection. Reduction of NK cell frequencies and increased NKG2A might result in low cytotoxic activity through decreased IFN-γ secretion in E. multilocularis infection. This result might be helpful to restore NK cell related immunity against E. multilocularis infection to treat alveolar echinococcosis.

Echinococcus multilocularis Infection, Southern Ontario, Canada.

Alveolar echinococcosis, the disease caused by infection with the intermediate stage of the Echinococcus multilocularis tapeworm, is typically fatal in humans and dogs when left untreated. Since 2012, alveolar echinococcosis has been diagnosed in 5 dogs, 3 lemurs, and 1 chipmunk in southern Ontario, Canada, a region previously considered free of these tapeworms. Because of human and animal health concerns, we estimated prevalence of infection in wild canids across southern Ontario. During 2015-2017, we collected fecal samples from 460 wild canids (416 coyotes, 44 foxes) during postmortem examination and analyzed them by using a semiautomated magnetic capture probe DNA extraction and real-time PCR method for E. multilocularis DNA. Surprisingly, 23% (95% CI 20%-27%) of samples tested positive. By using a spatial scan test, we identified an infection cluster (relative risk 2.26; p = 0.002) in the western-central region of the province. The cluster encompasses areas of dense human population, suggesting zoonotic transmission.

Intense Focus of Alveolar Echinococcosis, South Kyrgyzstan.

Human alveolar echinococcosis (AE) is a highly pathogenic zoonotic parasitic disease caused by Echinococcus multilocularis. An ultrasound study in southern Kyrgyzstan during 2012 revealed a prevalence of 4.2% probable or confirmed AE and an additional 2.2% possible AE, representing an emerging situation. The risk for probable or confirmed AE was significantly higher in dog owners.

Analysis of factors affecting outcomes of pregnancy complicated by Echinococcus: an algorithm for approach and management.

PURPOSE: The management of hydatid disease (HD) co-occurring with pregnancy remains a challenge for physicians. We aimed to determine factors that were related to fetal and maternal outcomes in HD complicated pregnancies and then develop an approach/treatment algorithm. METHOD: All patients at the participating hospitals were first analyzed to determine whether they had HD. Only patients diagnosed with HD during the course of their pregnancy were included. Certain cyst-related factors (diameter, localization, increase in size, and viability) and certain pregnancy-related factors (treatments, gestational week, maternal co-morbidities, and delivery type) were investigated. Nonlinear principal component analysis (NPCA) was performed to determine the relationships between the categories of variables. RESULTS: Out of 12,926 pregnancies, 27 cases were diagnosed with HD. In 13 cases, each developed at least one fetal problem. Using Albendazol in first trimester, presence of an active cyst, increased diameter by more than 1 cm during pregnancy and a cyst diagnosed in the second trimester were associated with at least one fetal problem. According to the NPCA results, cyst diameter when first diagnosed was related to fetal outcomes; a cyst greater than 10 cm was associated with "at least one fetal problem". Cysts 5-10 cm in diameter were in a neutral position, while 2-5 cm in diameter were in the "no problems" group. CONCLUSIONS: HD mostly affects fetus. If cyst-related and pregnancy-related variables are optimal, close follow-up on a monthly is the best course of action. However, in high-risk conditions, percutaneous interventions or surgery should be considered.

Time series analysis based on two-part models for excessive zero count data to detect farm-level outbreaks of swine echinococcosis during meat inspections.

