Biochemical and pathological evaluation of albendazole/thalidomide co-therapy against eosinophilic meningitis or meningoencephalitis induced by Angiostrongylus cantonensis
Department of Parasitology, Chung Shan Medical University, Taichung 402, Taiwan
* Corresponding author. Tel: +886-4-24730022-11641; Fax: +886-4-23823381; E-mail: shih{at}csmu.edu.tw
Received 19 May 2006; returned 18 October 2006; revised 9 November 2007; accepted 12 November 2006
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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-
, interleukin-1ß 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.
Keywords: parasites , angiostrongyliasis , thalidomide , proteinases , cytokines
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Angiostrongyliasis is caused by the rat lung worm, Angiostrongylus cantonensis, which is widely distributed in tropical and subtropical areas where rodents (the natural and definitive host) are known to be infected with this parasite. To date, this parasite has been identified throughout the island of Taiwan, where many cases of CNS disease caused by this parasite have been reported.1,2 Our previous study showed that the tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) may play an important role in eosinophilic meningitis of BALB/c mice.3 Additionally, matrix metalloproteinase-9 (MMP-9) was associated with eosinophilic meningitis and found to be a useful marker for angiostrongyliasis meningitis in BALB/c4 and ICR mice.5
The transport of compounds from the circulating blood into the CNS is restricted by blood–CNS barriers, such as the blood–brain barrier (BBB) and blood–CSF barrier, which are formed by tight junctions connecting the cerebral endothelial and epithelial cells of the choroid plexus, respectively.6 Breakdown of the BBB is a key feature of neuroinflammatory conditions and is associated with an influx of inflammatory cells, fluid and proteins, including complement and cytokines.7 MMPs have been implicated in the pathogenesis of BBB breakdown.7,8 Additionally, the ability of the uPA to recruit leucocytes and promote blood–CNS barrier breakdown, may play an important pathophysiological role in bacterial meningitis.9
Interleukin-5 (IL-5) is an important cytokine for the induction of A. cantonensis-induced eosinophilia.10 The CSF eosinophils of mice infected with A. cantonensis are resistant to apoptosis, and IL-5 is an important determinant in the survival and activation of eosinophils.11 The proinflammatory cytokine tumour necrosis factor- (TNF-) is a potent activator of neutrophils, modulating adherence, chemotaxis and degranulation; it is responsible for the severe cachexia that occurs in chronic infections.12 MMP-9 has been shown to cleave inactive TNF-, so that active TNF- is released.13 In addition, MMP-9 gene expression by macrophages and polymorphonuclear leucocytes, and hence the release of pro-MMP, can be induced by TNF- and interleukin-1ß (IL-1ß).14,15
Some effective anthelmintics have been proposed to treat A. cantonensis in humans. But dead worms in the brain may evoke a severe immune response resulting in brain damage. Though considered the drug of choice for treating angiostrongyliasis, albendazole is frequently used in combination with steroids to prevent the inflammatory reaction due to dead larvae. However, the use of steroid therapy is still controversial. The objectives of the present study were to assess the efficacy of the anthelmintic albendazole combined with the TNF- inhibitor thalidomide as treatment in a mouse model of parasitic meningitis caused by A. cantonensis infection, and to find out whether the TNF- inhibitor could substitute for steroid treatment.
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Experimental animals
Five-week-old male mice (weighing 20–25 g), BALB/c strain, were purchased from the National Laboratory Animal Center, Taipei, Taiwan. They were maintained on a regular 12:12 h light/dark cycle and provided with Purina Laboratory Chow and water ad libitum. All of the procedures involving animals and their care in this study were approved by the Institutional Animal Care and Use Committee of Chung-Shan Medical University in accordance with institutional guidelines for animal experiments.
The infective larvae (L3) of A. cantonensis, originally obtained from wild giant African snails (Achatina fulica), were propagated for several months in our laboratory by cycling through rats and snails (Biomphalaria glabrata). The larvae within tissues were recovered using a modification of the method of Parsons and Grieve.16 Briefly, the shells were crushed, and the tissues were homogenized, digested in a pepsin–HCl solution (pH 1–2, 500 IU pepsin/g tissue) and incubated with agitation in a 37°C waterbath for 2 h. Host cellular debris was removed from the digest by centrifugation at 1400 g for 10 min. The larvae in the sediment were collected by serial washing in double-distilled water and counted under a microscope.
