Synergism therapeutic and immunoregulatory effects of Albendazole + rAd-mIL-28B against Echinococcosis in experiment-infected mice with protoscoleces

The metacestode stage of Echinococcus granulosus can cause cystic echinococcosis (CE), which still widely occurs around the world. Since the early 1970s, benzimidazoles have been shown to inhibit the growth of cysts and used to treat CE. However, benzimidazoles are still ineffective in 20%-40% of cases. In order to explore the new agents against CE, we have investigated the therapeutic effect of the recombinant adenoviral vector expressing mouse IL-28B (rAd-mIL-28B) on protoscoleces-infected mice. In our study, we successfully established the model mice which infected with protoscoleces intraperitoneally. At 18 weeks post-infection, the mice received rAd-mIL-28B (1×10⁷ PFU) weekly by intramuscular injection for 6 weeks. Compared with the untreated control (13.1 ± 2.2 g), there was a significant reduction in cysts wet weight in rAd-mIL-28B group (8.3 ± 3.5 g) (P < 0.05), especially in Albendazole (ABZ) + rAd-mIL-28B group (5.8 ± 1.4 g) (P < 0.01). We also observed the severe damage of the germinal layer and the laminated layer of cysts after treatment. rAd-mIL-28B group showed a prominent increase in the level of Th1 type cytokines (such as IFN-γ, IL-2 and TNF-α). Meanwhile, the frequency of Foxp3⁺ T cells was decreased in the rAd-mIL-28B group (4.83 ± 0.81%) and ABZ + rAd-mIL-28B group (4.60 ± 0.51%), comparing with the untreated group (8.13 ± 2.60%) (P < 0.05). In addition, compared with the untreated control (122.14 ± 81.09 pg/ml), the level of IFN-γ significantly increased in peritoneal fluid in the rAd-mIL-28B group (628.87 ± 467.16 pg/ml) (P < 0.05) and ABZ + rAd-mIL-28B group (999.76 ± 587.60 pg/ml) (P < 0.001). Taken together, it suggested that ABZ + IL-28B may be a potential therapeutic agent against CE.     

Granulocyte-Macrophage Colony Stimulatory Factor Enhances the Pro-Inflammatory Response of Interferon-γ-Treated Macrophages to Pseudomonas aeruginosa Infection

Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe infections at compromised epithelial surfaces, such those found in burns, wounds, and in lungs damaged by mechanical ventilation or recurrent infections, particularly in cystic fibrosis (CF) patients. CF patients have been proposed to have a Th2 and Th17-biased immune response suggesting that the lack of Th1 and/or over exuberant Th17 responses could contribute to the establishment of chronic P. aeruginosa infection and deterioration of lung function. Accordingly, we have observed that interferon (IFN)-γ production by peripheral blood mononuclear cells from CF patients positively correlated with lung function, particularly in patients chronically infected with P. aeruginosa. In contrast, IL-17A levels tended to correlate negatively with lung function with this trend becoming significant in patients chronically infected with P. aeruginosa. These results are in agreement with IFN-γ and IL-17A playing protective and detrimental roles, respectively, in CF. In order to explore the protective effect of IFN-γ in CF, the effect of IFN-γ alone or in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), on the ability of human macrophages to control P. aeruginosa growth, resist the cytotoxicity induced by this bacterium or promote inflammation was investigated. Treatment of macrophages with IFN-γ, in the presence and absence of GM-CSF, failed to alter bacterial growth or macrophage survival upon P. aeruginosa infection, but changed the inflammatory potential of macrophages. IFN-γ caused up-regulation of monocyte chemoattractant protein-1 (MCP-1) and TNF-α and down-regulation of IL-10 expression by infected macrophages. GM-CSF in combination with IFN-γ promoted IL-6 production and further reduction of IL-10 synthesis. Comparison of TNF-α vs. IL-10 and IL-6 vs. IL-10 ratios revealed the following hierarchy in regard to the pro-inflammatory potential of human macrophages infected with P. aeruginosa: untreated < treated with GM-CSF < treated with IFN-γ < treated with GM-CSF and IFN-γ.     

