Inflammatory responses to Opisthorchis viverrini infection in animal models: A comparison between susceptible and nonsusceptible hosts in different anatomical locations.

Background: Inflammation caused by Opisthorchis viverrini infection increases the risk of cholangitis, cholecystitis, and leads to bile duct cancer (cholangiocarcinoma or CCA). However, only certain infected individuals are susceptible to CCA, suggesting the involvement of host factors in cancer development. In addition, there are reports indicating differences in the locations of CCA. Aim: This study aims to investigate cellular inflammatory responses in the common bile duct (CB), intrahepatic bile duct (IHB), and gallbladder (GB) in susceptible and non-susceptible hosts following O. viverrini infection. Methods: Thirty Syrian golden hamsters (a susceptible host) and 30 BALB/c mice (a non-susceptible host) infected with O. viverrini were studied at six time points (five animals per group). Histopathological evaluations were conducted on samples from the IHB, CB, and GB. Inflammatory cell infiltration was quantitatively assessed and compared between groups and time points. Statistical analysis was performed using one-way ANOVA, with a significance level of p < 0.05. Results: Inflammation was significantly more pronounced in the IHB compared to the other two biliary locations. In comparison between susceptible and non-susceptible hosts, the intensity of inflammation was higher in the OV+H group than in the OV+M group (p < 0.05). Conclusion: This study highlights the association between host response to inflammation, tissue location, and host susceptibility, with the IHB showing particular susceptibility to inflammation and pathological changes. These findings contribute to our understanding of the increased risk of CCA in susceptible hosts.

Inflammatory cell responses in biliary mucosa during Opisthorchis viverrini infection: Insights into susceptibility differences among hosts.

Background: Individual host susceptibility is believed to be a risk factor in the interaction between the host and the parasite. Since studying time series in humans is limited, animal models are replaced. Aim: This study aims to explore and compare the pattern of inflammatory cell types along the biliary tract and their association with proliferative lesions in the early development of cholangiocarcinoma from susceptible and nonsusceptible animal models. Methods: Thirty male Syrian golden hamsters and 30 BALB/c mice, serving as the susceptible and nonsusceptible animal models, were used in this comparative study. The animals were infected with 50 Opisthorchis viverrini metacercariae via gastric intubation. At days 1, 2, 7, 14, 28, and 56 postinfection (p.i.), five animals were randomly selected from each group and humanely sacrificed. The hepatobiliary tissues were collected and processed for histopathological study. Histochemical and immunohistochemical staining were applied to differentiate the inflammatory cell types. Kruskal-Wallis and Mann-Whitney tests were applied to assess all semi-quantitative and quantitative variables. The correlation between each variable was also analyzed using Spearman rank at a p-value < 0.05. Results: The results demonstrated that mice had different patterns of infiltrating cell types when compared to hamsters. This suggested that the cellular response to the infection in mice occurred earlier than that in hamsters. The response in mice reached its peak at D7 to D14 and then rapidly declined at D28. In contrast, although the inflammatory response in hamsters started slowly, the response reached the peak at D28 and maintained a high level until D56. Significant differences in the number of inflammatory cells between mice and hamsters were seen at D1 (p = 0.047), D7 (p = 0.049), D28 (p = 0.040), and D56 (p < 0.040). Conclusion: The inflammatory responses to O. viverrini infection in the nonsusceptible animal model occurred and declined earlier while the response in the susceptible animal model occurred later in a gradual manner. Both rodents are suitable animal models for the studies of opisthorchiasis susceptibility.

Elevated circulating TLR4+ monocytes in patients with liver fluke Opisthorchis viverrini is associated with advanced periductal fibrosis.

INTRODUCTION: Opisthorchis viverrini (Ov) infection can lead to several disease manifestations of the bile duct including advanced periductal fibrosis (APF) and the most severe complication, cholangiocarcinoma (CCA). Monocytes migrate to the infection site and differentiate into tissue macrophages to express and release molecules such as cytokines, reactive oxygen species, and growth factors. TLR4+ monocytes are classified as having a pro-tumor phenotype and secrete tumor-promoting factors. The aim of this study is to investigate the role of monocytes in the pathogenesis of opisthorchiasis. METHODOLOGY: We used flow cytometry to measure the number of TLR4+ monocytes in the circulating blood of Ov infected patients with or without APF compared to healthy, non-Ov-infected controls. RESULTS: We found, for the first time, that patients with AFP have elevated numbers of circulating TLR4+ monocytes when compared to patients without fibrosis and healthy individuals. Intriguingly, when we measured ROS from these monocytes, we found increased ROS production in patients with APF. CONCLUSIONS: We propose that excessive production of ROS from these TLR4+ monocytes may lead to excessive injury of surrounding tissue and hence contribute to the pathological processes that lead to the development of advanced periductal fibrosis.

