TPL-2 restricts Ccl24-dependent immunity to Heligmosomoides polygyrus.

TPL-2 (COT, MAP3K8) kinase activates the MEK1/2-ERK1/2 MAPK signaling pathway in innate immune responses following TLR, TNFR1 and IL-1R stimulation. TPL-2 contributes to type-1/Th17-mediated autoimmunity and control of intracellular pathogens. We recently demonstrated TPL-2 reduces severe airway allergy to house dust mite by negatively regulating type-2 responses. In the present study, we found that TPL-2 deficiency resulted in resistance to Heligmosomoides polygyrus infection, with accelerated worm expulsion, reduced fecal egg burden and reduced worm fitness. Using co-housing experiments, we found resistance to infection in TPL-2 deficient mice (Map3k8-/-) was independent of microbiota alterations in H. polygyrus infected WT and Map3k8-/-mice. Additionally, our data demonstrated immunity to H. polygyrus infection in TPL-2 deficient mice was not due to dysregulated type-2 immune responses. Genome-wide analysis of intestinal tissue from infected TPL-2-deficient mice identified elevated expression of genes involved in chemotaxis and homing of leukocytes and cells, including Ccl24 and alternatively activated genes. Indeed, Map3k8-/-mice had a significant influx of eosinophils, neutrophils, monocytes and Il4GFP+ T cells. Conditional knockout experiments demonstrated that specific deletion of TPL-2 in CD11c+ cells, but not Villin+ epithelial cells, LysM+ myeloid cells or CD4+ T cells, led to accelerated resistance to H. polygyrus. In line with a central role of CD11c+ cells, CD11c+ CD11b+ cells isolated from TPL-2-deficient mice had elevated Ccl24. Finally, Ccl24 neutralization in TPL-2 deficient mice significantly decreased the expression of Arg1, Retnla, Chil3 and Ear11 correlating with a loss of resistance to H. polygyrus. These observations suggest that TPL-2-regulated Ccl24 in CD11c+CD11b+ cells prevents accelerated type-2 mediated immunity to H. polygyrus. Collectively, this study identifies a previously unappreciated role for TPL-2 controlling immune responses to H. polygyrus infection by restricting Ccl24 production.

Soluble antigen derived from IV larva of Angiostrongylus cantonensis promotes chitinase-like protein 3 (Chil3) expression induced by interleukin-13.

Angiostrongyliasis caused by Angiostrongylus cantonensis (A. cantonensis) is an emerging food-borne parasitic disease, which refers basically to eosinophilic meningitis. Chitinase-like protein 3 (Chil3), a member of chitinase-like protein family which has chemotactic activity for eosinophils, is reported to be highly upregulated in brain of mouse infected with A. cantonensis. The mechanisms of high expression of Chil3 and the association between A. cantonensis and Chil3 are rarely reported. In order to understand the mechanism of high expression of Chil3 in A. cantonensis-infected mouse, we measured the level of Chil3 in RAW 264.7 and BV2 cell lines stimulated with soluble antigen of A. cantonensis by qPCR and ELISA. To explore the role of Chil3 in inflammation caused by A. cantonensis, we extracted and cultured brain mononuclear cells (BMNCs) and detected the eosinophil chemotactic activity of Chil3 using transwell assay and flow cytometer. Furthermore, we treated the infected mice by injection with rmChil3 and then counted the number of larvae in brains of infected mice and treated mice to examine the association between the worm and Chil3. Our results showed the soluble antigen from A. cantonensis could promote the Chil3 expression in macrophage and microglial cell lines induced by interleukin-13. In conclusion, we supposed that high expression of Chil3 enhanced by soluble antigens from A. cantonensis might be the reason of serious eosinophil infiltration in mouse brain after A. cantonensis infection.

Angiostrongylus cantonensis cathepsin B-like protease (Ac-cathB-1) is involved in host gut penetration.

Although the global spread of the emerging zoonosis, human angiostrongyliasis, has attracted increasing attention, understanding of specific gene function has been impeded by the inaccessibility of genetic manipulation of the pathogen nematode causing this disease, Angiostrongylus cantonensis. Many parasitic proteases play key roles in host-parasite interactions, but those of A. cantonensis are always expressed as the inactive form in prokaryotic expression systems, thereby impeding functional studies. Hence, a lentiviral system that drives secreted expression of target genes fused to a Myc-His tag was used to obtain recombinant Ac-cathB-1 with biological activity. Although this class of proteases was always reported to function in nutrition and immune evasion in parasitic nematodes, recombinant Ac-cathB-1 was capable of hydrolysis of fibronectin and laminin as well as the extracellular matrix of IEC-6 monolayer, so that the intercellular space of the IEC-6 monolayer increased 5.15 times as compared to the control, while the shape of the adherent cells partly rounded up. This suggests a probable role for this protease in intestinal epithelial penetration. The inhibition of Ac-cathB-1 enzymatic activity with antiserum partly suppressed larval penetration ability in the isolated intestine. Thus, an effective system for heterologous expression of parasite proteases is presented for studying gene function in A. cantonensis; and Ac-cathB-1 was related to larval penetration ability in the host small intestine.