Albendazole (Zentel®, GlaxoSmithKline, NC, USA) is a benzimidazole derivative. The molecular formula is C12H15N3O2S and the molecular mass is 265.34.17 It is widely used in veterinary medicine and has been found to be a safe broad-spectrum anthelmintic in animals and humans. Albendazole acts by binding to the parasite's ß-tubulin, inhibiting its polymerization and impairing glucose uptake, causing death.18 Thalidomide (TTY Biopharm, Taiwan) has the empirical formula C13H10N2O4 and a molecular mass of 258.23.19 Thalidomide affects cytokine production and T-lymphocyte proliferation. Thalidomide suppresses TNF- production by macrophages and may thereby reduce the inflammatory response.20
A total of 300 mice were randomly divided into five treated groups (60 mice/group), and subdivided into three different time points of administration (20 mice per time point). Food and water were withheld for 12 h before infection. The uninfected control mice were administered only distilled water orally on days 5, 10 and 15 post-infection (PI). All other groups were infected with 50 larvae, including the infected–untreated control mice, which were treated with only distilled water orally on days 5, 10 and 15 PI. The albendazole-treated mice were treated with albendazole only (5, 10 or 20 mg/kg per day) for 7 consecutive days on days 5, 10 and 15 PI, respectively; the thalidomide treated mice were treated with thalidomide only (25, 50 or 100 mg/kg per day) for 7 consecutive days on days 5, 10 and 15 PI, respectively; and the albendazole/thalidomide co-therapy mice received a treatment combining albendazole (10 mg/kg per day) and thalidomide (50 mg/kg per day) for 7 consecutive days on days 5, 10 and 15 PI, respectively. All groups were sacrificed on day 22 PI; the brains and CSF were collected for histopathological study and biochemical analysis.
Worm recovery and assessment of efficacy
Treated and control mice were killed by cervical dislocation on day 22 PI. Each brain was sliced into small pieces, digested in a pepsin–HCl solution (pH 1–2, 500 IU pepsin/g tissue) and incubated with agitation in a 37°C waterbath for 2 h. Host cellular debris was removed from the digest by centrifugation at 1400 g for 10 min. Larval counts were measured under 25x magnification using a dissecting microscope. The mean number of larvae was compared with that of the positive control (infected–untreated) group to assess drug efficacy.
Histopathological examinations
The mouse brains were fixed separately in 10% neutral buffered formalin for 24 h. The fixed specimens were dehydrated in a graded ethanol series (50%, 75% and 100%) and xylene, and then embedded in paraffin at 55°C for 24 h. Serial sections (5 µm thick) were cut for each brain from each mouse. Paraffin was removed by heating the sections for 5 min at 65°C. These sections were dewaxed by washing three times for 5 min each in xylene, then rehydrated through 100%, 95% and 75% ethanol for 5 min each, and finally rinsed with distilled water. After staining with haematoxylin (Muto, Tokyo, Japan) and eosin (Muto, Tokyo, Japan), pathological changes were examined under a light microscope.
The mice were sacrificed and their brains removed into a 35 mm dish. The cranial cavity and cerebral ventricles (lateral, third and fourth ventricles) were rinsed with 1 mL of PBS. The washing solution was collected in a centrifuge and spun at 400 g for 10 min. The resultant sediments were then gently mixed with 100 µL of Unopette buffer (Vacutainer System, Becton Dickinson, Franklin Lakes, NJ, USA) and 2 µL of acetic acid and placed in a haemocytometer cell-counting chamber (Paul Marienfeld, Lauda-Koenigshofen, Germany) to count eosinophils.
Production of cytokines (TNF-, IL-1ß and IL-5) in the CSF was assayed using commercial enzyme-linked immunosorbent assay (ELISA) kits (Biosource, Camarillo, CA, USA) according to the manufacturer's instructions. CSF samples for cytokine assays were pooled since the amounts of CSF harvested from each mouse were frequently insufficient for individual assays.
