Immunomodulatory action of excretory-secretory products of Angiostrongylus cantonensis in a mouse tumour model.

Excretory-secretory products (ESPs) of parasitic helminths are well known to exert immunostimulation and immunomodulation in hosts. Immune regulation plays a key role in anti-tumour therapy. The present study explored the anti-tumour effect of ESPs released by Angiostrongylus cantonensis. In Hepa1-6 mouse tumour models, ESPs significantly reduced tumour growth. Tumour-bearing mice treated with ESPs had significantly higher CD3+, CD4+, and CD8+ T cell counts than those treated with Freund's adjuvant. In vitro, human hepatocarcinoma HepG2 cells, human lung cancer A549 cells, and normal human liver HL-7702 cells were co-incubated with ESPs for 24 h and 48 h. ESPs significantly accelerated HepG2 apoptosis but had no inhibitory effect on the proliferation of A549 and HL-7702 cells. Apoptotic HepG2 cells displayed condensed nuclei, apoptotic bodies, and swollen endoplasmic reticulum (ER). Expression of the endoplasmic reticulum stress (ERS)-related factors activating transcription factor 6 (ATF6) and C/EBP-homologous protein (CHOP) in HepG2 cells increased with increasing ESP concentration and treatment time. Calreticulin (CRT) is a key effector protein of ESPs, and recombinant calreticulin (rCRT) was produced in BL21 Escherichia coli (E. coli). In contrast to ESPs, rCRT markedly reduced the proliferation of HepG2 cells. The expression levels of ATF6 and CHOP in HepG2 cells treated with 30 µg/mL rCRT significantly increased at 48 h. Notably, these findings synergistically suggest that ESPs and rCRT are promising candidates for anti-tumour immunotherapy.

Tanshinone IIA attenuates demyelination and promotes remyelination in A. cantonensis-infected BALB/c mice.

BACKGROUND: Angiostrongylus cantonensis infection can cause demyelination in the central nervous system, and there is no effective treatment. METHODS: We used dexamethasone, Tanshinone IIA (TSIIA) and Cryptotanshinone(Two traditional Chinese medicine monomers) in combination with albendazole (AB, a standard anti-helminthic compound) to observe their therapeutic effect on demyelination in A. cantonensis-infected mice. Luxol fast blue staining and electron microscope of myelin sheath, Oligodendrocyte (OL) number and myelin basic protein (MBP) expression in brain was detected in above groups. RESULTS: TSIIA+AB facilitated OL proliferation and significantly increased both myelin sheath thickness and the population of small-diameter axons. In addition, TSIIA treatment inhibited the expression of inflammation-related factors (interleukin [IL]-6, IL-1ß, tumor necrosis factor [TNF]-α, inducible nitric oxide synthase [iNOS]) rather than inhibiting eosinophil infiltration in brain. TSIIA also decreased microglial activation and shifted their phenotype from M1 to M2. CONCLUSIONS: Taken together, these results provide evidence that TSIIA combined with AB may be an effective treatment for demyelination caused by A. cantonensis infection and other demyelinating diseases.

Effects of albendazole combined with TSII-A (a Chinese herb compound) on optic neuritis caused by Angiostrongylus cantonensis in BALB/c mice.

