Ex vivo Immuno-modulatory effect of Echinococcus granulosus laminated layer during allergic rhinitis and allergic asthma: A study in Algerian Patients.

The effect of helminthic infections on allergic diseases and asthma is still inconclusive. Moreover, there is considerable evidence suggesting that nitric oxide (NO), metalloproteinases and pro-inflammatory cytokines play a significant role in the physiopathology of these diseases. In this sense, the aim of our study is to investigate the ex vivo immunomodulatory effect of the laminated layer (LL, outside layer of parasitic cyst) of the helminth Echinococcus granulosus on NO, IL-17A and IL-10 production. In the first step of our study, we evaluated in vivo the NO, MMP-9, IL-17A, IL-10 levels in Algerian patients with allergic asthma and allergic rhinitis and their changes in relation with exacerbation status of the patients. In the principal part of our work, we assessed NO, IL-10 and IL-17A levels in supernatants of patients PBMC cultures before and after stimulation with LL. Our results indicate a significant reduction in NO production by PBMC of patients with allergic rhinitis and allergic asthma whether mild, moderate or severe after stimulation with LL. Interestingly, LL induces a significant decrease in the production of NO and IL17-A levels as well as an increase in the production of IL-10 in the cultures performed with PBMC of patients with severe allergic asthma. Importantly, our data indicate that LL exert a down-modulatory effect on inflammatory mediators (NO, IL-17A) and up immune-regulatory effect on IL-10 production. Collectively, our study supports the hygiene hypothesis suggesting that Echinococcus granulosus infection like other helminths could prevent and/or modulate inflammation responses during inflammatory diseases.

Protoscolicidal activity of Atriplex halimus leaves extract against Echinococcus granulosus protoscoleces.

Cystic echinococcosis, an endemic zoonosis in Algeria, is caused by the development of the helminth Echinococcus granulosus. Surgery remains the main treatment despite inducing relapse and several adverse reactions. In this context, natural scolicidal agents seem to be promising tools to overcome these reactions. In our study, we evaluated the phytochemical contents, antioxidant activity and scolicidal effect of Atriplex halimus. In this context, the aqueous extract from AH leaves (AHE) was subjected to preliminary phytochemical screening by HPLC. The in vitro antioxidant activity was determined by DPPH test. The cytotoxicity of AHE was evaluated in murine peritoneal macrophages and cell viability was examined by MTT assay. Moreover, different concentrations of AHE (20, 40, 50, 60 and 100 mg/ml) were tested on E. granulosus protoscoleces (PSC) cultures, during different times of incubation (15, 30, 60, 90, 120 and 180 min). The viability was evaluated by eosin exclusion test. The morphological and ultrastructural damages were evaluated by SEM. Our results indicate that total phenolic and flavonoids contents were 37.93 µg of Gallic acid equivalent per mg of extract (GAE/mg E) and 18.86 µg of Quercetin equivalent per mg (QE/mg E) respectively. Furthermore, AHE has an antioxidant activity with an IC50 of 0.95 mg/ml. Interestingly, the extracts did not exhibit any cytotoxic effect against murine peritoneal macrophages. Moreover, our study indicated a significant scolicidal activity time- and dose-dependent. At 60 and 100 mg/ml; and after 120 min of incubation; the mortality rate was 99.36 and 100%, respectively. The parasite's tegument is one of the plant's targets as demonstrated by SEM. Our findings show the benefits of Atriplex halimus extract as a new promising scolicidal tool in hydatid cyst treatment.

Beneficial role of Pistacia lentiscus aqueous extract in experimental colitis: anti-inflammatory and potential therapeutic effects.