Pectin- Derived Acidic Oligosaccharides Improve the Outcome of Pseudomonas aeruginosa Lung Infection in C57BL/6 Mice

The administration of prebiotics as oligosaccharides (OS), by acting on intestinal microbiota, could modulate the immune and inflammatory response and represent a new strategy to improve the outcome of bacterial infection. The aim of this study was to determine whether pectin-derived acidic oligosaccharides (pAOS) could modulate the outcome of pulmonary P. aeruginosa (PA) infection in C57BL/6 mice, which develop a Th1 response to PA lung infection. Mice were randomized for 5 weeks to consume a control or a 5% pAOS diet and chronically infected by PA. Resistance to a second PA infection was also analyzed by reinfecting the surviving mice 2 weeks after the first infection. Compared with control mice, mice fed pAOS had reduced mortality (P<0.05). This improvement correlated with a better control of the inflammatory response with a lower neutrophil count on day 1 (P<0.05), a sustained neutrophil and macrophage recruitment on days 2 and 3 (P<0.01) a greater and sustained IL-10 release in lung (P<0.05) and a reduction of the Th1 response and M1 activation with a lower IFN-γ/IL-4 (P<0.01) and nos2/arg1 (P<0.05) ratios. These results coincided with a modulation of the intestinal microbiota as shown by an increased butyric acid concentration in feces (P<0.05). Moreover, pAOS decreased the bacterial load (P<0.01) in mice reinfected 2 weeks after the first infection, suggesting that pAOS could reduce pulmonary exacerbations. In conclusion, pAOS improved the outcome of PA infection in C57BL/6 mice by modulating the intestinal microbiota and the inflammatory and immune responses.     

The Role of Cryptococcus in the Immune System of Pulmonary Cryptococcosis Patients

Objectives

To investigate the role of Cryptococcus in the immune system of immunocompetent patients with pulmonary cryptococcosis (PC) by analysing the dynamic changes of patients’ immune status before and after antifungal therapy.

Methods

The level of the serum interferon-γ (IFN-γ) and interleukin (IL)-2, -4, -10 and -12 was measured before and after 6-months of treatment. Peripheral blood samples were obtained from 30 immunocompetent PC patients and 30 age- and gender-matched healthy controls. Peripheral blood mononuclear cells (PBMCs) were isolated and incubated with recombinant human IL-12 (rhIL-12) for 48 h. Then the concentrations of IFN-γ and IL-4 in the supernatant were analysed.

Results

Baseline serum IFN-γ level was significantly lower in the PC patients as compared with the control group (P < 0.001). The serum IL-2 and IFN-γ of PC patients were significantly increased after appropriate treatments (P < 0.05 and P < 0.001 when compared to their baseline levels). The productions of IFN-γ in the culture supernatant of PBMCs showed no significant difference between the control and PC patients both before and after antifungal treatments. RhIL-12 is a potent stimulus for IFN-γ production. Culture PBMCs collected from PC patients before treatments had a smaller increase of IFN-γ production in the present of rhIL-12 than the control (P < 0.01); PBMCs from PC patients completing 6-months of treatment showed a comparable increase of IFN-γ production by rhIL-12 stimulation to the control group.

Conclusions

In apparently immunocompetent patients with PC, a normalization of serum IFN-γ was achieved after recovery from infection. This suggests that Cryptococcus infection per se can suppress the immune system and its elimination contributes to the reestablishment of an immune equilibrium.     

Cytokine Profiling in Immigrants with Clinical Malaria after Extended Periods of Interrupted Exposure to Plasmodium falciparum

Immunity to malaria is believed to wane with time in the absence of exposure to Plasmodium falciparum infection, but immunoepidemiological data on longevity of immunity remain controversial. We quantified serum cytokines and chemokines by suspension array technology as potential biomarkers for durability of immunity in immigrants with clinical malaria after years without parasite exposure. These were compared to serum/plasma profiles in naïve adults (travelers) and semi-immune adults under continuous exposure, with malaria, along with immigrant and traveler patients without malaria. Immigrants had higher levels of IL-2, IL-5 and IL-8 compared to semi-immune adults with malaria (P≤0.0200). Time since immigration correlated with increased IL-2 (rho=0.2738P=0.0495) and IFN-γ (rho=0.3044P=0.0282). However, immigrants did not show as high IFN-γ concentrations as travelers during a first malaria episode (P<0.0001). Immigrants and travelers with malaria had higher levels of IFN-γ, IL-6, and IL-10 (P<0.0100) than patients with other diseases, and IL-8 and IL-1β were elevated in immigrants with malaria (P<0.0500). Therefore, malaria patients had a characteristic strong pro-inflammatory/Th1 signature. Upon loss of exposure, control of pro-inflammatory responses and tolerance to P. falciparum appeared to be reduced. Understanding the mechanisms to maintain non-pathogenic effector responses is important to develop new malaria control strategies.     