BACKGROUND: Angiostrongylus cantonensis (A. cantonensis) infection can lead to optic neuritis, retinal inflammation, damage to ganglion cells, demyelination of optic nerve and visual impairment. Combined therapy of albendazole and dexamethasone is a common treatment for the disease in the clinic, but it plays no role in vision recovery. Therefore, it has been necessary to explore alternative therapies to treat this disease. Previous studies reported the neuro-productive effects of two constituents of Danshen (a Chinese herb)-tanshinone II-A (TSII-A) and cryptotanshinone (CPT), and this study aims to evaluate the impacts of TSII-A or CPT combined with albendazole on optic neuritis caused by A. cantonensis infection in a murine model. METHODS: To assess the effects of TSII-A or CPT combined with albendazole on optic neuritis due to the infection, mice were divided into six groups, including the normal control group, infection group and four treatment groups (albendazole group, albendazole combined with dexamethasone group, albendazole combined with CPT group and albendazole combined with TSII-A group). The infection group and treatment groups were infected with A. cantonensis and the treatment groups received interventions from 14 dpi (days post infection), respectively. At 21 dpi, the visual acuity of mice in each group was examined by visual evoked potential (VEP). The pathologic alteration of the retina and optic nerve were observed by hematoxylin and eosin (H&E) staining and transmission electronic microscopy (TEM). RESULTS: Infection of A. cantonensis caused prolonged VEP latency, obvious inflammatory cell infiltration in the retina, damaged retinal ganglions and retinal swelling, followed by optic nerve fibre demyelination and a decreasing number of axons at 21 dpi. In treatment groups, albendazole could not alleviate the above symptoms; albendazole combined with dexamethasone lessened the inflammation of the retina, but was futile for the other changes; however, albendazole combined with CPT and albendazole combined with TSII-A showed obvious effects on the recovery of prolonged VEP latency, destruction and reduction of ganglion cells, optic nerve demyelination and axon loss. Compared with albendazole-CPT compound, albendazole combined with TSII-A was more effective. CONCLUSIONS: The current study demonstrates that albendazole combined with TSII-A plays a more effective role in treating optic neuritis caused by A. cantonensis in mice than with dexamethasone, as applied in conventional treatment, indicating that albendazole combined with TSII-A might be an alternate therapy for this parasitic disease in the clinic.

A severe case of rat lungworm disease in Hawa'i.

A 23-year-old man living on the island of Hawa'i developed a life threatening case of eosinophilic meningitis caused by infection with Angiostrongylus cantonensis (rat lungworm disease: RLWD). He was comatose for 3 months, incurring brain and nerve damage sufficiently extensive that he was not expected to recover. The case was complicated by secondary infections of methicillin-resistant Staphylococcus aureus, Clostridium difficile, and pneumonia, which resulted in an empyema requiring a thoracoscopy and decortication. He was treated with prednisone, mebendozal, and pain medication for RLWD, and antibiotics and antifungal medications for the secondary infections. The administration of herbal supplements was requested by the family and approved, and these were administered through a gastric tube. Less than a month after being declared in a persistent vegetative state the man was able to talk, eat, and had regained some muscle functions. After release from the hospital he continued the use of supplements and received treatments of intravenous vitamin therapy. Four years after onset of the illness he is able to ride a bicycle, is a part time student, plays guitar, and is fluent in two foreign languages. RLWD is an emerging tropical disease of growing importance in Hawa'i.

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.

Encephalitis caused by Angiostrongylus cantonensis after eating raw frogs mixed with wine as a health supplement.

Angiostrongylus cantonensis also known as the rat lungworm, is prevalent in the Pacific Islands and southeast Asia and is the most common cause of eosinophilic meningitis in humans. Although frogs and toads are known as paratenic hosts of A. cantonensis, they are rarely reported as the infectious source of human angiostrongyliasis. We report a case of encephalitis caused by Angiostrongylus cantonensis after eating raw frogs mixed with wine as a health supplement. Prednisolone at a dose of 1 mg/kg/day was prescribed for 14 days successfully. We advise that travelers and residents of endemic areas should avoid eating raw frogs and a public caution on the danger of eating raw wild animal products or the whole animal is recommended to alleviate such accidental infection.

The effects of different plant extracts on nematodes.

The anthelminthic efficacy of some differently obtained extracts of several plants was tested in vivo in laboratory animals and in vitro. The extracts were obtained by ethanolic, methanolic, aqueous, or chloroform, respectively, acetonitrile polyethylenglycol (PEG) and/or propylencarbonate (PC) elution at room temperature or at 37°C. The plants used were bulbs of onions, garlic, chives, coconut, birch tree, ananas, cistrose, banana, chicory, date palm fruit, fig, pumpkin, and neem tree seeds. The worm systems tested both in vivo and in vitro were Trichuris muris and Angiostrongylus cantonensis but only in vivo Toxocara cati. The tests clearly showed that the different extraction methods eluted different components and different mass amounts, which had different efficacies against the above-cited worms. In vitro effects against A. cantonensis and T.muris were best with aqueous extracts, followed by chloroform extracts. The other plant extracts showed only low or no effects on A. cantonensis in vitro. In the case of T. muris, best results were obtained in vivo and in vitro with PEG/PC extracts of the onion followed by the aqueous extract of coconut. The complete elimination of worms in the in vivo experiments with T. muris was obtained when infected mice were treated with a 1:1 mixture of extracts of coconut and onion being produced by elutions with a mixture of 1:1 PEG and PC and fed daily for 8 days. T. cati in a naturally infected cat was eliminated by daily oral application of 6 ml coco's fluid for 5 days. This study shows that a broad spectrum of plants has anti-nematodal activities, the intensity of which, however, depends on the mode of extraction. This implicates that, if results should be really comparable, the same extraction methods at the same temperatures have to be used. Furthermore, efficacy in in vitro systems does not guarantee as good--if at all--efficacy in vivo.