BACKGROUND: Pistacia lentiscus L. (PL) is a flowering plant traditionally used in the treatment of gastrointestinal disorders. The extracts of this plant are endowed with strong pharmacological activities. The aim of our current study was to investigate the anti-inflammatory and potential therapeutic effects of PL leaves aqueous extract (PLAE) against Dextran Sulfate Sodium (DSS)-induced acute colitis. MATERIALS AND METHODS: The therapeutic effect of PLAE was evaluated after orally administration of 3% DSS alone or concomitantly with PLAE (50, 100 or 200 mg/Kg). Mucosal lesions were assessed by macroscopic and histopathological examination. In this context, hemorrhage, diarrhea, weight loss, and disease activity index (DAI) were determined daily throughout the experiment. In the same way, hematoxylin-eosin and Alcian blue staining of colonic mucosal were used to evaluate, respectively, mucosal damages and mucus production. Furthermore, the levels of nitric oxide (NO), and pro-inflammatory cytokines [tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)] were measured in plasma, as well as in colonic explants and peritoneal macrophages cultures supernatants. RESULTS: Administration of DSS + PLAE indicated a significant reduction in clinical score of acute colitis DAI compared to DSS alone administration. Interestingly, histological analysis of the mucosa showed that DSS + PLAE-treated groups exhibited almost normal histology evidenced by an intact epithelium structure and less inflammatory cell infiltration in the mucosa. Alcian bleu staining revealed that DSS + PLAE-treated groups displayed almost normal mucus production. Importantly, a significant decrease in pro-inflammatory mediators (NO, IL-6 and TNF-α) levels in dose-dependent manner was reported in plasma, and culture supernatants of colonic explants and peritoneal macrophages from DSS + PLAE-treated mice compared to the DSS group. CONCLUSION: Our results showed that the systemic and local anti-inflammatory activities of aqueous leaves extract of PL improve the clinical signs of acute colitis. Our data suggest that PLAE has beneficial effects and could constitute a promising approach against acute ulcerative colitis by targeting the deregulated immune response.

Comparative study of nitric oxide (NO) production during human hydatidosis: relationship with cystic fluid fertility.

Human hydatidosis is characterized by a prolonged coexistence of Echinococcus granulosus and its host without effective rejection of the parasite. This parasitic infection constitutes a major health problem in Algeria. In this study, we investigated in vivo production of nitrite (NO(2)(-) + NO(3)(-)) in sera of Algerian patients carrying different cyst locations. Nitrite (NO(2)(-) + NO(3)(-)) levels were evaluated by the Griess method. Our results indicated that the levels of nitrite were significantly higher in the sera of hydatic patients than those of healthy controls supporting the involvement of nitric oxide (NO) in antihydatic action. The levels of nitrite in sera of the patients with hepatic hydatidosis were significantly higher than those with pulmonary infection. The lower serum (NO(2)(-) + NO(3)(-)) levels were observed in the relapsing cases. In addition, (NO(2)(-) + NO(3)(-)) levels of fertile hydatic fluids were significantly higher compared to infertile fluids. Our results suggest that the presence of NO products in hydatic fluids seems to be related to the location and the fertility of hydatic cysts. The assessment of protein concentration in hydatic fluids showed that the concentration of proteins was not exclusively dependent on the fertility but on the cyst locations. The assessment of (NO(2)(-) + NO(3)(-)) production in hydatic patients may be a useful tool to evaluate effector mechanisms of NO and clinical manifestations.

Increased Salivary Cytokines and Nitric Oxide Levels in Behçet's Disease: Interleukin-32, a Novel Player in Disease Prognosis.

BACKGROUND: Oral aphthosis is one of the major manifestations of Behçet's disease (BD), a chronic, multisystemic vasculitis. BD etio-pathogenicity related to oral health lack. OBJECTIVE: This study investigated the possible relationships between poor oral hygiene, oral activity, disease severity and saliva's Interleukin (IL)-32, IL-6, IL-10 and nitric oxide (NO) levels in Behçet's patients to determine their role in disease prognosis and their potential therapeutic interest. METHODS: Fifty-six patients with BD (22 orally active; 34 orally inactive) and 31 healthy subjects have been included in our study. Salivary levels of IL-32, IL-6, and IL-10 were measured using ELISA, while NO levels were assessed by modified Griess's method. Oral health status and disease severity scores were recorded for each participant. Kruskal-Wallis test and Spearman's test were performed for statistical analyses. RESULTS: We observed that the tested molecules were increased in BD patients compared to healthy controls (p˂0.05). Moreover, only IL-32 levels were associated with oral activity in patients (p˂0.05). Interestingly, the disease severity score was noted to be correlated positively and significantly with both IL-32 saliva levels (p˂0.01) and plaque index (p˂0.05) in BD patients. Furthermore, IL-32 levels were correlated with plaque index (p˂0.0001). CONCLUSION: Our results suggest that IL-32, IL- 6, IL-10 and NO were increased in saliva during BD. Our study indicated that IL-32 was associated with the genesis of oral ulcers in response to dental plaque. Ultimately, salivary IL-32 may serve as a prognostic biomarker and a possible therapeutic target for managing Behçet's disease severity.