An important role for A20-binding inhibitor of nuclear factor-kB-1 (ABIN1) in inflammation-mediated endothelial dysfunction: an in vivo study in ABIN1 (D485N) mice

IntroductionThe link between cardiovascular disease (CVD) and patients with chronic inflammation is not clearly understood. We examined a knock-in mouse expressing a poly-ubiquitin-binding-defective mutant of the protein ABIN1 (ABIN1(D485N)), which develops a systemic lupus erythematosus-like autoimmune disease because of the hyperactivation of IκB kinases (IκKs) and mitogen-activated protein kinases (MAPKs). These mice were used to determine the potential role of these signaling pathways in inflammation-mediated CVD development.MethodsLaser Doppler imaging in combination with the iontophoresis of vasoactive chemicals were used to assess endothelium-dependent vasodilatation in vivo in ABIN1 (D485N)) mutant defective (n = 29) and wild-type (WT) control (n = 26) mice. Measurements were made at baseline, and animals were subdivided to receive either chow or a proatherogenic diet for 4 weeks, after which, follow-up assessments were made. Paired and unpaired t tests, and ANOVA with post hoc Bonferroni correction were used for statistical significance at P <0.05.ResultsEndothelium-dependent vasodilatation to acetylcholine was attenuated at 4 weeks in ABIN1(D485N)-chow-fed mice compared with age-matched WT-chow-fed mice (P <0.05). The magnitude of attenuation was similar to that observed in WT-cholesterol-fed animals (versus WT-chow, P <0.01). ABIN1(D485N)-cholesterol-fed mice had the poorest endothelium-dependent responses compared with other groups (P <0.001). ABIN1(D485N)-chow-fed mice had increased plasma interleukin-6 (IL-6) levels (versus WT-chow, P <0.001), and this was further elevated in ABIN1(D485N)-cholesterol-fed mice (versus ABIN1(D485N)-chow; P <0.05). IL-1α was significantly greater in all groups compared with WT-chow (P <0.01). ABIN1(D485N) mice showed significant cardiac hypertrophy (P <0.05).ConclusionsThe ABIN(D485N) mice display endothelial dysfunction and cardiac hypertrophy, which is possibly mediated through IL-6 and, to a lesser degree, IL-1α. These results suggest that the ABIN1-mediated hyperactivation of IKKs and MAPKs might mediate chronic inflammation and CVD development.     

Selenoproteins and heat shock proteins play important roles in immunosuppression in the bursa of Fabricius of chickens with selenium deficiency

Selenium (Se) is necessary for the immune system in chicken and mediates its physiological functions through selenoproteins. Heat shock proteins (Hsps) are indispensable for maintaining normal cell function and for directing the immune response. The aim of the present study was to investigate the effects of Se deficiency on the messenger ribonucleic acid (mRNA) expression levels of selenoproteins and Hsps as well as immune functions in the chicken bursa of Fabricius. Two groups of chickens, namely the control and Se-deficient (L group) groups, were reared for 55 days. The chickens were offered a basal diet, which contained 0.15 mg Se/kg in the diet fed to the control group and 0.033 mg Se/kg in the diet fed to the L group. We performed real-time quantitative polymerase chain reactionto detect the mRNA expression levels of selenoproteins and Hsps on days 15, 25, 35, 45 and 55. Western blotting was used to determine the protein expression levels of Hsps on days 35, 45 and 55, and immune functions were assessed through an enzyme-linked immunosorbent assay on days 15, 35, and 55. The data showed that the mRNA expression levels of selenoproteins, such as Txnrd1, Txnrd2, Txnrd3, Dio1, Dio2, Dio3, GPx1, GPx2, GPx3 GPx4, Sepp1, Selo, Sel-15, Sepx1, Sels, Seli, Selu, Selh, and SPS2, were significantly lower (P < 0.05) in the L group compared with the control group. Additionally, the mRNA and protein expression levels of Hsps (Hsp27, Hsp40, Hsp60, Hsp70, and Hsp90) were also significantly higher (P < 0.05) in the L group. The expression levels of IL-2, IL-6, IL-8, IL-10, IL-17, IL-1β, IFN-α, IFN-β, and IFN-γ were significantly lower (P < 0.05) and TNF-α was significantly higher (P < 0.05) in the L group compared with the control group. Our results show that immunosuppression was accompanied by a downregulation of mRNA expression levels of selenoproteins and an upregulation of the Hsp mRNA expression levels. Thus, Se deficiency causes defects in the chicken bursa of Fabricius, and selenoproteins and Hsps play important roles in immunosuppression in the bursa of Fabricius of chickens with Se deficiency.     