Efficacy of combined treatment with albendazole and baicalein against eosinophilic meningitis induced by Angiostrongylus cantonensis in mice.

Angiostrongylus cantonensis infection causes eosinophilic meningitis in humans. Baicalein is a flavonoid originally isolated from the roots of Scutellaria baicalensis Georgi. In this study we evaluated the efficacy of the combination of albendazole and baicalein for treating eosinophilic meningitis in BALB/c mice. Therapeutic efficacy included the survival time, body weight, neurological function, leucocyte and eosinophil counts, eotaxin concentration, matrix metalloproteinase-9 (MMP-9) activity, larval recovery and histopathological examination. The results showed that the combination of albendazole and baicalein was more effective than either drug administered singly. Combination therapy increased the survival time, decreased body weight loss, neurological dysfunction, leucocyte response, eotaxin concentration and MMP-9 activity. Our results suggest that the combination of albendazole and baicalein may exhibit synergistic beneficial effects in the treatment of eosinophilic meningitis induced by A. cantonensis.

Larvicidal activities of ginger (Zingiber officinale) against Angiostrongylus cantonensis.

In this study, we investigated the anthelmintic activity of [6]-gingerol, [10]-shogaol, [10]-gingerol, [6]-shogaol and hexahydrocurcumin, a constituent isolate from the roots of ginger (Zingiber officinale), for the parasite Angiostrongylus cantonensis. This study found that the above constituents killed A. cantonensis larvae or reduced their spontaneous movements in a time- and dose-dependent manner. The larvicidal effect or ability to halt spontaneous parasite movement of [10]-shogaol, [6]-gingerol, [10]-gingerol, [6]-shogaol and hexahydrocurcumin at various concentrations was reached from 24 to 72 h, respectively. Further investigation to determine minimal effective doses of [10]-gingerol and hexahydrocurcumin revealed [10]-gingerol to have a greater maximum larvicidal effect and loss of spontaneous movements than hexahydrocurcumin, mebendazole and albendazole. These constituents of ginger showed effects against DPPH and peroxyl radical under larvicidal effect. Together, these findings suggest that these constituents of ginger might be used as larvicidal agents against A. cantonensis.

Comparative efficacies of albendazole and the Chinese herbal medicine long-dan-xie-gan-tan, used alone or in combination, in the treatment of experimental eosinophilic meningitis induced by Angiostrongylus cantonensis.

Angiostrongylus cantonensis, the rat lungworm, is the principal cause of human eosinophilic meningitis or meningoencephalitis world-wide. In the present study, the efficacies of early-stage treatment with the Chinese herbal medicine long-dan-xie-gan-tan (LDXGT) and albendazole, used alone or in combination, were evaluated in BALB/c mice with A. cantonensis-induced dysfunction of the blood-central-nervous-system barrier and eosinophilic meningo-encephalitis. Indicators of the therapeutic effect included worm recovery, histopathological scores for the meningitis, assays of tissue-type plasminogen activator (PA), urokinase-type PA and matrix metalloproteinase-9 (MMP-9) in the brain, the ratio between albumin concentrations in the cerebrospinal fluid (CSF) and serum, and counts of eosinophils in the CSF. Combined treatment with albendazole and LDXGT gave better results than monotherapy based on either drug, apparently inhibiting eosinophilic meningitis via antagonists of the PA/MMP-9 system. LDXGT may have a therapeutic role in reducing inflammatory reaction in the subarachnoid space. Monotherapy with such an anti-inflammatory drug may relieve the symptoms of mild infection and the host's immune responses to A. cantonensis larvae. In severe infection, however, co-therapy with an anthelmintic (to kill the larvae) and an anti-inflammatory agent (to provide symptomatic relief) is probably a better approach. The therapeutic strategy should be tailored to the severity of the illness and the numbers of eosinophils in the CSF.