Nitric Oxide, Neutrophil/Lymphocyte, and Platelet/Lymphocyte Ratios as Promising Inflammatory Biomarkers in Complicated Crohn's Disease: Outcomes of Corticosteroids and Anti-TNF-α Therapies.

Crohn's disease (CD) is a relapsing-remitting inflammatory bowel disease with a progressive course. The aim of our study was to evaluate the relationship between nitric oxide (NO), pro-inflammatory cytokines, and blood count-based ratios in patients with complicated Crohn's disease as well as the outcome of corticosteroid or anti-TNF-α therapy. In this context, we evaluated the NLR as the ratio of neutrophils count to lymphocytes count, PLR as the ratio of platelets count to lymphocytes count, and MLR as the ratio of monocytes count to lymphocytes count in patients and controls. Furthermore, we assessed NO production by the Griess method in plasma along with iNOS and NF-κB expression by immunofluorescence method in intestinal tissues of patients and controls. In the same way, we evaluated plasma TNF-α, IL-17A, and IL-10 levels using ELISA. Our results indicate that blood count-based ratios NLR, PLR, and MLR were significantly higher in patients compared to controls. In addition, increased systemic levels of NO, TNF-α, and IL-17A and colonic expression of iNOS and NF-κB were observed in the same patients. Interestingly, the high ratio of NLR and MLR as well as NO production were significantly decreased in treated patients. Collectively, our findings suggest that nitric oxide as well as the blood count-based ratios (NLR, PLR, MLR) could constitute useful biomarkers in complicated Crohn's disease, predicting the response to treatments.

In vitro immunoregulatory activity and anti-inflammatory effect of Echinococcus granulosus laminated layer.

The Laminated layer of Echinococcus granulosus (LL) is the outer layer of the hydatic cyst. It plays a pivotal role in protecting the metacestode from host immunity. In our current study, we investigated the immunomodulatory effect of the LL on mouse spleen cells in presence of Lipopolysaccharide (LPS). Mouse spleen cells were cultured with or without LL in presence of LPS. After 24 h, the nitrites level representative of Nitric oxide (NO) production was measured in the culture supernatant by Griess-modified method. In addition, the mRNA expression levels of cytokines (IFN-γ, IL-1ß, TGF-ß, IL-10), Foxp3, and CTLA-4 were measured by quantitative Real-Time Polymerase chain reaction (qRT-PCR). Interestingly, our results showed a significant decrease (p< 0.01) in NO production and IFN-γ mRNA level (p< 0.001) from LPS- induced spleen cells in response to LL after 24h of culture. Moreover, LPS induced high level of IL-1ß that was significantly (p<0.05) down regulated by LL. Importantly, mRNA levels of TGF-ß (p< 0.01), Foxp3 and IL-10 (p< 0.05) were significantly upregulated by LL. In conclusion, our data indicated the in vitro immuno-regulatory and anti-inflammatory effects of the hydatic Laminated Layer on mouse spleen cells. These effects are related to an innate response implicating up-regulation of Foxp3, IL-10 and TGF-ß expression and down-regulation of IFN-γ and IL-1ß expression. LL could constitute a potential candidate for controlling inflammation during inflammatory disease.