Matrix metalloproteinase-12 leads to elastin degradation in BALB/c mice with eosinophilic meningitis caused by Angiostrongylus cantonensis.

The rat lugworm Angiostrongylus cantonensis can cause eosinophilic meningitis. The purpose of this study was to determine whether matrix metalloproteinase (MMP)-12 and its substrate elastin participate in this inflammatory response. We showed that the MMP-12/tissue inhibitor of metalloproteinase-1 ratio was significantly increased in the CSF of A. cantonensis-infected mice from day 10 p.i., and reached high levels on days 20 and 25 p.i. MMP-12 production was correlated with elastin degradation, eosinophil count, blood-CSF barrier permeability and pathological changes in the subarachnoid space. Also, MMP-12 might contribute to elastin degradation in the meningeal vessel of the subarachnoid space. Simultaneous administration of albendazole and doxycycline significantly reduced the levels of MMP-12, elastin and Evans blue in mice with meningitis. These results imply that MMP-12 contributes to the elastin degradation that occurs in angiostrongyliasis meningitis, and doxycycline can reverse related inflammatory events by inhibition of MMP-12.

Th2-specific immunity and function of peripheral T cells is regulated by the p56Lck Src homology 3 domain.

T cell activation and effector function is essential for robust immunity. Ag TCR signals are known to regulate T lymphocyte differentiation, but the mechanisms involved in this regulation remain unclear. Recent work has demonstrated that the Src family protein tyrosine kinase p56Lck specifically links TCR signaling to activation of the MAPK pathway through the function of its Src homology 3 (SH3) domain. The MAPK pathway is involved in T cell activation and has previously been implicated in Th2 immunity. We have used Lck SH3 mutant knockin mice (LckW97A) to investigate the potential role of this regulatory mechanism in T lymphocyte activation and effector function. Our results demonstrate that Lck SH3 domain function regulates activation of T lymphocytes as indicated by reduced IL-2 production, CD69 induction, and proliferation of LckW97A T cells following TCR stimulation. Biochemical studies confirm that activation of the MAPK pathway is selectively altered following TCR ligation in LckW97A T lymphocytes. Phospho-ERK induction is reduced, but phospho-phospholipase Cgamma1 induction and calcium mobilization are largely unaffected. Immunization with DNP-keyhole limpet hemocyanin, heat-killed Brucella abortus, or infection with Nippostrongylus brasiliensis demonstrates selectively impaired Th2 immunity with reduced serum levels of IgG1, IgE, and IL-4. In vitro studies show that LckW97A T cells can differentiate into Th2-type cells, but they form IFN-gamma-producing cells under conditions that normally favor Th2 development. These data indicate that the Lck SH3 domain controls T lymphocyte activation by regulating MAPK pathway induction and demonstrate a novel role for Lck in the regulation of Th2-type immunity.

Cerebrospinal fluid u-plasminogen activator and matrix metalloproteinase-9 levels in human eosinophilic meningitis associated with angiostrongyliasis.

Cerebrospinal fluid (CSF) urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9) levels were measured in patients with eosinophilic meningitis associated with angiostrongyliasis (EOMA) by quantitative sandwich enzyme immunoassays. The CSF concentrations of uPA and MMP-9 were evaluated in 30 EOMA patients and 10 controls. The CSF uPA and MMP-9 levels of the EOMA patients were significantly higher than those of the controls (p<0.001). The positive detection values were 73% (22/30) and 86.7% (26/30) for uPA and MMP-9, respectively. The uPA detection was in correlation with headache duration (p=0.008) and stiff neck (p=0.048), while the MMP-9 was in correlation with CSF total protein (p=0.006), CSF leukocytosis (p=0.004) and CSF eosinophil numbers (p=0.02). CSF uPA and MMP-9 levels are potentially useful for the understanding of immunologic pathogenesis, for therapeutic targets, for the diagnosis of EOMA and for monitoring treatment efficacy.