Echinococcus multilocularis is a parasite that causes highly pathogenic zoonoses and is maintained in foxes and rodents on Hokkaido Island, Japan. Detection of E. multilocularis infections in swine is epidemiologically important. In Hokkaido, administrative information is provided to swine producers based on the results of meat inspections. However, as the current criteria for providing administrative information often results in delays in providing information to producers, novel criteria are needed. Time series models were developed to monitor autocorrelations between data and lags using data collected from 84 producers at the Higashi-Mokoto Meat Inspection Center between April 2003 and November 2015. The two criteria were quantitatively compared using the sign test for the ability to rapidly detect farm-level outbreaks. Overall, the time series models based on an autoexponentially regressed zero-inflated negative binomial distribution with 60th percentile cumulative distribution function of the model detected outbreaks earlier more frequently than the current criteria (90.5%, 276/305, p<0.001). Our results show that a two-part model with autoexponential regression can adequately deal with data involving an excessive number of zeros and that the novel criteria overcome disadvantages of the current criteria to provide an earlier indication of increases in the rate of echinococcosis.

Tubercular infection of hydatid cyst.

Tubercular infection of hydatid cyst of the chest wall in an immunocompetent individual is rare. Immune modulation for symbiosis between host cells and the parasite - Echinococcus granulosus favors tubercular infection. In this case report, we describe a case of both these chronic diseases coexisting together, to present as chest wall mass.

Molecular Characteristics and Serodiagnostic Potential of Dihydrofolate Reductase from Echinococcus granulosus.

The larval stage of Echinococcus granulosus causes cystic echinococcosis (CE), a neglected tropical disease that leads to morbidity and mortality in humans and livestock worldwide. Here, we identified and characterized dihydrofolate reductase (Eg-DHFR) from E. granulosus, and evaluated its potential as a diagnostic antigen for sheep CE. Comparison between mammalian (host) DHFR and Eg-DHFR indicates that 45.7% of the 35 active site residues are different. Immunolocalisation analysis showed that native Eg-DHFR was widely distributed in all life-cycle stages of E. granulosus. Recombinant Eg-DHFR (rEg-DHFR) showed typical DHFR enzymatic parameters towards substrate, and was very sensitive to inhibition by methotrexate (IC50 = 27.75 ± 1.03 nM) and aminopterin (IC50 = 63.67 ± 6.76 nM). However, inhibition of DHFR exhibited little protoscolicidal effect in vitro. As there is no reliable method to monitor sheep CE, the immunogenicity of rEg-DHFR was detected, and we developed an indirect ELISA (iELISA) for CE serodiagnosis. The iELISA exhibited diagnostic specificity of 89.58%, diagnostic sensitivity of 95.83%, and the diagnostic accuracy was 91.67% compared with necropsy. Cross-reactivity assay showed analytical specificity of 85.7%. These suggest that rEg-DHFR is an effective antigen for the diagnosis of sheep CE.

Spinal cord compression due to a primary vertebral hydatid disease: A rare case report in metropolitan France and a literature review.

INTRODUCTION: Bone echinococcosis or bone hydatidosis is mainly caused by the larva of a dog taenia, Echinococcus granulosus. We described a rare imported case in metropolitan France of spinal cord compression from a primary vertebral hydatidosis. CASE: A 25-year-old woman, native of a rural area in the South of Romania, was admitted for backache and slight weakness of both legs. Radiological findings showed a paravertebral pluricystic lesion invading the spinal canal with spinal cord compression at the T9 level, without associated visceral localization. We performed an urgent surgical decompression using a posterior approach. The whole extradural cysts were carefully excised with irrigation of the cystic fluid with hypertonic saline. Treatment was completed with long-term anti-parasitic chemotherapy. DISCUSSION: Bone echinococcosis is rare and represents about 2% of hydatidosis. The spine localization is found in half of the cases. This pathology particularly occurs in the Eastern and Southern countries of Mediterranean sheep breeding areas, but still rare in metropolitan France. Spinal cord compression is a frequent presentation of spinal hydatidosis but neurological symptoms are various and non-specific. The reference treatment is removal surgery with particular precautions, followed by an anti-parasitic chemotherapy (albendazole) to limit recurrences. However, a long-term follow-up is mandatory due to later recurrences.

Molecular characterization of Echinococcus granulosus sensu lato from farm animals in Egypt.