Gastro-protective, therapeutic and anti-inflammatory activities of Pistacia lentiscus L. fatty oil against ethanol-induced gastric ulcers in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Pistacia lentiscus L. (Anacardiaceae) (PL) is a flowering plant that grows in the Mediterranean area. It is traditionally used in the treatment of various skin, respiratory and gastrointestinal disorders AIM OF THE STUDY: In the present study, we investigated the anti-ulcerogenic activity of Pistacia lentiscus fatty oil (PLFO) on ethanol-induced gastric ulcers in Wistar rats MATERIAL AND METHODS: PLFO was orally administered to two experimental groups of rats before or after ethanol induction of gastric ulcer. The lesions of the gastric mucosa were evaluated by macroscopic and histopathological examination. In addition, the amount of nitric oxide (NO) and pro-inflammatory cytokines [tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)] in the supernatant from cultures of gastric mucosa explants were assessed. Finally, the mucus production and iNOS (inducible NO synthase) expression were determined by histochemical and immunohistochemical analysis, respectively RESULT: Our results indicated that the PLFO pretreatment or PLFO treatment significantly reduced ulcerated and hemorrhagic areas. Additionally, pretreatment or treatment with PLFO after ethanol-induced ulceration significantly reduced the plasma concentration of NO. Furthermore, a significant decrease of NO, IL-6 and TNF-α levels was observed in explant culture supernatants. iNOS expression was also reduced in the gastric mucosa. In contrast, mucus production by goblet cells was enhanced. Interestingly, histological analysis of the gastric mucosa has indicated that PLFO- pretreated and treated groups displayed normal histology CONCLUSION: Our results demonstrate that PLFO display significant prophylactic and therapeutic effects against gastric ulcers. Importantly, the mechanism underlying PLFO activities might implicate inhibition of inflammatory responses during gastric ulcer.

The involvement of neuroinflammation and necroptosis in the hippocampus during vascular dementia.

The prevalence of vascular dementia is increasing at an alarming rate. The Confirmation of the clinical diagnosis of vascular dementia depends on post-mortem examination of the brain. In our study, we investigated the vascular disease and neuroinflammation during vascular dementia. Our results showed a ß-amyloid deposits, neovascularization, neuronal hypertrophy and neuroinflammation in the hippocampus tissue. Interestingly, the neuroinflammation was characterized by a higher expression of TNF-α, IL-1ß, TGF-ß and iNOS which are TLR4/RelA pathway dependent. Finally, the finding of necroptosis by impaired blood supply and inflammation state suggests that the cognitive impairment was caused by vascular disease and neuroinflammation.

In vitro antihydatic action of IFN-gamma is dependent on the nitric oxide pathway.

Hydatidosis is a widely endemic helminthic disease vectored in human by the larval stage of the metacestode Echinococcus granulosus. It is characterized by the long-term coexistence of chronic infection with detectable humoral and cellular responses against the macroparasite. Previous studies demonstrated interferon-gamma (IFN-gamma) and nitric oxide (NO) production (in vivo and in vitro) during hydatidosis. In this study, we tested the hypothesis that NO production after IFN-gamma induction may constitute a host defense against E. granulosus. We also investigated the IFN-gamma effect on protoscolices (larval form of the parasite) viability in coculture with hydatid patients' peripheral blood mononuclear cells (PBMC). PBMCs from hydatic patients incubated with IFN-gamma (100 U/mL) alone are effective in the killing of protoscolices. This scolicidal activity is concomitant with elevation of nitrite levels. NO release and cytotoxic activity are inhibited by N-monomethyl-L-arginine (L-NMMA), a specific inhibitor of the NO pathway and increased by L-arginine, an NO precursor, and tetrahydrobiopterin (BH4), a nitric oxide synthase (NOS) cofactor. Our results indicate that IFN-gamma mediated iNOS induction as one of host defense mechanism against human E. granulosus infection.

In vitro anti-hydatic and immunomodulatory effects of ginger and [6]-gingerol.

