Neuroprotective effects of CysLT2R antagonist on Angiostrongylus cantonensis-induced edema and meningoencephalitis.

Cysteinyl leukotrienes (CysLTs) can induce a disruption of the blood-brain barrier (BBB), and this reaction is mediated by cysteinyl-leukotriene receptors. In this study, we used A. cantonensis-induced eosinophilic meningoencephalitis as a model to investigate whether the CysLT2 receptor involved in the pathogenesis of angiostrongyliasis meningoencephalitis. The present study provides evidence that the CysLT2 receptor antagonist HAMI3379 reduced the number of infiltrated eosinophils and brain edema in eosinophilic meningoencephalitis. Additionally, we found that HAMI3379 significantly decreased the protein levels of M1 polarisation markers (CD80, iNOS, IL-5 and TNF-α), increased the expression of M2 polarisation markers (CD206, IL-10 and TGF-ß) both in vivo and in vitro. Matrix metalloproteinase-9, S100B, GFAP, fibronectin, and claudin-5 were markedly lower after HAMI3379 treatment. Therefore, HAMI3379 reduced the BBB dysfunction in angiostrongyliasis meningoencephalitis. We have identified microRNA-155 as a BBB dysfunction marker in eosinophilic meningoencephalitis. The results showed that microRNA-155 was 15-fold upregulated in eosinophilic meningoencephalitis and 20-fold upregulated after HAMI3379 treatment. Our results suggest that CysLT2R may be involved in A. cantonensis-induced brain edema and eosinophilic meningoencephalitis and that down-regulation of CysLT2R could be a novel and potential therapeutic strategy for the treatment of angiostrongyliasis meningoencephalitis.

Calycosin attenuates Angiostrongylus cantonensis-induced parasitic meningitis through modulation of HO-1 and NF-κB activation.

Angiostrongylus cantonensis causes a form of parasitic meningitis in humans. Albendazole (ABZ) kills nematode larvae in the brain. However, dead larvae can trigger a severe inflammatory response, resulting in brain damage. Accumulating evidence suggests that calycosin represents a potential anti-inflammatory therapeutic candidate. In this study, we investigated the combined effects of ABZ and calycosin in angiostrongyliasis caused by A. cantonensis in BALB/c mice. Inflammatory mediators (such as phospho-nuclear factor-κB, cyclooxygenase-2, matrix metalloproteinase-9, tumour necrosis factor-α and interleukin-1ß) are associated with the development of meningitis and immune inflammatory reactions. We found that A. cantonensis significantly induces inflammatory mediator production and increases the blood­brain barrier (BBB) permeability. However, co-administration of both ABZ and calycosin markedly suppressed meningitis and inflammatory mediator production and decreased the BBB permeability compared to treatment with a single drug. Furthermore, calycosin and ABZ plus calycosin treatment facilitated production of the antioxidant haem oxygenase-1 (HO-1). Moreover, co-therapy with ABZ and calycosin failed to mitigate angiostrongyliasis in the presence of tin-protoporphyrin IX, an HO-1-specific inhibitor. This finding suggests that the beneficial effects of ABZ plus calycosin treatment on the regulation of inflammation are mediated by the modulation of HO-1 activation. The present results provide new insights into the treatment of human angiostrongyliasis using co-therapy with ABZ and calycosin.

Heme oxygenase-1 modulates brain inflammation and apoptosis in mice with angiostrongyliasis.

The rat nematode lungworm Angiostrongylus cantonensis undergoes obligatory intracerebral migration in its hosts and causes eosinophilic meningitis or meningoencephalitis. Heme oxygenase 1 (HO-1) has several cytoprotective properties such as anti-oxidative, anti-inflammatory, and anti-apoptotic effects. HO-1 in brain tissues was induced in A. cantonensis-infected group and showed positive modulation in cobalt protoporphyrin (CoPP)-treated groups. Assay methods for the therapeutic effect include western blot analysis, enzyme-linked immunosorbent assay, gelatin zymography, blood-brain barrier permeability evaluation and eosinophil count in cerebrospinal fluid. The combination of albendazole (ABZ) and CoPP significantly decreased pro-inflammatory cytokines, tumor necrosis factor-α, interleukin (IL)-1ß, IL-5, and IL-33 but significantly increased anti-inflammatory cytokines IL-10 and transforming growth factor-ß. In addition, worm recovery, matrix metalloproteinase-9, BBB permeability, and eosinophil counts were decreased in the ABZ and CoPP co-treated groups. Induction of HO-1 with CoPP strongly inhibited the protein levels of caspase-3 and increased the induction of annexin-V and B-cell leukemia 2. Thus, co-treatment with ABZ and CoPP prevented A. cantonensis-induced eosinophilic meningoencephalitis and its anti-apoptotic effect by promoting HO-1 signaling prior to BBB dysfunction. HO-1 induction might be a therapeutic modality for eosinophilic meningoencephalitis.