Oxidative stress in mice infected with Angiostrongylus cantonensis coincides with enhanced glutathione-dependent enzymes activity.

This study aimed to estimate reactive oxygen species (ROS) production, antioxidants activity, and biomarkers level of oxidative damage to protein and DNA in the cerebrospinal fluid (CSF) of C57BL/6 mice infected with Angiostrongylus cantonensis. The mean ROS concentration in the CSF of infected mice increased gradually, and the increase in ROS in CSF became statistical significance at days 12-30 post-infection compared to that before infection (P<0.001), and then ROS returned to normal level at day 45 after infection. In parallel with the increase in ROS in the CSF, infected mice showed similar of changes in reduced glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx), and glutathione S-transferase (GST) as that in ROS in the CSF. GSH, GR, GPx, and GST in the CSF of infected mice were all significantly higher than they were before infection during days 12-30 post-infection. However, protein carbonyl content and 8-hydroxy-2'-deoxyguanosine, biomarkers of oxidative damage to protein and DNA, respectively, were also significantly higher in the CSF of infected mice during this period. These results suggest that oxidative stress occur in the cells of central nervous system of mice infected with A. cantonensis during days 12-30 after infection due to ROS overproduction in CSF despite the increase in antioxidants during this period.

5-lipoxygenase and cyclooxygenase-2 in porcine parasitic bronchopneumonia: immunohistochemical and biochemical investigations.

Eicosanoids are products of arachidonic acid metabolism and have numerous biological roles. The present study aimed to investigate the role of 5-lipoxygenase (5-LOX)- and cyclooxygenase-2 (COX-2)- dependent enzymatic pathways in the pathogenesis of porcine parasitic bronchopneumonia caused by Metastrongylus spp. Pulmonary tissue samples from healthy control and parasitized pigs were processed for histopathological, immunohistochemical and biochemical investigations. In control animals, immunohistochemistry demonstrated that 5-LOX and COX-2 expression was almost exclusively limited to the bronchiolar epithelial cells. Parasitized pigs had greater 5-LOX- and COX-2- specific immunoreactivity, involving a wide range of cell types within foci of granulomatous and eosinophilic bronchopneumonia. Biochemical investigations demonstrated the presence of 5-LOX (and the related product Leukotriene B(4)) and COX-2 (and the related product prostaglandin E(2); PGE(2)) in all tissues under study. COX-2 activity and PGE(2) concentration were significantly higher in diseased lungs compared with normal healthy controls. These findings demonstrate that 5-LOX and COX-2 are differentially expressed in normal versus lungworm-infected lungs and therefore suggest that both biochemical pathways are likely to be involved in the pathogenesis of porcine parasitic bronchopneumonia.

Differences of proteolytic enzymes and pathological changes in permissive and nonpermissive animal hosts for Angiostrongylus cantonensis infection.

In this study, mice (nonpermissive hosts) infected with Angiostrongylus cantonensis showed significant worm degeneration and eosinophil degranulation, whereas infected rats (permissive hosts) showed lower worm degeneration and eosinophil degranulation. Pathophysiological changes developed to a lesser extent in rat than in the mouse strains. Neurological evaluation of A. cantonensis-infected mice showed mechanical damage caused by worms migrating to the brain. A significant correlation between the proteolytic enzymes, plasminogen activator (PA) and matrix metalloproteinase-9 (MMP-9), and pathological changes was found in hosts with eosinophilic meningitis or meningoencephalitis. Also, the ratio of cerebrospinal fluid (CSF) to serum albumin was consistently increased in hosts with angiostrongyliasis as compared with control. These data clearly indicate that PA and MMP-9 proteolytic enzymes as well as pathological changes are different in permissive and nonpermissive hosts.

Nippostrongylus brasiliensis: infection decreases plasma butyrylcholinesterase activity in rats.

Plasma butyrylcholinesterase (BChE) hydrolyzes ester-containing compounds such as succinylcholine, as well as acting as a scavenger against neurotoxic organophosphates (OPs). We previously found that Nippostrongylus brasiliensis infection makes rats more susceptible to OP toxicity by decreasing serum paraoxonase-1 (PON1) activity. In the present study, we examined the effects of N.brasiliensis infection on acetylcholinesterase (AChE) activity in plasma, red blood cells (RBCs), brain and diaphragm, as well as serum PON1 activity, in rats at day 7 after infection. N.brasiliensis infection significantly decreased plasma BChE and PON1 activities without significantly altering AChE activity in RBCs, brain and diaphragm. These results provide further insight into the unusual deleterious effects of intestinal nematode infections on body homeostasis.