Neutrophils form extracellular traps in response to Opisthorchis viverrini crude antigens, which are elevated in neutrophils from opisthorchiasis patients with hepatobiliary abnormalities.

Opisthorchis viverrini (Ov) infection can cause several disease conditions of the bile duct including hepatobiliary abnormalities (HBAs) and the most severe, cholangiocarcinoma (CCA). Fibrosis occurs when tissues are damaged and normal wound-healing responses are dysregulated. Neutrophils are the first cells to migrate to an infection site to protect the host from intruding extracellular pathogens through a wide range of effector mechanisms such as phagocytosis, production of reactive oxygen species, proteases, or release of neutrophil extracellular traps (NETs). In this work, we used confocal microscopy to assess whether Ov crude antigens can cause release of NETs from neutrophils from Ov-free individuals. We demonstrated for the first time that these antigens could induce release of NETs ex vivo in a dose-dependent manner from neutrophils isolated from Ov-free individuals. Intriguingly, when we measured NETs from neutrophils isolated from Ov-infected patients, we found increased spontaneous production of NETs in patients with HBAs. Interestingly, exposure to Ov crude antigens lowered the level of NETs released by neutrophils from patients with active Ov infection regardless of HBA status. We propose that in the case of acute Ov infection, even when concentration of Ov antigens is relatively low, neutrophils can form NETs. However, when this infection becomes chronic, manifesting as a definite HBA, the levels of NET production are reduced when treated with Ov crude antigens. Excessive production of proinflammatory mediators from these NETs might have effects on the parasites, but may also lead to excessive injury of surrounding tissues resulting in HBAs and may lead eventually to the most severe complications such as CCA.

Distinct antibody response in susceptible and non-susceptible hosts of the carcinogenic liver fluke Opisthorchis viverrini infection.

Opisthorchis viverrini is a carcinogenic parasite that can cause bile duct cancer called cholangiocarcinoma. A study of the immune response of this parasite in susceptible and non-susceptible hosts may provide a clue to develop vaccines and immunodiagnostic markers, which are currently not available. Here, we compared the antibody response in susceptible Golden Syrian hamsters and non-susceptible BALB/c mice infected by the liver fluke. In mice, the antibody was detected between 1 and 2 weeks post-infection, whereas it was positive between 2 and 4 weeks post-infection in hamsters. Immunolocalization revealed that the antibody from mice reacts strongly with the tegumental surface and gut epithelium of the worm, while hamster antibody showed a weak signal in the tegument and a comparable signal in the gut of the worm. Immunoblot of the tegumental proteins demonstrated that while hamster antibody showed a broad specificity, mice strongly reacted with a single protein band. Mass spectrometry revealed these immunogenic targets. Recombinant proteins of the reactive targets were produced in the bacterial expression system. The immunoblot of these recombinant proteins confirm the reactivity of their native form. In summary, there is a different antibody response against O. viverrini infection in susceptible and non-susceptible hosts. The non-susceptible host reacts quicker and stronger than the susceptible host.

Increased IL-31 expression in serum and tissue protein in prurigo nodularis.

