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.

A two-arm analysis of the immune response to heterologous boosting of inactivated SARS-CoV-2 vaccines.

Several vaccine programs were introduced during the COVID-19 pandemic, which included inactivated virus, DNA viral vectors and mRNA vaccines. Booster programs are recommended, especially for those in high-risk groups. However, many of these booster programs involve heterologous vaccines. This study enrolled volunteers who first received two full-dose CoronaVac vaccinations before receiving heterologous boosters with DNA- and/or mRNA-vaccines for an additional 2 doses (n = 40) or an additional 3 doses (n = 16). Our results showed no difference in side effects, neutralizing antibodies, or T-cell responses for any of the heterologous vaccination programs. However, the neutralizing capacity and IFN-γ responses against the Omicron variant in volunteers who received 4 or 5 doses were improved. Polarization of peripheral memory T cells after stimulation in all booster groups with Omicron peptide showed an increased trend of naïve and central memory phenotypes of both CD4+ and CD8+ T cells, suggesting that exposure to Omicron antigens will drive T cells into a lymphoid resident T cell phenotype. Our data support a continuous vaccination program to maximize the effectiveness of immunity, especially in people at high risk. Furthermore, the number of boosting doses is important for maintaining immunity.

Helicobacter pylori extract induces purified neutrophils to produce reactive oxygen species only in the presence of plasma.

H. pylori is a bacterial pathogen infecting over half of the world's population and induces several gastric and extra-gastric diseases through its various virulence factors, especially cagA. These factors may be released from the bacteria during interactions with host immune cells. Neutrophils play key roles in innate immunity, and their activity is regulated by plasma factors, which can alter how these cells may interact with pathogens. The aim of the present study was to determine whether purified neutrophils could produce reactive oxygen species (ROS), one of the key functions of their anti-microbial functions, in response to extracts of cagA+ and cagA- H. pylori. Extracts from either cagA+ or cagA- H. pylori were co-cultured with human neutrophils in the presence or absence of plasma, and the neutrophil ROS production was measured. In the absence of plasma, extracts from cagA+ and cagA- H. pylori did not induce neutrophil ROS production, whereas in the presence of plasma, extracts from both cagA+ and cagA- H. pylori-induced ROS production. Furthermore, when peripheral blood mononuclear cells (PBMCs) were added to the purified neutrophils in the absence of plasma, there was no neutrophil ROS production after challenging with extracts from either cagA+ or cagA- H. pylori. Thus, it is suggested that plasma contains immunological components that change the responsiveness of neutrophils, such that when neutrophils encounter the bacterial antigens in H. pylori extracts, they become activated and produce ROS. This study also revealed a potential novel immunopathogenic pathway by which cagA activation of neutrophils contributed to inflammatory damage.

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.

Phenotypic and functional changes of T cell subsets after CoronaVac vaccination.

BACKGROUND: The pandemic coronavirus disease 2019 (COVID-19) is a major global public health concern and several protective vaccines, or preventive/therapeutic approaches have been developed. Sinovac-CoronaVac, an inactivated whole virus vaccine, can protect against severe COVID-19 disease and hospitalization, but less is known whether it elicits long-term T cell responses and provides prolonged protection. METHODS: This is a longitudinal surveillance study of SARS-CoV-2 receptor binding domain (RBD)-specific IgG levels, neutralizing antibody levels (NAb), T cell subsets and activation, and memory B cells of 335 participants who received two doses of CoronaVac. SARS-CoV-2 RBD-specific IgG levels were measured by enzyme-linked immunosorbent assay (ELISA), while NAb were measured against two strains of SARS-CoV-2, the Wuhan and Delta variants. Activated T cells and subsets were identified by flow cytometry. Memory B and T cells were evaluated by enzyme-linked immune absorbent spot (ELISpot). FINDINGS: Two doses of CoronaVac elicited serum anti-RBD antibody response, elevated B cells with NAb capacity and CD4+ T cell-, but not CD8+ T cell-responses. Among the CD4+ T cells, CoronaVac activated mainly Th2 (CD4+ T) cells. Serum antibody levels significantly declined three months after the second dose. INTERPRETATION: CoronaVac mainly activated B cells but T cells, especially Th1 cells, were poorly activated. Activated T cells were mainly Th2 biased, demonstrating development of effector B cells but not long-lasting memory plasma cells. Taken together, these results suggest that protection with CoronaVac is short-lived and that a third booster dose of vaccine may improve protection.

Burkholderia pseudomallei biofilm phenotypes confined but surviving in neutrophil extracellular traps of varying appearance.