Little is known on the diversity and public health significance of Echinococcus species in livestock in Egypt. In this study, 37 individual hydatid cysts were collected from dromedary camels (n=28), sheep (n=7) and buffalos (n=2). DNA was extracted from protoscoleces/germinal layer of individual cysts and amplified by PCR targeting nuclear (actin II) and mitochondrial (COX1 and NAD1) genes. Direct sequencing of amplicons indicated the presence of Echinococcus canadenesis (G6 genotype) in 26 of 28 camel cysts, 3 of 7 sheep cysts and the 2 buffalo derived cysts. In contrast, Echinococcus granulosus sensu stricto (G1 genotype) was detected in one cyst from a camel and 4 of 7 cysts from sheep, whereas Echinococcus ortleppi (G5 genotype) was detected in one cyst from a camel. This is the first identification of E. ortleppi in Egypt.

Th17 cells are associated with the Th1/Th2­cell balance during Echinococcus multilocularis infection.

The present study investigated the immunopathological effect of Echinococcus multilocularis (Em) using cytokine detection. Expression of the T­helper (Th) 17­cytokine, interleukin 17 (IL­17), was observed using immunohistochemical staining, and levels of cytokines, including IL­17, transforming growth factor ß1 (TGF­ß1), IL­6, interferon Î³ (IFN­Î³) and IL­4, were assessed using ELISA at different stages of infection. IL­17 expression occurred in hepatic cells at 1 month post­infection, reached a maximum at 3 months post­infection and then decreased gradually. Compared with the uninfected control, levels of the cytokines IL­17, TGF­ß1, IL­6, IFN­Î³ and IL­4 exhibited different dynamic patterns when infected with Em. In the immune response during the whole infection period, Th17 cells play an important role by secreting IL­17, which may be involved in the Th1/Th2­cell balance during the immune response. Th17 cells are associated with immunopathology in Em infection.

First case of Echinococcus multilocularis infection in a zoo-housed flying squirrel (Pteromys volans orii).

A 33 month-old male flying squirrel kept in a zoo developed progressive dyspnea and died. Macroscopically, the liver and lung were enlarged with numerous nodular vesicles. Histologically, these organs were replaced by numerous collapsed vesicles demarcated by fibrous tissues. The cysts lined by a cellular, germinal layer contained numerous brood capsules with abundant production of well-developed protoscolices. Protoscolices were about 80-100 µm in diameter, and had hooks being visible as refractive structures. This zoo locates in the east of Hokkaido where is an endemic area of Echinococcus multilocularis infection. From epidemiology and pathological findings, this animal was diagnosed as E.multilocularis infection. This report describes the pathology of the first case of E. multilocularis infection in a flying squirrel.

Differential expressions of MHC-DQB1 mRNA in Chinese merino sheep infected with Echinococosus granuclosus.

The purpose of the present study was to investigate the dynamic changes of MHC-DQB1 mRNA expression in sheep infected with Echinococosus granuclosus. A total of 14 healthy Chinese merino sheep were experimentally infected with E. granuclosus. The blood samples were collected on days 0 (initiation of the infection), 7, 21, 30, and 60 post-infection, respectively. On day 60 post-infection, when the experiment was terminated, all sheep were euthanized to make a diagnosis of cystic echinococcosis (CE) using routine meat inspection and microscopical examination, respectively. The sheep were then divided into two groups according to the diagnostic results: group A (n = 8) consisted of sheep which were diagnosed as CE infection, while group B (n = 6) comprised sheep diagnosed as self-cured or healthy controls. Blood samples obtained during the period of the study were correspondingly divided into groups A and B. The mRNA expression levels of DQB1 revealed significant alterations detected at different stages of E. granuclosus infection in the two groups. Results showed that in group A, DQB1 mRNA expression underwent a progressive increase from day 0 to day 21 post-infection (P = 0.073), and suddenly, suffered from a dramatic drop until day 30 post-infection, and then jumped rapidly and peaked on day 60 post-infection (P = 0.004). Meanwhile, in group B, DQB1 mRNA expression displayed a sharp increase from day 0 to day 7 post-infection (P = 0.000), which thereafter showed a marked decrease until day 30 post-infection, and experienced a plateau from day 30 to day 60 post-infection, remaining at or above that on day 0. It is concluded that DQB1 mRNA expression levels varied in different stages of E. granuclosus infection in sheep. In addition, it appears that the ability to eliminate the parasites possibly depends, at least in part, on the DQB1 expression in the early stage of infection, especially in the first week post-infection.