Proteasome serves as pivotal regulator in Angiostrongylus cantonensis-induced eosinophilic meningoencephalitis.

Proteasome primarily degrades the unneeded or damaged proteins by proteolysis. Disruption of the brain barrier and its resulting meningoencephalitis caused by Angiostrongylus cantonensis are important pathological events in non-permissive hosts. In this study, the results showed upregulated proteasome during A. cantonensis infection. Occludin degradation and matrix metalloproteinase-9 (MMP-9) activity were significantly increased in infected mice than in uninfected mice. Moreover, confocal immunoflourescence microscopy showed that occludin was co-localized with MMP-9. The infected-mice were treated with proteasomal activity inhibitor MG132 by 1.5 and 3.0 mg/kg/day, which resulted in significantly reduced protein levels of phosphorylated IκBα (P<0.05) compared with the untreated control. The phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) showed similar result. In addition, MMP-9 activity and occludin degradation were reduced because of MG132 treatment. These results suggested that the proteasome in A. cantonensis infection degraded phosphorylated IκBα, modulated phosphorylated NF-κB, and then regulated the activation of MMP-9 and occludin degradation. Proteasome alterations were presented in eosinophilic meningitis of BALB/c mice and may contribute to the pathophysiology of eosinophilic meningitis by increasing occludin degradation. This molecule would serve as pivotal regulator in A. cantonensis-induced eosinophilic meningoencephalitis.

Matrix metalloproteinase-2 and matrix metalloproteinase-9 in mice with ocular toxocariasis.

In ocular toxocariasis, Toxocara canis-induced inflammatory reaction can lead to eye destruction and granuloma, which is formed by immune cell infiltration and concurrent extensive remodeling tissue. Herein, the histomorphology of granuloma and proteinase production in the eye of T. canis-infected BALB/c mice were investigated. Pathological effects substantially increased after the infection culminated in a severe leukocyte infiltration and granuloma formation from days 4 to 56 post-inoculation. The matrix metalloproteinase (MMP)-2 and MMP-9 activities remarkably increased, compared with those of uninfected control, by gelatin zymography and Western blot analysis in ocular toxocariasis. Granuloma formation had a remarkably positive correlation with MMP-2 and MMP-9 levels. We suggested that T. canis larvae and leukocytes infiltrated from blood vessel both migrated into corpus adiposum orbitae. Activated leukocytes secreted MMP-2 and MMP-9, leading to fibronectin degradation. The imbalance of MMP-2/TIMP-2 and MMP-9/TIMP-1 may play a role in inflammatory cell infiltration and extracellular matrix degradation, forming granuloma, in ophthalmological pathogenesis of T. canis infection.

Resveratrol relieves Angiostrongylus cantonensis - Induced meningoencephalitis by activating sirtuin-1.

Resveratrol, a natural herbal compound found in high levels in grapes and red wine, is frequently used as activator of sirtuin-1. This study investigated the potential function of sirtuin-1 in regulating angiostrongyliasis meningoencephalitis in resveratrol-treated mice. Mice were subjected to meningoencephalitis to study the protective effect of resveratrol against meningoencephalitis and investigate the effects of sirtuin-1 activation on brain. Results demonstrated that sirtuin-1 level decreased in mice with meningoencephalitis and significantly increased in resveratrol-treated mice. Moreover, resveratrol treatment significantly reduced eosinophil counts, p65, Interferon-γ, interleukin (IL)-5, IL-33, and tumor necrosis factor-α levels, matrix metalloproteinase-9 activity, claudin-5 degradation, and blood-brain barrier permeability. By contrast, the anti-inflammatory factor IL-10 was significantly increased in resveratrol-treated mice. Resveratrol treatment was partially beneficial in controlling the pathological processes of angiostrongyliasis meningoencephalitis. The results demonstrate the neuroprotective and anti-inflammatory effects of resveratrol against Angiostrongylus cantonensis-induced eosinophilic meningoencephalitis in mice. Treatment with sirtuin-1 agonist was given within a therapeutic window after A. cantonensis infection.

Naringenin inhibits migration of lung cancer cells via the inhibition of matrix metalloproteinases-2 and -9.