IL-12p40 Deficiency Leads to Uncontrolled Trypanosoma cruzi Dissemination in the Spinal Cord Resulting in Neuronal Death and Motor Dysfunction

Chagas’ disease is a protozoosis caused by Trypanosoma cruzi that frequently shows severe chronic clinical complications of the heart or digestive system. Neurological disorders due to T. cruzi infection are also described in children and immunosuppressed hosts. We have previously reported that IL-12p40 knockout (KO) mice infected with the T. cruzi strain Sylvio X10/4 develop spinal cord neurodegenerative disease. Here, we further characterized neuropathology, parasite burden and inflammatory component associated to the fatal neurological disorder occurring in this mouse model. Forelimb paralysis in infected IL-12p40KO mice was associated with 60% (p<0.05) decrease in spinal cord neuronal density, glutamate accumulation (153%, p<0.05) and strong demyelization in lesion areas, mostly in those showing heavy protein nitrosylation, all denoting a neurotoxic degenerative profile. Quantification of T. cruzi 18S rRNA showed that parasite burden was controlled in the spinal cord of WT mice, decreasing from the fifth week after infection, but progressive parasite dissemination was observed in IL-12p40KO cords concurrent with significant accumulation of the astrocytic marker GFAP (317.0%, p<0.01) and 8-fold increase in macrophages/microglia (p<0.01), 36.3% (p<0.01) of which were infected. Similarly, mRNA levels for CD3, TNF-α, IFN-γ, iNOS, IL-10 and arginase I declined in WT spinal cords about the fourth or fifth week after infection, but kept increasing in IL-12p40KO mice. Interestingly, compared to WT tissue, lower mRNA levels for IFN-γ were observed in the IL-12p40KO spinal cords up to the fourth week of infection. Together the data suggest that impairments of parasite clearance mechanisms in IL-12p40KO mice elicit prolonged spinal cord inflammation that in turn leads to irreversible neurodegenerative lesions.     

Remifentanil up‐regulates HIF1α expression to ameliorate hepatic ischaemia/reperfusion injury via the ZEB1/LIF axis

Ischaemia/reperfusion (I/R)‐induced hepatic injury is regarded as a main reason of hepatic failure after transplantation or lobectomy. The current study aimed to investigate how the opioid analgesic remifentanil treatment affects I/R‐induced hepatic injury and explore the possible mechanisms related to HIF1α. Initially, an I/R‐induced hepatic injury animal model was established in C57BL/6 mice, and an in vitro hypoxia‐reoxygenation model was constructed in NCTC‐1469 cells, followed by remifentanil treatment and HIF1α silencing treatment. The levels of blood glucose, lipids, alanine transaminase (ALT) and aspartate transaminase (AST) in mouse serum were measured using automatic chemistry analyser, while the viability and apoptosis of cells were detected using CCK8 assay and flow cytometry. Our results revealed that mice with I/R‐induced hepatic injury showed higher serum levels of blood glucose, lipids, ALT and AST and leukaemia inhibitory factor (LIF) expression, and lower HIF1α and ZEB1 expression (P < .05), which were reversed after remifentanil treatment (P < .05). Besides, HIF1α silencing increased the serum levels of blood glucose, lipids, ALT and AST (P < .05). Furthermore, hypoxia‐induced NCTC‐1469 cells exhibited decreased HIF1α and ZEB1 expression, reduced cell viability, as well as increased LIF expression and cell apoptosis (P < .05), which were reversed by remifentanil treatment (P < .05). Moreover, HIF1α silencing down‐regulated ZEB1 expression, decreased cell viability, and increased cell apoptosis (P < .05). ZEB1 was identified to bind to the promoter region of LIF and inhibit its expression. In summary, remifentanil protects against hepatic I/R injury through HIF1α and downstream effectors.     