Gelatin and casein substrate zymography
Activity of MMP-9 was determined by gelatin zymography,21 and plasminogen activator (PA) activities by casein zymography.3 Briefly, the CSF was centrifuged at 12000 g for 10 min to remove debris. The protein contents of supernatants were analysed on SDS/polyacrylamide gels copolymerized with 0.1% gelatin (Sigma, St Louis, MO, USA) for gelatinase activities, 0.1% casein (Sigma) for plasmin activities and plasminogen (13 mg/L, Sigma) for PA activities. Electrophoresis was performed in running buffer (25 mM Tris, 250 mM glycine and 1% SDS) at room temperature at 120 V for 1 h. The gel was washed twice at room temperature for 30 min each in 2.5% Triton X-100, and then washed twice with double-distilled water for 10 min each. The gel was incubated in reaction buffer at 37°C for 18 h. The gel was stained with 0.25% Coomassie Brilliant Blue R-250 (Sigma) for 1 h and destained in 15% methanol/7.5% acetic acid. The final gel had a uniform background except in regions to which MMP-9, tPA or uPA had migrated and cleaved their respective substrates. Quantitative analysis of the gelatinolytic and caseinolytic enzymes was performed with a computer-assisted imaging densitometer system, UN-SCAN-ITTM gel Version 5.1 (Silk Scientific, Orem, UT, USA).
Measurement of albumin in CSF and serum
The CSF and serum were centrifuged at 12000 g at 4°C for 10 min, and the concentration of albumin in CSF and serum was measured by the Dade Behring assay system using the automated Dade Behring Nephelometer (BN ProSpec, Newark, NJ, USA).
Results in the different groups of mice were compared using the non-parametric Kruskal–Wallis test followed by post-testing using Dunn's multiple comparison of means. All results are presented as mean ± standard deviation (SD). P values of <0.05 were considered statistically significant.
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Influence of treatment on larvae recovery
The larvicidal effect of treatment with three doses of albendazole beginning on days 5, 10 and 15 PI was lower on day 15 PI than on days 5 or 10 PI (Table 1). There was no significant difference (P > 0.05) in worm recovery between the non-treated group and the thalidomide-treated group (Table 2). Larvae recovered from brain tissue were virtually absent in infected mice treated with albendazole/thalidomide beginning on days 5 and 10 PI (Table 3). The numbers of recovered larvae were less after albendazole alone or albendazole/thalidomide than no treatment.
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Influence of treatment on pathological changes
Optical microscopic examination of haematoxylin and eosin stained tissues revealed the mechanical damage caused by migrating larvae and meningeal inflammation characterized by eosinophilic infiltration. In the subarachnoid space, large-scale infiltration by leucocytes was evident 22 days after the inoculation of the larvae. Treatment with albendazole alone or albendazole/thalidomide beginning on days 5 and 10 PI significantly decreased the number of infiltrating leucocytes (P < 0.05) and resulted in larval degeneration (Figure 1), but thalidomide alone caused only mild reduction in leucocyte number. Treatment on day 15 PI: leucocytes were only mildly reduced by albendazole alone or albendazole/thalidomide and not significantly affected by thalidomide alone. Evidence of brain parenchyma and ventricle infection included haemorrhage, perivascular cuffing, nodular lesions, reactive proliferation of glial cells, diffuse infiltration by inflammatory cells, and a spongy appearance of parenchyma in all areas of the brain. Treatment on days 5 and 10 PI: albendazole alone or albendazole/thalidomide co-therapy significantly decreased (P < 0.05) haemorrhage and infiltrating leucocytes, whereas these were only mildly reduced by thalidomide. Treatment on day 15 PI: albendazole alone or albendazole/thalidomide mildly reduced haemorrhage and infiltrating leucocytes, and thalidomide alone had no significant effect. Additionally, albendazole treatment alone or albendazole/thalidomide caused larval degeneration (Figure 2).
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Influence of treatment on eosinophil counts
The CSF eosinophils were significantly increased (P < 0.05) in mice infected with A. cantonensis compared with uninfected controls. Treatment on days 5 and 10 PI: albendazole alone or albendazole/thalidomide significantly reduced eosinophil counts (P < 0.05), whereas thalidomide treatment mildly reduced these counts (P < 0.05). Treatment on day 15 PI: albendazole/thalidomide mildly reduced eosinophils (P < 0.05), but albendazole alone or thalidomide alone had no significant effect (P > 0.05) (Figure 3).