[Immunorehabilitation in patients with paecilomycosis-complicated echinococcosis and asthma].

Paecilomycosis is a new type of systemic mycosis caused by different species of fungi of the genus Paecilomyces. Paecilomycosis-complicated echinococcosis and asthma run a severe course. A complication of mycosis is accompanied by secondary immunodeficiency. A good result was obtained in the treatment of ill children by using the fungicide diflucan and the immunomodulator polyoxidonium. In the examinees with paecilomycosis-complicated echinococcosis, secondary immunodeficiency was characterized by a statistical significant reduction in the blood levels of the lymphoid cells CD3+, CD4+, CD8+, CD16+, CD21+, by phagocytosis, a decrease in its quantitative parameters, and an increase in the counts of immunoglobulins and circulating immunocomplexes. To normalize the immune status in patients with paecilomycosis-complicated echinococcosis, it is expedient to postsurgicallyuse fungicides, such as nizoral, diflucan, orungal, mycosyst, and the immunomodulators polyoxidonium and irillen.

Time course of gene expression profiling in the liver of experimental mice infected with Echinococcus multilocularis.

BACKGROUND: Alveolar echinococcosis (AE) is a severe chronic parasitic disease which behaves like a slow-growing liver cancer. Clinical observations suggest that the parasite, Echinococcus multilocularis (E. multilocularis) influences liver homeostasis and hepatic cell metabolism. However, this has never been analyzed during the time course of infection in the common model of secondary echinococcosis in experimental mice. METHODOLOGY/PRINCIPAL FINDINGS: Gene expression profiles were assessed using DNA microarray analysis, 1, 2, 3 and 6 months after injection of E. multilocularis metacestode in the liver of susceptible mice. Data were collected at different time points to monitor the dynamic behavior of gene expression. 557 differentially expressed genes were identified at one or more time points, including 351 up-regulated and 228 down-regulated genes. Time-course analysis indicated, at the initial stage of E. multilocularis infection (month 1-2), that most of up-regulated pathways were related to immune processes and cell trafficking such as chemokine-, mitogen-activated protein kinase (MAPK) signaling, and down-regulated pathways were related to xenobiotic metabolism; at the middle stage (month 3), MAPK signaling pathway was maintained and peroxisome proliferator-activated receptor (PPAR) signaling pathway emerged; at the late stage (month 6), most of up-regulated pathways were related to PPAR signaling pathway, complement and coagulation cascades, while down-regulated pathways were related to metabolism of xenobiotics by cytochrome P450. Quantitative RT-PCR analysis of a random selection of 19 genes confirmed the reliability of the microarray data. Immunohistochemistry analysis showed that proliferating cell nuclear antigen (PCNA) was increased in the liver of E. multilocularis infected mice from 2 months to 6 months. CONCLUSIONS: E. multilocularis metacestode definitely exerts a deep influence on liver homeostasis, by modifying a number of gene expression and metabolic pathways. It especially promotes hepatic cell proliferation, as evidenced by the increased PCNA constantly found in all the experimental time-points we studied and by an increased gene expression of key metabolic pathways.

Cranial hydatid abscess.

A woman of age 20 years presented with a tender cystic swelling over the right temporal region, with proptosis of the right eye and purulent discharge from the right ear. A computed tomography scan revealed a mixed density lesion involving both extra- and intracranial spaces, which was not enhanced with contrast. Following an operation, histopathology revealed the structure to be a hydatid abscess (infected hydatid cyst).