Reovirus Induction of and Sensitivity to Beta Interferon in Cardiac Myocyte Cultures Correlate with Induction of Myocarditis and Are Determined by Viral Core Proteins

Reovirus-induced acute myocarditis in mice serves as a model to investigate non-immune-mediated mechanisms of viral myocarditis. We have used primary cardiac myocyte cultures infected with a large panel of myocarditic and nonmyocarditic reassortant reoviruses to identify determinants of viral myocarditic potential. Here, we report that while both myocarditic and nonmyocarditic reoviruses kill cardiac myocytes, viral myocarditic potential correlates with viral spread through cardiac myocyte cultures and with cumulative cell death. To address the role of secreted interferon (IFN), we added anti-IFN-α/β antibody to infected cardiac myocyte cultures. Antibody benefited nonmyocarditic more than myocarditic virus spread (P < 0.001), and this benefit was associated with the reovirus M1 and L2 genes. There was no benefit for a differentiated skeletal muscle cell line culture (C2C12 cells), suggesting cell type specificity. IFN-β induction in reovirus-infected cardiac myocyte cultures correlated with viral myocarditic potential (P = 0.006) and was associated with the reovirus M1, S2, and L2 genes. Sensitivity to the antiviral effects of IFN-α/β added to cardiac myocyte cultures also correlated with viral myocarditic potential (P = 0.004) and was associated with the same reovirus genes. Several reoviruses induced IFN-β levels discordant with their myocarditic phenotypes, and for those tested, sensitivity to IFN-α/β compensated for the anomalous induction levels. Thus, the combination of induction of and sensitivity to IFN-α/β is a determinant of reovirus myocarditic potential. Finally, a nonmyocarditic reovirus induced cardiac lesions in mice depleted of IFN-α/β, demonstrating that IFN-α/β is a determinant of reovirus-induced myocarditis. This provides the first identification of reovirus genes associated with IFN induction and sensitivity and provides the first evidence that IFN-β can be a determinant of viral myocarditis and reovirus disease.     

IL-17RA Signaling Reduces Inflammation and Mortality during Trypanosoma cruzi Infection by Recruiting Suppressive IL-10-Producing Neutrophils

Members of the IL-17 cytokine family play an important role in protection against pathogens through the induction of different effector mechanisms. We determined that IL-17A, IL-17E and IL-17F are produced during the acute phase of T. cruzi infection. Using IL-17RA knockout (KO) mice, we demonstrate that IL-17RA, the common receptor subunit for many IL-17 family members, is required for host resistance during T. cruzi infection. Furthermore, infected IL-17RA KO mice that lack of response to several IL-17 cytokines showed amplified inflammatory responses with exuberant IFN-γ and TNF production that promoted hepatic damage and mortality. Absence of IL-17RA during T. cruzi infection resulted in reduced CXCL1 and CXCL2 expression in spleen and liver and limited neutrophil recruitment. T. cruzi-stimulated neutrophils secreted IL-10 and showed an IL-10-dependent suppressive phenotype in vitro inhibiting T-cell proliferation and IFN-γ production. Specific depletion of Ly-6G+ neutrophils in vivo during T. cruzi infection raised parasitemia and serum IFN-γ concentration and resulted in increased liver pathology in WT mice and overwhelming wasting disease in IL-17RA KO mice. Adoptively transferred neutrophils were unable to migrate to tissues and to restore resistant phenotype in infected IL-17RA KO mice but migrated to spleen and liver of infected WT mice and downregulated IFN-γ production and increased survival in an IL-10 dependent manner. Our results underscore the role of IL-17RA in the modulation of IFN-γ-mediated inflammatory responses during infections and uncover a previously unrecognized regulatory mechanism that involves the IL-17RA-mediated recruitment of suppressive IL-10-producing neutrophils.     

Direct Transformation of Lung Microenvironment by Interferon-α Treatment Counteracts Growth of Lung Metastasis of Hepatocellular Carcinoma

Background

Interferon (IFN)-α is effective in inhibiting tumor growth and metastasis of hepatocellular carcinoma (HCC). However, the biologic mechanisms of IFN-α treatment in lung metastasis are not yet clear.

Methods

The effect of IFN-α treatment was studied by using an orthotopic xenograft model and measuring tumor size and lung metastasis. Pretreatment with IFN-α before implantation of tumor was done to explore the effect of IFN-α on lung tissues. Cytokines and macrophages were measured by immunohistochemistry and/or PCR assay, using human origin or mouse origin primers to differentiate the sources. Circulating tumor cells (CTCs) were also assayed by flow cytometry.