Lung cancer is among the most common causes of cancer-related mortality. It has a high mortality rate and resistance to chemotherapy due to its high metastatic potential. Naringenin, a bioactive compound identified in several fruits, displays anti-inflammatory and antitumor effects. Furthermore, naringenin mitigates the migration of several human cancer cell types. However, the effects of naringenin on lung cancer remain unclear. The current study investigated the mechanisms of naringenin on the migration of lung cancer A549 cells. The results indicate that significant alteration in A549 cell proliferation was observed in response to naringenin (0-300 µM) treatment for 24 and 48 h. Furthermore, a dose-dependent migration inhibition of A549 in the presence of naringenin was observed by healing and transwell migration assays. In addition, a zymography assay revealed that naringenin exhibited a concentration-dependent inhibition of matrix metalloproteinase (MMP)-2 and -9 activities. Furthermore, naringenin also inhibited the activities of AKT in a dose-dependent manner. These observations indicated that naringenin inhibited the migration of lung cancer A549 cells through several mechanisms, including the inhibition of AKT activities and reduction of MMP-2 and -9 activities.

Apoptosis in meningoencephalitis of Angiostrongylus cantonensis-infected mice.

A hallmark of eosinophilic meningoencephalitis is infiltration of leukocytes into brain parenchyma and subarachnoid space infected by Angiostrongylus cantonensis. Apoptosis, a process that eliminates useless cells and counterbalances tissue homeostasis, is important for homeostasis of the immune system. In this study, we investigated the characteristics of cell death induced in BABL/c mice infected with A. cantonensis. We observed increased expression of the apoptotic proteins, caspase-3, caspase-8, caspase-9, and cytochrome c, and decreased expression of anti-apoptotic proteins, B-cell leukemia 2 and inhibitor of apoptosis protein 1. On immunohistochemistry, apoptotic proteins were localized within the leukocytes infiltrate. A terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling assay to detect DNA fragmentation confirmed these observations. The infiltration of leukocytes present in the brain parenchyma and subarachnoid space in vivo may also express these apoptotic regulatory molecules, which demonstrates the capacity of these cells to undergo apoptosis.

Angiostrongylus cantonensis: induction of urokinase-type PA and degradation of type IV collagen in rat lung granulomatous fibrosis.

Granuloma formation and subsequent fibrosis around Angiostrongylus cantonensis larvae in the lungs were induced experimentally in Sprague-Dawley strain rats. Casein zymogram analysis demonstrated that urokinase-type plasminogen activator (uPA) activity was increased during lung inflammation and fibrosis. Granulomatous fibrosis, type IV collagen degradation and activation of uPA occur simultaneously. Furthermore, the present study demonstrated that collagen avidly binds uPA. Immunohistochemical observations showed localization of uPA within the infiltrating leucocytes. We propose that uPA may participate in A. cantonensis-induced granulomatous fibrosis.

Matrix metalloproteinase-2, -9 and -13 are involved in fibronectin degradation of rat lung granulomatous fibrosis caused by Angiostrongylus cantonensis.

Pulmonary granuloma formation and fibrosis were experimentally induced in Sprague-Dawley strain rats by Angiostrongylus cantonensis. Increased protein levels of matrix metalloproteinase (MMP)-2, -9, -13 and the imbalance between these enzymes and metalloproteinase inhibitors, tissue inhibitors of MMPs (TIMP-1 and -2), occur during granulomatous fibrosis. Activation of proteolytic enzymes (MMP-2, -9 and -13) and fibronectin degradation occur simultaneously. Furthermore, the present study demonstrated that fibronectin avidly binds MMP-2, -9 or -13. Immunohistochemical observations also showed the localization of MMP-13, TIMP-1 and -2 within the infiltrating leucocytes. These results suggest that MMP-2, -9 and -13 may participate in the fibronectin degradation of A. cantonensis-induced granulomatous fibrosis.

Biochemical and pathological evaluation of albendazole/thalidomide co-therapy against eosinophilic meningitis or meningoencephalitis induced by Angiostrongylus cantonensis.