Efficacy of curcumin therapy against Angiostrongylus cantonensis-induced eosinophilic meningitis.

Angiostrongylus cantonensis can invade the central nervous system, leading to human eosinophilic meningitis or eosinophilic meningoencephalitis. Curcumin is a natural product which has the effects of anti-inflammation, anti-oxidation and anti-carcinogensis, while the administration of curcumin has been reported to possibly relieve the symptoms of meningitis. The present study tested the potential efficacy of curcumin in A. cantonensis-induced eosinophilic meningitis of BALB/c mice. Assay indicators for the therapeutic effect included the larvicidal effect, eosinophil counts and matrix metalloproteinase-9 (MMP-9) activity in angiostrongyliasis. Eosinophils were mildly reduced in treatment groups compared with infected-untreated mice. However, there were no significant differences in larvicidal effects or MMP-9 activity. This study suggests that anti-inflammatory treatment with curcumin alone has low efficacy, but the treatment does not interfere with MMP-9 expression and is not useful for larvicidal effects. The possible reasons include low curcumin across the blood-brain barrier and also those larvae that survive stimulate MMP-9 production, which promotes blood-brain barrier damage, with leukocytes then crossing the blood-brain barrier to cause meningitis. Further studies will be required to test these possibilities.

Angiostrongylus cantonensis: apoptosis of inflammatory cells induced by treatment with mebendazole or/and interleukin 12 in mice.

Angiostrongylus cantonensis is the major cause of human eosinophilic meningoencephalitis. ICR mice were infected orally with 35 infective larvae and sacrificed at 4-14 days, 25 days or 32 days post infection (dpi) for pathological and immunocytochemical examinations. In the non-treated group, no apoptosis signal was found in the meninges or parenchyma of the brains (4-14 dpi). Only a few apoptotic cells were noticed at 25 dpi (3%) and 32 dpi (10%). In the groups, the animals were given a single dose of mebendazole (20 mg/kg, per os at various times) or injections of interleukin 12 (IL-12) (10 ng/daily, intraperitoneally), all the animals were sacrificed at 14 dpi; the number of apoptotic cells was increased (17-21%). In the group that received a single dose of mebendazole (4 dpi) in combination with IL-12 injections (4-13 dpi), mild meningitis was observed, and most of the infiltrated inflammatory cells were in the apoptotic program (55%). Taken together, apoptosis of the inflammatory cells (most were eosinophils) could be induced when the infected mice were treated with mebendazole or/and IL-12.

The influence of zinc in mice on infection with Angiostrongylus cantonensis.

BALB/cByJ mice were divided into zinc-deficient (ZD), high zinc-supplemented (ZH), adequately zinc-replete (ZA) and normal control groups by daily dietary control. The body weight in ZD and ZH mice became significantly less than that of normal control mice from 4 weeks (P < 0.002) until 7 weeks (P < 0.0001) after consecutive zinc control. The kinetics of change in body weight of ZD and ZH mice after infection with Angiostrongylus cantonensis were similar to that of uninfected groups mentioned above. The worms recovered at days 7, 14 and 21 after infection from ZD and ZH mice were significantly more in number than that in control mice (P < 0.01). However, the worms from day 21 after infection were shorter in size than those in control mice (P < 0.01). The production of interleukin (IL)-5 was significantly depressed in cultured spleen cells from uninfected and infected ZD and ZH mice, compared with that from respective control group (P < or = 0.02). Furthermore, significantly lower eosinophil counts were observed in the cerebral spinal fluid(CSF) of ZD and ZH mice 3 weeks after infection than in the CSF of control mice (P < 0.001). The levels of immunoglobulins IgG, IgM, IgA and IgE in the supernatant of cultivated spleen cells and serum from ZD and ZH mice were all lower than those of control mice. Thus, the level of zinc correlated with the defence against infection by A. cantonensis, due to the influence of zinc on the growth of mice and their production of IL-5, eosinophils and immunoglobulins.