Results

IFN-α treatment did not decrease the number of CTCs (0.075%±0.020% versus 0.063%±0.018%, P = 0.574, IFN-α–treated versus control groups), but did decrease the number and size of lung metastasis (number: 1.75±1.0 versus 28.0±6.3, P = 0.008; size [pixels]: 116.8±72.2 versus 5226.4±1355.7, P = 0.020), and inhibited macrophage infiltration (0.20%±0.04% versus 1.36%±0.21%, P = 0.0058) and alteration of matrix metalloproteinase (MMP)-9 expression (mean integrated optical density (IOD): 5.1±1.7 versus 21.9±0.4, P<0.000) in the lung, which was independent of the primary tumor.

Conclusion

IFN-α inhibited lung metastasis by directly modulating the lung microenvironment.     

F3MB(PANDER) Decreases Mice Hepatic Triglyceride and Is Associated with Decreased DGAT1 Expression

ObjectivePancreatic-derived factor (PANDER, also named as FAM3B) is secreted by pancreatic α and β cells. Increasing evidence suggests that it may serve a hormonal function related to glycemic and lipid metabolism. In this study, we investigated the effects of PANDER overexpression on hepatic and adipose triglyceride metabolism in high-fat diet-fed male C57BL/6 mice.MethodsPANDER overexpression was achieved by tail-vein injection of recombinant Ad-PANDER and Ad-GFP injected mice served as a control. The TG metabolism in both groups were compared.ResultsAdenoviral-mediated overexpression of PANDER did not affect body weight, food consumption, or liver enzymes. The triglyceride (TG) content of both liver and adipose tissue was significantly decreased in Ad-PANDER mice (liver: 6.16±1.89 mg/g vs. control 14.95±2.27 mg/g, P<0.05; adipose: 39.31±1.99 mg/100mg vs. 47.22±2.21 mg/100mg, P<0.05). The free fatty acid (FFA) content of adipose tissue in Ad-PANDER mice was also decreased (1.38±0.18 mg/g vs. 2.77±0.31 mg/g, P<0.01). The investigation of key enzymes of triglyceride hydrolysis and FFA oxidation in liver and adipose tissue showed that p-HSL/HSL was significantly increased and that DGAT1 gene and protein expression were significantly reduced in the liver of PANDER-overexpressing mice. PKA phosphorylation was also significantly increased in the livers of Ad-PANDER mice. No differences in ATGL, CPT1, ACOX1, or DGAT2 expression were observed.ConclusionOverexpression of PANDER is associated with observable decreases in TG, increases in PKA phosphorylation, and decreased DGAT1 expression, suggesting a possible interrelationship. The mechanisms by which this occurs remain to be elucidated.     

Pituitary Adenylate Cyclase-Activating Polypeptide Ameliorates Experimental Acute Ileitis and Extra-Intestinal Sequelae

Background

The neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) plays pivotal roles in immunity and inflammation. So far, potential immune-modulatory properties of PACAP have not been investigated in experimental ileitis.

Methodology/Principal Findings

Mice were perorally infected with Toxoplasma (T.) gondii to induce acute ileitis (day 0) and treated daily with synthetic PACAP38 from day 1 to 6 post infection (p.i.; prophylaxis) or from day 4 to 6 p.i. (therapy). Whereas placebo-treated control mice suffered from acute ileitis at day 7 p.i. and succumbed to infection, intestinal immunopathology was ameliorated following PACAP prophylaxis. PACAP-treated mice exhibited increased abundance of small intestinal FOXP3+ cells, but lower numbers of ileal T lymphocytes, neutrophils, monocytes and macrophages, which was accompanied by less ileal expression of pro-inflammatory cytokines such as IL-23p19, IL-22, IFN-γ, and MCP-1. Furthermore, PACAP-treated mice displayed higher anti-inflammatory IL-4 concentrations in mesenteric lymph nodes and liver and higher systemic anti-inflammatory IL-10 levels in spleen and serum as compared to control animals at day 7 p.i. Remarkably, PACAP-mediated anti-inflammatory effects could also be observed in extra-intestinal compartments as indicated by reduced pro-inflammatory mediator levels in spleen (TNF-α, nitric oxide) and liver (TNF-α, IFN-γ, MCP-1, IL-6) and less severe histopathological sequelae in lungs and kidneys following prophylactic PACAP treatment. Strikingly, PACAP prolonged survival of T. gondii infected mice in a time-of-treatment dependent manner.

