Matrix metalloproteinases activation in Toxocara canis induced pulmonary pathogenesis.

BACKGROUND: Toxocara canis, a source of visceral larva migrans, causes toxocariasis and induces respiratory symptoms. The reasons by which the pulmonary pathological alteration in the lungs infected with T. canis remain unclear. METHODS: The involvement of the pulmonary pathological alteration by histology, enzyme activity, and Western blot analysis in the lungs of BALB/c mice after the infection of 2000 embryonated eggs. RESULTS: The pathological effects gradually increased after the infection culminated in severe leukocyte infiltration and hemorrhage from days 4-14 post-inoculation. Gelatin zymography using substrate showed that the relative activity of matrix metalloproteinase (MMP) -9 and MMP-2 significantly increased in T. canis-infected mice. Western blot analysis indicated that the MMPs protein level of fibronectin monomer significantly increased in T. canis-infected mice compared with that in uninfected control. T. canis larvae mainly initiated leukocyte infiltration and hemorrhage in the lungs. CONCLUSION: These phenomena subsequently induced the activities of MMPs in parallel with the pathological changes in early stage pulmonary inflammation. In conclusion, T. canis larval migration activated the MMPs and caused pulmonary pathogenesis.

Risk factors for and prevalence of clonorchiasis in Miaoli County, Taiwan.

The objective of this study was to investigate the risk factors for and prevalence of clonorchiasis in Miaoli County, Taiwan. In 2009, 6,929 subjects were randomly selected in Miaoli County and given a questionnaire to fill out regarding risk factors for clonorchiasis; the response rate was 69%. Stool sample was obtained from each participant who filled out the questionnaire and examined using the merthiolate-iodine-formaldehyde concentration (MIFC) technique to determine the presence and concentration of Clonorchis sinensis eggs. Fifty-one subjects gave a history of clonorchiasis (prevalence rate 0.7%). Seven stool samples were positive for C. sinensis (prevalence rate 0.1%). Shihtan Township (5.0%) in Miaoli County had the highest prevalence of clonorchiasis. Using logistic regression, we found people who often fished (OR: 3.65, p=0.013) or who had a family member with a history of clonorchiasis (OR: 18.7, p<0.001) were more likely to have it. We also found tourists who traveled to China and ate fish there (OR: 2.46, p=0.105) or who owned a fish pond (OR: 1.93, p=0.128) were more likely to get clonorchiasis. The prevalence of clonorchiasis in Miaoli County was relatively low, which can be explained by good sanitation and personal hygiene. The Public Health Department of Taiwan should warn Taiwanese travelers about high risk areas for contracting clonorchis infection and encourage these travelers to avoid going fishing or eating raw fish in high risk areas.

Macrophage migration inhibitory factor induces ICAM-1and thrombomobulin expression in vitro.

Macrophage migration inhibitory factor (MIF) is an important cytokine in the modulation of inflammatory and immune responses, but its role in coagulation remains to be elucidated. In this study, we investigated the potential role of MIF in coagulation through its influence on two factors, thrombomodulin (TM) and intercellular adhesion molecule-1 (ICAM-1). Recombinant human MIF was added to human microvascular endothelial cell line (HMEC-1) to investigate its influence on the expression of TM and ICAM-1. The results showed that both TM and ICAM-1 were induced with MIF addition in a dose-dependent and time-dependent manner. The expression of ICAM-1 and TM was increased as MIF doses were increased, with the highest expression seen at 12 hr after 400 ng/ml of MIF treatment. Besides, anti-MIF antibody treatment reduced the TM expression in HMEC-1 cells. In conclusion, our data support a role of MIF as an important factor in the regulation of coagulation.

Curcumin alleviates eosinophilic meningitis through reduction of eosinophil count following albendazole treatment against Angiostrongylus cantonensis in mice.

Angiostrongylus cantonensis (A. cantonensis) is the most common cause of parasitic eosinophilic meningitis worldwide. By using an animal model of BALB/c mice infected with A. cantonensis, previous studies indicated that the anthelmintic drug, albendazole, could kill A. cantonensis larvae and prevent further infection. However, the dead larvae will induce severe immune responses targeting at brain tissues. To alleviate the detrimental effects caused by the dead larvae, we administered curcumin, a traditional anti-inflammatory agent, as a complementary treatment in addition to albendazole therapy, to determine whether curcumin could be beneficial for treatment. The results showed that although curcumin treatment alone did not reduce worm number, combined treatment by albendazole and curcumin helped to reduce eosinophil count in the cerebrospinal fluid, better than using albendazole alone. This alleviating effect did not affect albendazole treatment alone, since histological analysis showed similar worm eradication with or without addition of curcumin. Nevertheless, curcumin treatment alone and combined albendazole-curcumin treatment did not inhibit MMP-9 expression in the brain tissue. In conclusion, curcumin, when used as a complementary treatment to albendazole, could help to alleviate eosinophilic meningitis through suppression of eosinophil count in the cerebrospinal fluid.

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.