Gastrointestinal nematode infection increases organophosphate toxicity in rats.

Serum paraoxonase-1 (PON1) is an esterase associated with high-density lipoproteins in plasma and is involved in the detoxification of organophosphates (OP). We have previously reported a significant decrease in serum PON1 activity following Nippostrongylus brasiliensis infection in Wistar rats. In the present study we investigated the effects of decreased serum PON1 activity due to N. brasiliensis infection on acute toxicity induced by chlorpyrifos oxon (CPO) and paraoxon (PO) in rats. CPO and PO were dermally applied at doses of 8 mg/kg and 0.2 mg/kg body weight, respectively, to infected (on day 7 post-infection) and uninfected rats, after which acetylcholinesterase (AChE) activity was measured within the brain, diaphragm, plasma, and red blood cells, 4h after administration as a measure of toxicity. In addition, serum PON1 activity was measured immediately prior to administration of CPO and PO. N. brasiliensis infection significantly increased the degree of inhibition of AChE in the brain and diaphragm after treatment with CPO and PO in association with a significant reduction in PON1 activity. Likewise, similar findings were observed in the blood (plasma and RBCs) ChE activity after treatment with PO, but not CPO. These results indicate that N. brasiliensis infection makes rats more susceptible to CPO and PO toxicity, suggesting that gastrointestinal nematode infection might be a potential factor affecting OP toxicity.

Nippostrongylus brasiliensis infection leads to the development of emphysema associated with the induction of alternatively activated macrophages.

Chronic obstructive pulmonary disease (COPD) is the 5(th) most prevalent disease worldwide leading to severe morbidity and mortality in developed countries. The disease is strongly associated with smoking, and can be characterized by progressive and irreversible deterioration in lung function and destruction of the lung parenchyma. We show here that infection with the hookworm Nippostrongylus brasiliensis results in deterioration in lung function, destruction of alveoli and long-term airways hyperresponsiveness, consistent with COPD and emphysema. N. brasiliensis infection leads to chronic low level hemorrhaging in the lung and the presence of hemosiderin-laden macrophages in the absence of an overt inflammatory infiltrate. Microarray analysis of gene expression in diseased lungs and quantitative RT-PCR analysis of purified macrophages revealed a state of prolonged tissue injury and the presence of alternatively activated macrophages producing MMP-12. Taken together, these data show that lung tissue damage caused by hookworm infection can result in the development of COPD and emphysema.

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.

Re-feeding rapidly restores protection against Heligmosomoides bakeri (Nematoda) in protein-deficient mice.

This study determined whether the timing of re-feeding of protein-deficient mice restored functional protection against the gastrointestinal nematode, Heligmosomoides bakeri. Balb/c mice were fed a 3% protein-deficient (PD) diet and then transferred to 24% protein-sufficient (PS) diet either on the day of primary infection, 10 days after the primary infection, on the day of challenge infection, or 7 days after the challenge infection. Control mice were fed either the PD or PS diet. Onset of challenge, but not primary, infection caused short-term body weight loss, anorexia and reduced feed efficiency. Weight gain was delayed in mice when re-feeding commenced on the day of challenge infection; alkaline phosphatase (ALP) was also elevated in these mice on day 28 post-challenge. In contrast, other re-feeding groups attained similar body weights to PS mice within 4 days and had similar ALP at day 28. Serum leptin was higher in PD than PS mice and positively associated with food intake. As expected, worm survival was prolonged in mice fed the PD diet. However, egg production and worm burdens were similar in all re-feeding groups to the PS mice, indicating that protein re-feeding during either the primary or challenge infection rapidly restored normal parasite clearance.

Induction of tumour necrosis factor, interleukin-1beta and matrix metalloproteinases in pulmonary fibrosis of rats infected with Angiostrongylus cantonensis.

In angiostrongyliasis, chronic parasite-induced granuloma formation can lead to tissue destruction and fibrosis. Here, the histomorphology of granulomatous fibrosis and proteinase production in the lungs of Angiostrongylus cantonensis-infected Sprague-Dawley rats were investigated. The relationship between metalloproteinases and granulomatous fibrosis was investigated following infection of each rat with 60 infective larvae. Granulomata and fibrosis were marked in the lungs of rats on day 180 post-inoculation. Reverse transcriptase polymerase chain reaction of lung mRNA showed an up-expression of proinflammatory cytokine including tumour necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1beta). According to Western blot analysis, matrix metalloproteinase-2 (MMP-2) proenzyme was presented in the lungs of uninfected and infected rats, and partial conversion of 72 kDa proenzyme to the 64 kDa active form occurred in infected rats. In addition, increased protein levels of MMP-9 and MMP-13 were detected in infected lungs, but were undetectable in controls. The results suggest that TNF-alpha, IL-1beta, MMP-2, -9, and -13 may be associated with the granulomatous fibrosis.