Background: Prurigo nodularis (PN) is a chronic pruritic skin disease which can greatly impact patients' quality of life. Moreover, the pathogenesis remains unclear, making it a difficult-to-treat condition. Aims: To investigate the expression of interleukin-31 (IL-31) in serum and skin biopsy specimens of PN patients and healthy subjects and identify its possible correlation to disease severity and itch intensity. Methods: Patients with PN and healthy volunteers were recruited for the study. Expression levels of IL-31 were measured by enzyme-linked immunosorbent assay and immunohistochemistry. Baseline characteristics, disease activity, itch intensity, and related laboratory results were collected. Results: Forty-three PN patients and 31 healthy subjects participated in our study. The PN patients had significantly higher mean serum IL-31 levels than the healthy subjects (52.9 ± 18.2 versus 36.3 ± 10.7 pg/ml, p < 0.001). Epidermal and dermal PN lesions also exhibited significantly higher IL-31 expression compared with the healthy skin (p < 0.001 and p = 0.01, respectively). However, there was no significant difference in serum or lesional expression of IL-31 by disease severity or itch intensity. Conclusion: Increased IL-31 expression in serum and PN lesions suggests that IL-31 has a potential role in the pathogenesis of PN.

Proteomic analysis of infected primary human leucocytes revealed PSTK as potential treatment-monitoring marker for active and latent tuberculosis.

Markers for monitoring clearance of Mycobacterium tuberculosis (Mtb) infection during anti-TB drug treatment could facilitate management of tuberculosis (TB) treatment, but are lacking. We aimed to screen for Mtb clearance markers from in-vitro-infected leucocytes and to evaluate these markers in followed-up active TB (ATB) patients and latent TB (LTBI) cases after anti-TB drug treatment. Extracellular proteins from primary leucocytes infected with each of the Mtb lineages (East-Asian, Indo-Oceanic, Euro-American and the laboratory strain H37Rv) were screened as possible clearance markers. Leucocytes infected with Staphylococcus aureus acted as controls. The proteomic analysis was performed using GeLC-MS/MS. Several quantitative and qualitative candidate clearance markers were found. These proteins were suppressed during the infection stage of all Mtb lineages and re-expressed after bacillary clearance. PSTK, FKBP8 and MGMT were common clearance markers among the four Mtb lineages in our model. Only PSTK was a potential clearance marker based on western blot validation analysis from culture supernatants. The PSTK marker was further validated with western blot analysis using serum samples (n = 6) from ATB patients and LTBI cases during anti-TB drug treatment, and from healthy controls (n = 3). Time-dependent increase of PSTK was found both in ATB and LTBI patients during the course of anti-TB drug treatment, but not in healthy controls. We have demonstrated that PSTK is a potential treatment-monitoring marker for active and latent TB.

Enhanced neutrophil functions during Opisthorchis viverrini infections and correlation with advanced periductal fibrosis.

Millions of people are infected with the liver fluke, Opisthorchis viverrini (OV), but only ~25% of those infected develop liver disease and even fewer develop cholangiocarcinoma. The reasons for these differential outcomes following infection are unknown but it has been proposed that differential immune responses to the parasite may play a role. We therefore measured granulocyte (neutrophil) function in OV-infected individuals, with and without advanced periductal fibrosis, to determine if these cells have a "pro-inflammatory" phenotype that may contribute to liver disease post-infection. A case-controlled study (n = 54 in each cohort) from endemic OV-infected areas of northeastern Thailand measured neutrophil functions in whole blood from non-infected (healthy controls) and OV-infected individuals with and without APF. We measured reactive oxygen species production, phagocytosis, receptor expression and apoptosis. Secreted products from OV cultures (obtained after in vitro culture of parasites) stimulated reactive oxygen species production in non-infected healthy controls, but levels were two-fold greater after OV infection (P < 0.0001); neutrophil reactive oxygen species production in individuals with APF was double that observed in those without APF (P < 0.0001). OV-infected neutrophils had elevated CD11b expression and greater phagocytic capacity, which was even three-fold higher in those with advanced periductal fibrosis (P < 0.0001). This "activated" phenotype of circulating neutrophils was further confirmed by the observation that isolated neutrophils had delayed apoptosis ex vivo. We believe this is the first study to show that circulating blood neutrophil function is enhanced following OV infection and is more activated in those with advanced periductal fibrosis. We propose that this activated phenotype could contribute to the pathology of liver disease. These data support the hypothesis of an activated innate inflammatory phenotype following OV infection and provide the first evidence for involvement of neutrophils in disease pathology.

High macrophage activities are associated with advanced periductal fibrosis in chronic Opisthorchis viverrini infection.