Melioidosis is a fatal infectious disease caused by Burkholderia pseudomallei. Complications following treatment are usually due to antibiotic resistance and relapse is mainly caused by B. pseudomallei biofilm. Although the release of neutrophil extracellular traps (NETs) is crucial to capture and eliminate bacterial pathogens, to date response of NETs to B. pseudomallei biofilm is poorly understood. Here we compare the NETs produced by neutrophils in response to B. pseudomallei H777 (a biofilm-producing strain containing the bpsl0618 gene), a biofilm-defect strain lacking this gene (B. pseudomallei M10) and a bpsl0618 biofilm-complemented strain, B. pseudomallei C17, in which function of bpsl0618 was restored. Co-cultivation of these strains with healthy human neutrophils at MOI 10 with or without cytochalasin D demonstrated that H777 significantly resisted neutrophil-mediated killing and non-phagocytotic mechanisms compared to M10 (p < 0.0001). Three distinct morphotypes of NETs were seen: "aggregated", "spiky" and "cloudy". These were induced in different proportions by the different bacterial strains. All types of NETs were shown to confine all B. pseudomallei strains. Strains H777 and C17 could stimulate production of twice as much extracellular DNA (234.62 ng/mL and 205.43 ng/mL, respectively) as did M10 (111.87 ng/mL). Cells of H777 and C17 were better able to survive in the presence of neutrophil killing mechanisms relative to M10 (p < 0.0001) and NET formation (p < 0.0001 and 0.05). These findings suggest that NET stimulation was insufficient to eradicate B. pseudomallei H777 and C17 despite their possession of bpsl0618, a sugar-transferase gene associated with biofilm formation ability. Our findings demonstrate that B. pseudomallei biofilm phenotype may be a key factor in assisting pathogens to escape killing by neutrophils. This work provides a better understanding of how B. pseudomallei biofilm-associated infections induce and survive NET formation, resulting in bacterial persistence and increased severity of disease.

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.

High rate of reinfection and possible transmission of Mycobacterium avium complex in Northeast Thailand.

The Mycobacterium avium complex (MAC) includes two main species of non-tuberculous mycobacteria (NTM), M. avium and Mycobacterium intracellulare. These can cause serious disease, especially in immunocompromised patients. Little information is available concerning genetic diversity of NTM. We used multilocus sequence typing (MLST) based on a highly discriminative gene set to analyze MAC serially isolated from patients to determine the rate of MAC reinfection. Genomic DNA was sequenced from 49 MAC isolates (15 cases comprised of 11 true infections and 4 instances of colonization). More than half of the MAC isolates tested were found to be multidrug resistant. The discriminatory power was assessed of 24 house-keeping genes (fusA, atpD, pheT, glnA, topA, secA, argH, glpK, murC, cya, pta, rrl, rrs, hsp65, rpoB, 16S-23S rRNA ITS, recF, lipT, pepB, gnd, aspB, groEL, sodA and est) previously used for genotyping of MAC and other NTM. Seven genes (fusA, secA, rpoB, hsp65, 16S rRNA, 23S rRNA, 16S-23S rRNA ITS) had a discriminatory power index higher than 0.9 and were included in the optimized set that we used. This set was significantly better for genotyping and diagnosis of MAC than previously used 4-gene, 5-gene and 9-gene sets. MLST using our 7-gene set indicated that the rate of reinfection was 54.55% (6/11 cases). Persistent infections (n = 5 cases, 45.45%) were found. A changing of clone in the same patient was found in 1/4 (25%) of the colonization cases. Two small clusters of possible MAC transmission between humans were found. Our study demonstrated that the high frequency of apparent treatment failure of MAC might be artefactual, as a consequence of a high rate of MAC reinfection in Thai population. Our useful highly discriminative gene set for MAC species and clonal strain analysis could be further applied for the diagnosis and patient management.

Characterisation of secretome-based immune responses of human leukocytes infected with various Mycobacterium tuberculosis lineages.

BACKGROUND: Differences in immune responses against different lineages of Mycobacterium tuberculosis (Mtb), and by different types of immune cell, are still poorly understood. We aimed to compare the secretome-based immune responses among three Mtb lineages and among immune-cell types. The immune responses were also investigated during infection and when the bacilli had been eliminated from the immune cells. METHODS: Human primary leukocytes were infected with strains representing three lineages of Mtb (East-Asian, Indo-Oceanic and Euro-American). Label-free GeLC MS/MS proteomic analysis of secretomes was performed. The response of each immune-cell type was compared with the appropriate interactome database for each. RESULTS: The expression pattern of proteins secreted by Mtb-infected leukocytes differed among Mtb lineages. The ancestral lineage (IO lineage) had a greater ability to activate MMP14 (associated with leukocyte migration) than did the more recent lineages (EA and EuA). During infection, proteins secreted by macrophages, dendritic cells, neutrophils and B-cells were associated with cell proliferation. Following clearance of Mtb, proteins associated with interferon signaling were found in macrophages, dendritic cells and neutrophils: proteins associated with antigen processing were found in B-cells and regulatory T-cells. Expression of immune response-related proteins from many immune-cell types might be suppressed by Mtb infection. Our study has provided a better insight into the host-pathogen interaction and immune response against different Mtb lineages.