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Influence of treatment on cytokine production
To determine the kinetics of cytokine production and its possible role in mouse resistance to A. cantonensis, sandwich ELISAs were used to assay TNF-, IL-1ß and IL-5 in CSF. The concentrations of TNF-, IL-1ß and IL-5 were significantly higher (P < 0.05) in infected mice than in uninfected mice, which had low levels of these. When compared with the corresponding cytokine concentrations in infected–untreated mice, TNF- concentration was significantly lowered (P < 0.05) in infected mice by albendazole alone, thalidomide alone or albendazole/thalidomide (Figure 4), IL-1ß concentration was significantly lowered (P < 0.05) by albendazole alone or albendazole/thalidomide but not by thalidomide alone, which had no effect (Figure 5), and IL-5 concentration was significantly lowered (P < 0.05) by albendazole alone, thalidomide alone or albendazole/thalidomide beginning on day 5 PI. For mice treated with albendazole alone or albendazole/thalidomide beginning on days 10 and 15 PI, IL-5 concentration was significantly lowered (P < 0.05), but thalidomide alone had no effect (P > 0.05) (Figure 6).
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Influence of treatment on tPA and uPA activity
Casein zymography measures the activity of enzymes such as tPA and uPA, which migrate electrophoretically as 70 kDa and 55 kDa species, respectively. These enzymes were significantly increased (P < 0.05) in the CSF of infected mice. For mice treated beginning on days 5 and 10 PI, albendazole alone, thalidomide alone or albendazole/thalidomide significantly reduced tPA and uPA activities (P < 0.05). Treatment on day 15 PI: albendazole alone or albendazole/thalidomide co-therapy mildly reduced these activities (P < 0.05), but thalidomide alone had no significant effect (P > 0.05) (Figures 7 and 8).
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Influence of treatment on MMP-9 activity
Gelatin zymography measures the activity of enzymes such as MMP-9, which migrates electrophoretically as a 94 kDa species. This metalloproteinase activity was significantly increased (P < 0.05) in the CSF of mice infected with A. cantonensis. For mice whose treatment began on day 5 PI, albendazole alone, thalidomide alone or albendazole/thalidomide significantly reduced MMP-9 activity (P < 0.05). Treatment on days 10 and 15 PI: albendazole alone or albendazole/thalidomide mildly reduced the MMP-9 activity (P < 0.05), and thalidomide alone had no significant effect (P > 0.05) (Figure 9).
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Influence of treatment on CSF/serum albumin ratio
The CSF/serum albumin ratio was significantly increased (P < 0.05) in infected mice and when albendazole alone, thalidomide alone or albendazole/thalidomide therapy began on day 5 PI, it was significantly decreased (P < 0.05) in infected mice. Treatment on days 10 and 15 PI: albendazole alone or albendazole/thalidomide mildly reduced the ratio (P < 0.05). In contrast, thalidomide alone had no significant effect (P > 0.05) (Figure 10).
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Albendazole is reported to be effective against most intestinal nematodes, with a single oral dose being adequate in most cases.22 Animal studies with albendazole showed that at least 50% of an orally administered single dose is absorbed.22 In this study, oral albendazole at 10 mg/kg per day for 7 consecutive days beginning on days 5 and 10 PI had remarkable larvicidal effect. The A. cantonensis larvae in the brains of mice were almost eliminated. However, less satisfactory effects were observed when medication was begun on day 15 PI. This may be explained by the fact that albendazole acts against the third or fourth but not fifth stage larvae of A. cantonensis. After the infective L3 larvae of A. cantonensis are ingested, they migrate from the gastrointestinal tract to the CNS via the circulatory system. Most of the larvae complete their third moult on day 5 PI and the fourth moult around day 10 PI. On day 15 PI, most are fifth stage larvae (young adult worms) that migrate to the surface of the brain23 and into meningeal spaces. Treatment at this time is more difficult than on days 5 and 10 PI. These data confirm the previous finding17 that the timing of drug administration affects anthelmintic efficacy and larvicidal activity in A. cantonensis-infected mice.
In the early stage of A. cantonensis infection, treatment is primarily supportive. Repeated lumbar punctures provide relief for patients with persistent headaches from increased intracranial pressure.24 Anthelminthics are not effective and often worsen symptoms, possibly because of the inflammatory reaction to antigens released by dying worms.25 In the present study, the degree of brain inflammation correlated with the number of larvae found in the CNS. We initiated treatment before (on day 5 PI) and after (on days 10 and 15 PI) the appearance of inflammatory signs in the experimental animals. The treatment reduced the number of worms, reducing the pathology associated with infection. Though albendazole alone could increase neurological damage by causing simultaneous death of a large number of A. cantonensis larvae in the early stage (5 to 10 PI) (data not shown), in fact the severity of meningitis was lessened owing to the lower number of worms.