Liver fluke infection caused by Opisthorchis viverrini induces several hepatobiliary conditions including advanced periductal fibrosis (APF) and cholangiocarcinoma (CCA), but >25% of the infected population develops APF and 1% develop CCA. The innate immune response is the first line of defence, and macrophages are critical regulators of fibrosis. We hypothesized that macrophages from infected individuals have different capacities to either promote or suppress periductal fibrosis. We compared phagocytic activities of macrophages of healthy individuals and O viverrini-infected individuals ± APF, and found that macrophages from infected individuals with APF ingested significantly higher numbers of beads compared with healthy controls and O viverrini-infected individuals without APF. To further investigate proteolytic activity, we monitored real-time phagosomal proteolysis of beads conjugated to DQ-BODIPY-BSA using live cell imaging. We show that macrophages from O viverrini-infected individuals with APF also have elevated phagosomal proteolysis activity, which is consistent with their increased phagocytic activity. Additionally, stimulated ROS production by blood monocytes was higher in individuals with APF compared with healthy controls and infected individuals without APF. These results suggest that during O viverrini infection, macrophages with high phagocytic and proteolytic activities together with elevated ROS production are the phenotypes that can promote tissue damage, which results in periductal fibrosis.

Opisthorchiasis-Induced Cholangiocarcinoma: How Innate Immunity May Cause Cancer.

Innate, inflammatory responses towards persistent Opisthorchis viverrini (OV) infection are likely to contribute to the development of cholangiocarcinoma (CCA), a liver cancer that is rare in the West but prevalent in Greater Mekong Subregion countries in Southeast Asia. Infection results in the infiltration of innate immune cells into the bile ducts and subsequent activation of inflammatory immune responses that fail to clear OV but instead may damage local tissues within the bile ducts. Not all patients infected with OV develop CCA, and so tumourigenesis may be dependent on multiple factors including the magnitude of the inflammatory response that is activated in infected individuals. The purpose of this review is to summarize how innate immune responses may promote tumourigenesis following OV infection and if such responses can be used to predict CCA onset in OV-infected individuals. It also hypothesizes on the role that Helicobacterspp., which are associated with liver fluke infections, may play in activation of the innate the immune system to promote tissue damage and persistent inflammation leading to CCA.

Synergistic Effect of Forbesione From Garcinia hanburyi in Combination with 5-Fluorouracil on Cholangiocarcinoma

Background: Chemotherapy for advanced cholangiocarcinoma (CCA) is largely ineffective; thus innovative combinations of chemotherapeutic agents and natural compounds represent a promising strategy. This study aimed to investigate the synergistic effects of forbesione combined with 5-fluorouracil (5-FU) in hamster cholangiocarcinoma (Ham-1) cells both in vitro and in vivo. The anti-tumor effects of 5-FU combined with forbesione in vitro were determined using the Sulforhodamine B (SRB) assay and the effects in vivo were assessed in transplanted Ham-1 allograph models. Using ethidium bromide/acridine orange (EB/AO) staining, the morphological changes of apoptotic cells was investigated. The expressions of apoptosis-related molecules after combined treatment with forbesione and 5-FU were determined using real-time RT-PCR and western blot analysis. Forbesione or 5-FU alone inhibited proliferation of Ham-1 cells in a dose-dependent manner and their combination showed a synergistic proliferation inhibitory effect in vitro. In vivo studies, forbesione in combination with 5-FU exhibited greater inhibition of the tumor in the hamster model compared with treatment using either drug alone. Forbesione combined with 5-FU exerted stronger apoptotic induction in Ham-1 cells than did single drug treatment. The combination of drugs strongly suppressed the expression of B-cell lymphoma 2 (Bcl-2) and procaspase-3 while enhancing the expression of p53, Bcl-2-associated X protein (Bax), apoptotic protease activating factor-1 (Apaf-1), caspase-9 and caspase-3, compared with single drug treatments. These results explained the decreased expression of cytokeratin 19 (CK19) positive cells and proliferation cell nuclear antigen (PCNA) positive cells in Ham-1 cell tumor tissues of the treated hamsters. There was no apparent systemic toxicity observed in the treated animals compared with the control groups. Forbesione combined with 5-FU strongly induced apoptosis in Ham-1 cells. The growth inhibitory effect of combined treatment using these two drugs was much greater than treatment with either drug alone, both in vitro and in vivo.