Increased serum IL-31 levels in chronic spontaneous urticaria and psoriasis with pruritic symptoms.

BACKGROUND: Chronic spontaneous urticaria (CSU) is a common pruritic skin condition, the pathogenesis of which remains unclear. Interleukin-31 (IL-31) is a major pruritogenic cytokine that plays a role in inducing pruritus in various skin diseases. AIM: To 1) compare serum IL-31 levels among CSU patients, psoriasis patients with pruritic symptoms, and healthy subjects, 2) examine the correlations between serum IL-31 levels and disease severity, and 3) compare IL-31 levels in patients with and without CSU-associated auto-antibodies. METHODS: Patients with CSU, psoriasis with pruritic symptoms, and healthy volunteers were recruited in the study. Serum IL-31 levels were measured with commercial kits. Baseline characteristics, urticaria activity score, psoriasis area severity index, pruritic intensity score, and related laboratory results were collected. RESULTS: Sixty-five CSU patients, 30 psoriasis patients who had pruritus, and 31 healthy subjects participated in our study. The CSU patients had significantly higher mean serum IL-31 levels than the psoriasis patients (252.4 ± 115.5 vs 121.4 ± 16.6 pg/mL, P < 0.001). Both CSU and psoriasis patients also had significantly higher mean serum IL-31 when compared with the healthy subjects. Serum IL-31 levels of CSU and psoriasis patients did not differ significantly according to disease or itching severity. Thyroid antibodies and antinuclear antibodies were positive in 22 (33.8%) and 28 (43.1%) CSU patients, respectively. The CSU patients with ANA titers ≥1:160 had significantly higher mean serum IL-31 levels than in those who were negative for ANA and those with titers of 1:80 (P < 0.003 and P < 0.008, respectively). CONCLUSION: Higher serum IL-31 levels were found in patients with CSU and psoriasis with pruritic symptoms. This suggests that IL-31 has a possible role in the pathogenesis of CSU and psoriasis with pruritic symptoms.

Interleukin-31 and Chronic Pruritus of Unknown Origin.

Chronic pruritus of unknown origin (CPUO) is a refractory condition. The expression of Interleukin-31 (IL-31), a major pruritogenic cytokine, in CPUO patients has not been investigated. This study aimed to investigate the potential association of IL-31 with CPUO. This was a cross-sectional, analytical study. Patients diagnosed with CPUO and healthy subjects were included at a ratio of 1:2. Serum IL-31 levels were measured in both groups and compared. There were 10 CPUO and 20 healthy subjects who participated in this study. The median IL-31 level in the CPUO group was significantly higher than in the healthy group (127.3 vs 34.4 pg/mL; P < .001). The presence of CPUO was independently associated with IL-31 levels with a coefficient of 89.678 (P < .001). The serum IL-31 cutoff point for CPUO was 56.8 pg/mL, with an area under the receiver operating characteristic curve (ROC) of 100%. Chronic pruritus of unknown origin was significantly and independently associated with higher IL-31 levels. Further clinical trials of IL-31-related treatment may be justified in CPUO patients.

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.

CLIC1 regulates dendritic cell antigen processing and presentation by modulating phagosome acidification and proteolysis.

Intracellular chloride channel protein 1 (CLIC1) participates in inflammatory processes by regulating macrophage phagosomal functions such as pH and proteolysis. Here, we sought to determine if CLIC1 can regulate adaptive immunity by actions on dendritic cells (DCs), the key professional antigen presenting cells. To do this, we first generated bone marrow-derived DCs (BMDCs) from germline CLIC1 gene-deleted (CLIC1(-/-)) and wild-type (CLIC1(+/+)) mice, then studied them in vitro and in vivo We found phagocytosis triggered cytoplasmic CLIC1 translocation to the phagosomal membrane where it regulated phagosomal pH and proteolysis. Phagosomes from CLIC1(-/-) BMDCs displayed impaired acidification and proteolysis, which could be reproduced if CLIC1(+/+), but not CLIC1(-/-) cells, were treated with IAA94, a CLIC family ion channel blocker. CLIC1(-/-) BMDC displayed reduced in vitro antigen processing and presentation of full-length myelin oligodendrocyte glycoprotein (MOG) and reduced MOG-induced experimental autoimmune encephalomyelitis. These data suggest that CLIC1 regulates DC phagosomal pH to ensure optimal processing of antigen for presentation to antigen-specific T-cells. Further, they indicate that CLIC1 is a novel therapeutic target to help reduce the adaptive immune response in autoimmune diseases.