Anaphylactic release of mucosal mast cell granule proteases: role of serpins in the differential clearance of mouse mast cell proteases-1 and -2.

The granule-derived mouse mast cell proteases-1 and -2 (mMCP-1 and -2) colocalize in similar quantities in mucosal mast cells but micrograms of mMCP-1 compared with nanograms of mMCP-2 are detected in peripheral blood during intestinal nematode infection. This differential systemic response was investigated both in vitro and in vivo. Bone marrow-derived mucosal mast cell homologs released similar quantities of mMCP-1 and-2 concomitantly with beta-hexosaminidase in response to calcium ionophore ( approximately 60% release) or IgE/DNP (25% release). In contrast, serum from mice sensitized by infection with Nippostrongylus brasiliensis 10 days earlier contained >1500-fold more mMCP-1 (10,130 +/- 1,609 ng/ml) than mMCP-2 (6.4 +/- 1 ng/ml), but, in gut lumen, the difference was approximately 8-fold. After OVA sensitization, >600-fold more mMCP-1 (7,861 +/- 2,209 ng/ml) than mMCP-2 (12.8 +/- 4.7 ng/ml) was present in blood 1 h after challenge, but, in gut lumen, there were relatively comparable levels of mMCP-1 and -2. To estimate the rates of systemic accumulation and clearance, 10 microg of mMCP-1 or -2 was injected i.p. Plasma levels of injected mMCP-2 peaked (1%) at 15 min then declined, whereas levels of mMCP-1 were maximal (approximately 25%) at 3 h. Inactivation of mMCP-1 with PMSF before injection resulted in mMCP-2-like kinetics, but inhibition of mMCP-1 by serum gave kinetics similar to that of native mMCP-1. mMCP-1 isolated from serum is complexed with serpins and we conclude that both the accumulation and the longevity of mMCP-1 in blood is due to complex formation, protecting it from a pathway that rapidly clears mMCP-2, which is unable to form complexes with serpins.

Biochemical serum profiles in dogs experimentally infected with Angiostrongylus vasorum (Baillet, 1866).

The biochemical profiles of crossbred dogs experimentally infected with the parasite Angiostrongylus vasorum were studied. Two groups of five dogs were experimentally inoculated with 50 and 100 third stage infective larvae (L3) of A. vasorum per kilogram of body weight. A third group of five uninfected animals were used as control. Serum from these animals were used for biochemical tests to measure total and fractioned proteins, urea, creatinine and to determine the activities of aspartate (AST), alanine (ALT) aminotransferase, gamma-glutamyl transferase (GGT), alkaline phosphatase (PAL) and creatine kinase isoenzyme MB (CK-MB). The alpha-1, alpha-2 and beta-globulins fractions showed alterations during acute phase of the infection. No modifications were observed in the biochemical profiles of ALT, AST, GGT, PAL, urea and creatinine. CK-MB was shown to be a good early indicator of cardiac injury in dogs experimentally infected with A. vasorum.

Matrix metalloproteinase-2 and -9 in the granulomatous fibrosis of rats infected with Angiostrongylus cantonensis.

The histomorphology of granuloma formation and gelatinase production were investigated in the brains, hearts, lungs and livers of Sprague-Dawley rats infected with Angiostrongylus cantonensis. The relationships between two gelatinases and granulomatous fibrosis were explored, following infection of each rat with 60 infective larvae of the nematode. Worm recovery from the brain was maximal on day 15 post-inoculation whereas peak recovery from the lungs was maximal 75 days later, on day 90. The granulomatous reactions and fibrosis were marked in the lungs but only mild, if present at all, in the brain, heart and liver. Gelatin zymography revealed that matrix metalloproteinase2 (MMP-2) was present, at all time-points, in the heart and lungs, although only in the lungs was there partial conversion of the 72-kDa pro-enzyme to the 64-kDa active form during granulomatous fibrosis. The activity of the MMP-9 pro-enzyme was significantly higher at the time-points when granuloma formation was observed than at other times. Immuno-histochemistry revealed MMP-2 and MMP-9 within the lung granulomas, around infiltrating leucocytes and the epithelial cells of the alveoli. As the granulomatous fibrosis appeared to be strongly associated with MMP-2 and MMP-9, these enzymes may be useful markers in the lungs of rats infected with A. cantonensis.