OBJECTIVE: To study in vitro anti-hydatic and immunomodulatory effects of ginger and [6]-gingerol as an alternative therapy for cystic echinococcosis. METHODS: Effect of a commonly used herbal product and ginger (Zingiber officinale) towards protoscoleces (PSC) and cyst wall in vitro was studied. The effect of [6]-gingerol, and the pungent constituent of ginger, was also evaluated on PSC culture. Furthermore, the activity of both extracts in association with interferon-gamma (IFN-γ) on PSC co-cultured with mononuclear cells of hydatic patients was evaluated. The nitric oxide (NO) production was measured in each co-culture. RESULTS: Ginger exhibited a concentration- and time-dependent cytotoxic effect against PSC and cyst wall. Interestingly, ginger was more effective than the [6]-gingerol. Moreover, additional parasitic effect between extracts and IFN-γ are also observed in co-cultures. Furthermore, both extracts attenuated the NO production elicited by this infection or by the IFN-γ. CONCLUSIONS: Ginger has an important anti-hydatic effect in vitro. This effect is amplified in the presence of IFN-γ. Moreover, this herbal product may protect against host's cell death by reducing the high levels of NO. Ginger may act, at least, through the [6]-gingerol. All our data suggest the promising use of ginger in the treatment of Echinococcus granulosus infection.

A protective effect of the laminated layer on Echinococcus granulosus survival dependent on upregulation of host arginase.

The role of nitric oxide (NO) in host defense against Echinococcus granulosus larvae was previously reported. However, NO production by NOS2 (inducible NO synthase) is counteracted by the expression of Arginase. In the present study, our aim is to evaluate the involvement of the laminated layer (external layer of parasitic cyst) in Arginase induction and the protoscoleces (living and infective part of the cyst) survival. Our in vitro results indicate that this cystic compound increases the Arginase activity in macrophages. Moreover, C-type lectin receptors (CLRs) with specificity for mannan and the TGF-ß are implicated in this effect as shown after adding Mannan and Anti-TGFß. Interestingly, the laminated layer increases protoscoleces survival in macrophages-parasite co-cultures. Our results indicate that the laminated layer protects E. granulosus against the NOS2 protective response through Arginase pathway, a hallmark of M2 macrophages.

Crude extract of hydatid laminated layer from Echinococcus granulosus cyst attenuates mucosal intestinal damage and inflammatory responses in Dextran Sulfate Sodium induced colitis in mice.

BACKGROUND: Inflammatory bowel disease is an immunologically mediated disease. Notably, it is less common in countries where there is a greater risk of exposure to helminths. In our study, we examined the modulatory effect of the laminated layer extracted from the cyst wall of a helminth parasite, Echinococcus granulosus, on dextran sulfate sodium (DSS)-induced colitis in mice. METHODS: An acute colitis was induced in BALB/c mice using 2.5% w/v DSS in drinking water. The crude extract of E. granulosus laminated layer was injected intraperitoneally daily, starting 3 days before colitis induction. The Disease Activity Index was monitored daily, colon length and weight were measured and histological scores were evaluated. Nitric oxide (NO) and cytokine levels (interferon γ (IFN-γ), tumor necrosis factor α (TNF-α) and interleukin 10 (IL-10)) were assessed by enzyme-linked immunosorbent assay. In addition, the colonic expression of inducible nitric oxide synthase (iNOS) and nuclear factor-κB (NF-κB) was examined. Statistical analyses were performed by one-way analysis of variance and the survival rate was analyzed by the long rank test. RESULTS: Hydatid laminated layer pretreatment significantly improved the clinical symptoms and histological scores (*** p < 0.01) observed during DSS-induced colitis and maintained mucus production by goblet cells. Furthermore, treatment with hydatid laminated layer caused a significant decrease in NO, IFN-γ (** p < 0.01) and TNF-α production (* p < 0.05) and an increase in IL-10 production. These results were associated with localized downregulation of iNOS and NF-κB expression. CONCLUSIONS: Our results demonstrate the potent anti-inflammatory effects of hydatid laminated layer. Furthermore, preventive treatment with the laminated layer played a beneficial role in maintaining the integrity of the intestinal mucosal barrier against DSS-induced injury.

Involvement of interferon-γ in bowel disease pathogenesis by nitric oxide pathway: a study in Algerian patients.