Association of plasminogen activators and matrix metalloproteinase-9 proteolytic cascade with blood-CNS barrier damage of angiostrongyliasis.

Blood-central nervous system (blood-CNS) barrier breakdown, an important pathophysiological event in meningitis, results in extravasation of leucocytes into subarachnoid space. The blood-CNS barrier disruption is mediated by primarily two enzyme systems, the plasminogen activators (PAs) and matrix metalloproteinases (MMPs). The present study showed that the activities of tissue-type PA (tPA), urokinase-type activator (uPA) and MMP-9 in cerebrospinal-like fluid (CSF-like fluid) were significantly increased in mice with eosinophilic meningitis compared with uninfected mice. Eosinophilia significantly correlated with tPA, uPA and MMP-9 activities, and albumin concentration. In addition, when GM6001, a specific matrix metalloproteinase blocker, was injected into infected mice, MMP-9 activity and total protein concentrations declined from their preinjection highs. These results suggest that the PAs and MMP-9 proteolytic cascade may be associated with blood-CNS barrier disruption in eosinophilic meningitis caused by Angiostrongylus cantonensis.

Upregulation of MMP-9/TIMP-1 enzymatic system in eosinophilic meningitis caused by Angiostrongylus cantonensis.

Proteolysis depends on the balance between the proteases and their inhibitors. Matrix metalloproteinase-9 (MMP-9) and its specific inhibitors, tissue inhibitors of metalloproteinases (TIMP), contribute to eosinophilic inflammatory reaction in the subarachnoid space of the Angiostrongylus cantonensis-infected mice. The expression of MMP-9 in cerebrospinal fluid (CSF) was significantly increased in mice with eosinophilic meningitis, compared to that in uninfected ones. However, the TIMP-1 levels were unchanged and remained at basal levels at all time points, even in uninfected mice. Elevated MMP-9 mRNA expression coincided with protein levels and proteolytic activity, as demonstrated by means of positive immunoreactivity and gelatin zymography. CSF protein contents correlated significantly with MMP-9 intensity and CSF eosinophilia. In addition, immunohistochemistry demonstrated MMP-9 and TIMP-1 localization in eosinophils and macrophages. When the specific MMP inhibitor, GM6001, was added, MMP-9 enzyme activity was reduced by 45.4%. The percentage of eosinophil increased significantly upon the establishment of infection, but subsided upon inhibition. These results show that MMP-9/TIMP-1 imbalance in angiostrongyliasis may be associated with eosinophilic meningitis.

Elevation of plasminogen activators in cerebrospinal fluid of mice with eosinophilic meningitis caused by Angiostrongylus cantonensis.

A hallmark of parasitic meningitis is the infiltration of eosinophils into the subarachnoid space. Infection with Angiostrongylus cantonensis in mice induced proteinase activity in parallel with the pathological changes of eosinophilic meningitis. Zymogram analysis demonstrated that 70 and 55 kDa proteinases from cerebrospinal fluid (CSF) were active against the casein/plasminogen substrate. The proteinase activities were clearly inhibited by phenylmethanesulphonyl fluoride but not by ethylenediamine tetraacetic acid, 1,10-phenanthroline or leupeptin. Western blotting confirmed these enzymes to be tissue-type plasminogen activator and urokinase-type plasminogen activator, respectively. High activities of tissue-type plasminogen activator and urokinase-type plasminogen activator were detected in the CSF of mice with eosinophilic meningitis, and correlated positively with CSF eosinophil numbers and total protein, respectively. Immunohistochemistry demonstrated that tissue-type plasminogen activator and urokinase-type plasminogen activator localised in the endothelial cells of blood vessels, in blood clots and in infiltrated leukocytes. These results suggest that tissue-type plasminogen activator and urokinase-type plasminogen activator may be play a role in the pathogenesis of eosinophilic meningitis of angiostrongyliasis.

Association of matrix metalloproteinase-9 and Purkinje cell degeneration in mouse cerebellum caused by Angiostrongylus cantonensis.

Angiostrongylosis is a neurological disorder caused by invasion of the central nervous system by developing larvae of Angiostrongylus cantonensis. Purkinje cells in infected mouse cerebellums are small and irregular with degenerative atrophy or partial loss. Ultrastructural changes in degenerative cells included enlarged vacuolar structures and swollen mitochondria within the cytoplasm. The matrix metalloproteinase-9 mRNA which is low in normal cerebellums was expressed in A. cantonensis-infected mice cerebellum prior to Purkinje cell degeneration. Matrix metalloproteinase-9 protein level and enzyme activity increased when the Purkinje cells appeared degenerated. Using immunohistochemistry, matrix metalloproteinase-9 was localised within degenerative Purkinje cells. In addition, when the specific matrix metalloproteinase inhibitor, GM6001, was added, matrix metalloproteinase-9 enzyme activity was reduced by 41.6%. The numbers of degenerative Purkinje cells increased significantly upon establishment of infection but subsided upon inhibition. These results suggested that the expression of matrix metalloproteinase-9 may be associated with degeneration of Purkinje cells in mouse cerebellum infected by A. cantonensis.