OBJECTIVES: To evaluate the curative effect of albendazole/thalidomide co-therapy on eosinophilic meningitis in BALB/c mice caused by Angiostrongylus cantonensis. METHODS: Male mice were infected with 50 A. cantonensis larvae and treated with albendazole (5, 10 or 20 mg/kg per day) alone, thalidomide (25, 50 or 100 mg/kg per day) alone, or a combination of albendazole (10 mg/kg per day) and thalidomide (50 mg/kg per day) for 7 consecutive days on days 5, 10 and 15 post-inoculation (PI), respectively. RESULTS: Indicators used to measure this effect included: (i) worm recovery; (ii) histopathological score of meningitis; (iii) eosinophil counts; (iv) level of pro-inflammatory cytokines, such as tumour necrosis factor-alpha, interleukin-1beta and interleukin-5; (v) activity of enzymes, such as tissue-type plasminogen activator, urokinase-type plasminogen activator and matrix metalloproteinase-9; and (vi) CSF/serum albumin ratio. The results showed that albendazole/thalidomide co-therapy significantly decreased (P < 0.05) these factors when treatment was initiated on days 5 or 10 PI compared with treatment initiated on day 15 PI. CONCLUSIONS: The timing of medication use is important and is closely related to the anthelmintic efficacy of a drug. For a given dosage, earlier medication use is more effective. This novel approach to treating parasitic meningitis may suggest other new methods of treatment.

Chinese herbal medicine Yin-Chen-Extract as an adjunct to anthelmintic albendazole used against Angiostrongylus cantonensis-induced eosinophilic meningitis or meningoencephalitis.

The effects of albendazole and Yin-Chen-Extract co-therapy on Angiostrongylus cantonensis-induced eosinophilic meningitis in BALB/c mice were evaluated. Assay indicators for the therapeutic effect include worm recovery, histopathological score of the fourth ventricle, tissue-type plasminogen activator, urokinase-type plasminogen activator, matrix metalloproteinase-9, cerebrospinal fluid total protein, leukocyte counts, and proinflammatory cytokines. As a result, albendazole and Yin-Chen-Extract co-therapy significantly decreased (P < 0.05) these factors. Although Yin-Chen-Extract may possess pharmacologic activities beneficial to the inhibition of parasite-induced inflammation, many of these hypothetical benefits await scientific testing and confirmation before application. This study suggests that the combination of Yin-Chen-Extract and albendazole has synergistic effects in managing eosinophilic meningitis or eosinophilic meningoencephalitis.

Induction of cysteine proteinase in the encapsulation of Hymenolepis diminuta eggs in the American cockroach, Periplaneta americana.

Haemocytes play an essential role in defending invertebrates against pathogens and parasites that enter their haemocoel. In the present study, the cockroach, Periplaneta americana was able to encapsulate Hymenolepis diminuta eggs within 24 h after injection. Proteolytic activity of egg capsules was determined by gelatin zymography. A gelatinase-type proteolytic enzyme with molecular weight about 65 kDa was present at the time of capsule formation. Enzyme activity was obviously inhibited by leupeptin but not by ethylenediamine tetraacetic acid (EDTA) or 1,10-phenanthroline or phenylmethanesulfonyl fluoride (PMSF). Hence, we tentatively characterized this enzyme as a cysteine proteinase. The specificity of the cellular immune response in vivo and the increased cysteine proteinase activity coincided with the capsule size and encapsulation process. The possible function of this cysteine proteinase activity during encapsulation of the H. diminuta eggs by P. americana is discussed.

Immunolocalization of prophenoloxidase in the process of wound healing in the mosquito Armigeres subalbatus (Diptera: Culicidae).

Hemolymph coagulation began almost immediately after wounding in mosquito, Armigeres subalbatus, (Coquillett) larvae. Immunocytochemical localization showed that prophenoloxidase (pro-PO) was distributed in the wound site. In the initial wounding, coagulation and wound plug formation occurred with granulocyte migration. The hemocytes lysed and released granular materials around the wound site, prophenoloxidase being mostly localized in granules and cuticle. In the second phase of wound healing, melanin accumulation occurred at the wound site along the margin of the cuticle and rapidly increased in thickness. Immunogold-labeled pro-PO was localized in vacuoles, melanins, and cuticle, with the gold particles labeled intensely on the undarkened cuticle and weakly on the darkened cuticle. It is believed that pro-PO is activated upon wound initiation to produce melanin product and deposited on the cuticle. In the final phase of healing, scab melanization and pro-PO immunogold localization were reduced and accompanied by epithelial cell regeneration. This proenzyme was localized in vesicles and flocculent materials, but was absent in the melanized scab. Our results further indicate that pro-PO was present in granules, cuticles, epithelial cells, vacuoles, and flocculent materials but not in melanized scab and coagulated clot. The pro-PO immunogold particles labeled intensely in the initial wounding but weakly in the final phase. Our observations also suggest that pro-PO is released from granulocytes by cell rupture, synthesized or stored in granulocytes, and then is released into the wound site via the cytoplasmic granules. This study indicates that the pro-PO is involved in numerous physiological roles in the process of wound healing in this mosquito.