Conclusion/Significance

Synthetic PACAP ameliorates acute small intestinal inflammation and extra-intestinal sequelae by down-regulating Th1-type immunopathology, reducing oxidative stress and up-regulating anti-inflammatory cytokine responses. These findings provide novel potential treatment options of inflammatory bowel diseases.     

Characterization and Clinical Implication of Th1/Th2/Th17 Cytokines Produced from Three-Dimensionally Cultured Tumor Tissues Resected from Breast Cancer Patients

OBJECTIVES: Several cytokines secreted from breast cancer tissues are suggested to be related to disease prognosis. We examined Th1/Th2/Th17 cytokines produced from three-dimensionally cultured breast cancer tissues and related them with patient clinical profiles. METHODS: 21 tumor tissues and 9 normal tissues surgically resected from breast cancer patients were cultured in thermoreversible gelatin polymer–containing medium. Tissue growth and Th1/Th2/Th17 cytokine concentrations in the culture medium were analyzed and were related with hormone receptor expressions and patient clinical profiles. RESULTS: IL-6 and IL-10 were expressed highly in culture medium of both cancer and normal tissues. However, IFN-γ, TNF-α, IL-2, and IL-17A were not detected in the supernatant of the three-dimensionally cultured normal mammary gland and are seemed to be specific to breast cancer tissues. The growth abilities of hormone receptor–negative cancer tissues were significantly higher than those of receptor-positive tissues (P = 0.0383). Cancer tissues of stage ≥ IIB patients expressed significantly higher TNF-α levels as compared with those of patients with stage < IIB (P = 0.0096). CONCLUSIONS: The tumor tissues resected from breast cancer patients can grow in the three-dimensional thermoreversible gelatin polymer culture system and produce Th1/Th2/Th17 cytokines. Hormone receptor–positive cancer tissues showed less growth ability. TNF-α is suggested to be a biomarker for the cancer stage.     

Sex Bias in Experimental Immune-Mediated,Drug-Induced Liver Injury in BALB/c Mice: Suggested Roles for Tregs,Estrogen, and IL-6

Background and Aims

Immune-mediated, drug-induced liver injury (DILI) triggered by drug haptens is more prevalent in women than in men. However, mechanisms responsible for this sex bias are not clear. Immune regulation by CD4+CD25+FoxP3+ regulatory T-cells (Tregs) and 17β-estradiol is crucial in the pathogenesis of sex bias in cancer and autoimmunity. Therefore, we investigated their role in a mouse model of immune-mediated DILI.

Methods

To model DILI, we immunized BALB/c, BALB/cBy, IL-6–deficient, and castrated BALB/c mice with trifluoroacetyl chloride-haptenated liver proteins. We then measured degree of hepatitis, cytokines, antibodies, and Treg and splenocyte function.

Results

BALB/c females developed more severe hepatitis (p<0.01) and produced more pro-inflammatory hepatic cytokines and antibodies (p<0.05) than did males. Castrated males developed more severe hepatitis than did intact males (p<0.001) and females (p<0.05). Splenocytes cultured from female mice exhibited fewer Tregs (p<0.01) and higher IL-1β (p<0.01) and IL-6 (p<0.05) than did those from males. However, Treg function did not differ by sex, as evidenced by absence of sex bias in programmed death receptor-1 and responses to IL-6, anti-IL-10, anti-CD3, and anti-CD28. Diminished hepatitis in IL-6-deficient, anti-IL-6 receptor α-treated, ovariectomized, or male mice; undetectable IL-6 levels in splenocyte supernatants from ovariectomized and male mice; elevated splenic IL-6 and serum estrogen levels in castrated male mice, and IL-6 induction by 17β-estradiol in splenocytes from naïve female mice (p<0.05) suggested that 17β-estradiol may enhance sex bias through IL-6 induction, which subsequently discourages Treg survival. Treg transfer from naïve female mice to those with DILI reduced hepatitis severity and hepatic IL-6.

Conclusions

17β-estradiol and IL-6 may act synergistically to promote sex bias in experimental DILI by reducing Tregs. Modulating Treg numbers may provide a therapeutic approach to DILI.