The blood–CNS barrier breakdown is regarded as an important pathophysiological event in meningitis, since it allows extravasation of leucocytes into the subarachnoid space. MMPs are implicated in the disruption of the BBB during leucocyte diapedesis,8,26 and uPA promotes blood–CNS barrier breakdown.9 Excessive proteolytic activities of PAs and MMP-9 can be detrimental, leading to disruption of the blood–CNS barrier. Measuring ratios of CSF to serum albumin may be a good index to monitor blood-to-CNS-barrier integrity without the use of invasive methods. Yii24 reported that CSF protein levels were higher in patients with A. cantonensis-induced eosinophilic meningitis than in normal individuals. Our previous studies have suggested that the MMP-9 inhibitor GM6001 used as an adjunct to the anthelmintic albendazole significantly inhibited the proteolytic enzyme MMP-9 and decreased eosinophilia in angiostrongyliasis.27 In the present study, the CSF/serum albumin ratio and eosinophilia were significantly decreased when mice were treated with albendazole/thalidomide co-therapy. However, thalidomide alone only partially reduced CSF/serum albumin ratio and eosinophilia. These results suggest that CSF/serum albumin ratio might be an important indicator in eosinophilic meningitis and imply that treatment improves blood–CNS barrier damage by reducing activity of PAs/MMP-9 proteolytic enzymes and reducing the CSF/serum albumin ratio.
The anti-inflammatory and immunomodulatory effects of thalidomide have led scientists to assess its therapeutic value clinically. The efficacy of thalidomide is thought to be based on its ability to alter the phenotype of circulating immunological cells,28 inhibit TNF- synthesis by macrophages,29 alter the level of cytokine and lymphokine synthesis and release, alter lymphocyte trafficking and neutrophil migration, and induce alterations in cellular adhesion molecules, consequently changing the interactions of leucocytes with the endothelial cell layer and causing the modulation of inflammation and immunity.19 Thalidomide is safe and well tolerated in children with stage 2 tuberculous meningitis.30 Thalidomide is now being considered as an adjuvant treatment for angiostrongyliasis meningitis. However, thalidomide alone reduces cytokine synthesis, has no larvicidal effect, and only mildly reduces the number of CSF eosinophils. The mild reduction of inflammation might be explained by the persistence of larvae that stimulate the immune response.
Th2-type cytokine production, especially of IL-5, is prevalent during A. cantonensis infection in mice and is probably involved in the induction of CSF and peripheral eosinophilia,10 especially since it fails to elicit eosinophilia in IL-5 receptor knockout mice.31 In this study, albendazole/thalidomide co-therapy significantly lowered the IL-5 level and thereby strongly depressed CSF eosinophilia. Additionally, thalidomide, by interfering with the production of TNF-, can reduce the inflammatory response.19 Though basal levels are essential for normal growth and development, TNF- increases during A. cantonensis infection causing pathology. Co-therapy with albendazole and thalidomide significantly lowered but did not eliminate the TNF- concentration. Thus, albendazole/thalidomide co-therapy targeting cytokines (TNF-, IL-1ß and IL-5) may offer an effective treatment for eosinophilic meningitis.
In conclusion, the present study was designed to estimate the efficacy of combining the anthelmintic albendazole with the TNF- inhibitor thalidomide to treat eosinophilic meningitis in a mouse model. Albendazole/thalidomide co-therapy as early as 5 or 10 days after infection significantly reduced indicators of disease, such as worm recovery, eosinophil counts, cytokines, proteolytic enzymes and CSF/serum albumin ratio. The severity of meningitis was lessened to a large degree by reduction in the number of worms. The number of dead worms in the brain capable of stimulating inflammation may be reduced by thalidomide. This combination of drugs probably exploits the potential synergistic effects of lowering cytokines and PAs/MMP-9 proteolytic enzymes on eosinophilic meningitis.
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None to declare.
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This study was supported by a research grant (NSC-93-2314-B-040-031) from the National Science Council, Republic of China.
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