The pathogenesis of inflammatory bowel disease (IBD) is complex, involving a wide range of molecules including cytokines. Abnormalities in the expression of immunoregulatory cytokines such as interferon-γ (IFN-γ) and interleukin-12 (IL-12) may indicate a dysregulation of intestinal immunity probably associated with pathogenic events. The aim of this work was to study the implication of IFN-γ and nitric oxide (NO) in bowel disease pathogenesis. In this study, we investigated the circulating IFN-γ and IL-12 production in 2 groups of Algerian patients with IBD (Crohn's disease and ulcerative colitis). Moreover, systemic NO concentrations and NO generation by colonic mucosa were determined in these patients. Finally, we examined the effect of IFN-γ on NO production by peripheral blood mononuclear cells (PBMCs) of these patients. Our results indicate that IFN-γ/IL-12 production in IBD patients was increased in comparison to healthy donors. This strong production correlates with high levels of NO in sera and colonic mucosa culture. Interestingly, NO production was related to the clinical stage of IBD patients (inactive or active stage). The relationship between IFN-γ and NO production in IBD patients were confirmed by in vitro experiments and the role of IFN-γ in NO synthase induction in patients' PBMC culture was suggested. Collectively, our results show that IFN-γ plays a pivotal role in IBD pathogenesis through NO pathway.

Involvement of IL-10 and IL-4 in evasion strategies of Echinococcus granulosus to host immune response.

Human echinococcosis is one of the world's major zoonotic infections. In this study, our aim is to clarify one of the strategies used by the parasite for evasion and prolonged infestation in the host. We wished to provide further immunological evidence for the involvement of IL-10/IL-4 in these mechanisms. In this regard, we investigated the effects of IL-4 and IL-10 on protoscoleces (PSC: the larval form of the parasite), co-cultured with patient PBMC. Furthermore, we used IL-4 and IL-10 antibodies to confirm this effect. Our results showed that IL-4 and IL-10 reduced PSC killing. This reduction correlates with an decrease in NO production by PBMC. In conclusion, the results reported here suggest that IL-4/IL-10 impairs the Th1 protective response and allows the parasite to survive in hydatid patients.

In Vitro Study of Nitric Oxide Metabolites Effects on Human Hydatid of Echinococcus granulosus.

Hydatidosis is characterized by the long-term coexistence of larva Echinococcus granulosus and its host without effective rejection. Previous studies demonstrated nitric oxide (NO) production (in vivo and in vitro) during hydatidosis. In this study, we investigated the direct in vitro effects of NO species: nitrite (NO(2) (-)), nitrate (NO(3) (-)) and peroxynitrite (ONOO(-)) on protoscolices (PSCs) viability and hydatid cyst layers integrity for 24 hours and 48 hours. Our results showed protoscolicidal activity of NO(2) (-) and ONOO(-) 24 hours and 3 hours after treatment with 320 muM and 80 muM respectively. Degenerative effects were observed on germinal and laminated layers. The comparison of the in vitro effects of NO species on the PSCs viability indicated that ONOO(-) is more cytotoxic than NO(2) (-). In contrast, NO(3) (-) has no effect. These results suggest possible involvement of NO(2) (-) and ONOO(-) in antihydatic action and point the efficacy of these metabolites as scolicidal agents.

In vivo IL-12 and IL-8 production in hydatic patients and their possible diffusion in hydatid cysts.

Cystic echinococcosis (CE) is caused by infection with the larval stage of the cestode Echinococcus granulosus. It is one of the world's major zoonotic infections. Variability and severity of clinical expression of this parasitosis are associated with duration and intensity of infection. They are also related to the variety of human immunological responses to the hydatic antigens. The aim of this work is to study the inflammatory response associated with human hydatidosis by evaluating the possible roles of the proinflammatory cytokines in hydatic patients. We investigated the patterns of IL-12 and IL-8 in serum from Algerian hydatic patients. Serum IL-12 and IL-8 levels are significantly higher in patients with hydatidosis than in control subjects. Furthermore, cytokines secretion correlates with disease statues (cystic localizations and clinical stage). These data indicate that infection with E. granulosus is associated with high levels of circulating IL-12 and IL-8. Moreover, our data, to our knowledge, constitute the first report of IL-12 and IL-8 diffusion into the hydatid cyst. Our results underline the permeability of the cyst wall to the soluble immune system of the host. The relationship between cyst fertility and cytokine infiltration indicates a strong host-parasite interaction. All these findings have important implications for the diagnosis of hydatidosis in humans.