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Immunology. 2004 Mar; 111(3): 343–352.
PMCID: PMC1782416

Expression of matrix metalloproteinases and their inhibitors during the resorption of schistosome egg-induced fibrosis in praziquantel-treated mice

Abstract

Schistosomiasis mansoni is a tropical helminthic disease characterized by parasite egg-induced granulomatous inflammation and cumulative fibrosis. Because fibrosis is influenced by the imbalance between degradative matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), we analysed the resorption of fibrous tissue and MMP/TIMP expression in the livers of S. mansoni-infected and praziquantel-cured mice. Worm elimination significantly enhanced survival rate, ameliorated the granulomatous pathology and reduced collagen I, III and IV gene expression at 6 and 12 months post-treatment. Compared to 6 months infected, untreated controls, liver fibrous tissue was resorbed by 71·4% at 12 months after treatment. At 3 months post-treatment, expression of the MMP-2, -3, -8, -10, -13, -14 and -16 genes decreased compared with untreated controls. By 6 months, a highly significant increase in MMP-10 gene expression was manifest. At 12 months, messages for all MMP genes decreased in relation to untreated controls. TIMP-1, -2 and -3 gene expression drastically decreased between 3 and 6 months. At 1 year, only TIMP-1 expression was significantly diminished. Overall, profibrogenic tumour necrosis factor (TNF)-α, transforming growth factor (TGF)-β and inducible nitric oxide synthase (iNOS) gene expression decreased. Antigen-stimulated splenocytes secreted significantly higher levels of interleukin (IL)-4, IL-5, IL-10 and IL-13 cytokines between 3 and 12 months after treatment. Production of interferon (IFN)-γ was higher than in untreated controls 3 and 6 months after treatment. In conclusion, praziquantel-treated mice showed a slow resorption of liver fibrous tissue. Resorption is attributed to the precipitous drop in TIMP-1 gene expression level, which shifted the balance in favour of MMP message expression and presumed enhanced collagenase activity.

Introduction

Schistosomiasis mansoni is a tropical helminthic disease characterized by parasite egg-induced granulomatous inflammation and fibrosis.1 In a minority of infected humans, Symmers' periportal fibrosis develops in the liver, leading to portal hypertension, esophageal varices, hematemesis and occasional death.2,3 Although during the infection cumulative fibrosis is mostly irreversible, following eradication of the infection by chemotherapy Symmers' fibrosis regresses, as confirmed by ultrasound examination.46 The murine model of the infection shows close similarities in the pathological manifestations to that of the human disease. Whereas early in the granulomatous response hepatic collagen synthesis and collagenase activity occur simultaneously, with the progress of the inflammation collagenase activity decreases but collagen production and deposition continue, leading to pronounced fibrosis.7,8 Several histological and biochemical studies indicated that curative chemotherapy of mice at the acute,912 but not chronic,13 stage of the infection also induced slow resorption of the deposited fibrous tissue. At the microscopic and ultrastructural level, extracellular breakdown of collagen fibrils was observed.14,15 This was attributed to the action of both latent and active collagenase enzymes,16 the latter being attached to collagen fibres.17

Whereas during resorption qualitative changes in the distribution of collagen isotypes have been observed,18 no data are available on the molecular events that take place during the resorption of egg-induced postgranulomatous fibrosis. According to the prevailing observations, liver fibrosis is the outcome of an imbalance between the action of collagen-degradative matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs).19,20

To gain insight into such a balance, the present study analysed collagen isotypes, MMP and TIMP gene expression, and cytokine dynamics in drug-treated mice during the long-term resorption of liver fibrous tissue. Results indicate that during resorption gene expression of TIMPs strongly decreased compared with that of MMPs. Thus, an imbalance that favours MMP enzyme activity can account for matrix degradation.

Materials and methods

Mice, parasites and antigen preparation

Female CBA/Jk mice (Jackson Laboratories, Bar Harbor, ME), 6–8 weeks old, were used throughout the study. All mice were housed under specific-pathogen-free conditions in a Wayne State University School of Medicine animal facility, approved by the American Association for the Accreditation of Laboratory Animal Care. Cercariae of the Puerto Rican strain of Schistosoma mansoni worms were obtained from infected Biomphalaria glabrata snails (Biomedical Research Institute, Rockville, MD). Mice were infected subcutaneously with 30 cercariae. Soluble egg antigens (SEA) were prepared from homogenized eggs as previously described.21

Praziquantel treatment

Praziquantel (Sigma, St. Louis, MO) was dissolved in solvent consisting of 7·4 ml distilled water, 2·5 ml glycerol and 0·1 ml Cremophor EL (Sigma). Animals 8 weeks post-infection were given the drug by gavage twice at a concentration of 300 mg/kg body weight in 200 µl of solvent for 2 days. Control animals received solvent only. Animals from both groups were tested after 3, 6 and 12 months of praziquantel treatment. Animals were killed by cervical dislocation, and their livers and spleens were harvested aseptically and weighed. Some liver pieces were snap-frozen in liquid nitrogen for real-time polymerase chain reaction (PCR) and hydroxyproline assay, and others were fixed in 10% buffered formalin (Sigma) for histology.

Spleen cell culture and cytokine production

Spleens were removed aseptically and single cell suspensions were prepared. Cells were cultured in 24-well tissue culture plates at a final concentration of 5 × 106 cells/ml. The culture medium contained RPMI-1640 supplemented with 10% fetal calf serum, 2 mmol/l glutamine, 1 mmol/l sodium pyruvate, 50 µmol/l 2-mercaptoethanol, penicillin-streptomycin and antimycotic solution (Life Technologies, Gaithersburg, MD). Cultures were incubated at 37° in a humidified atmosphere of 5% CO2. Cells were stimulated with soluble egg antigens (SEA) (10 µg/ml) or medium alone. Supernatant culture media was harvested at 48 hr and assayed for cytokine production.

Cytokine assays

IL-4, IL-10, IL-5, IL-13, interferon (IFN)-γ and transforming growth factor (TGF)-β levels were determined by sandwich enzyme-linked immunosorbent assay with matched antibody pairs (Pharmingen, San Diego, CA) and developed with streptavidin-alkaline phosphatase and para-nitrophenyl phosphate (Sigma). IL-13 levels were measured using murine IL-13 enzyme-linked immunosorbent assay (ELISA) kits (R&D System Inc., Minneapolis, MN).

Liver histology and granuloma measurement

Formalin-fixed liver tissue was processed for histology. Three 5-µm-thick sections were taken at 250-µm-depth intervals from one another. Slides were stained with hematoxylin and eosin or Masson's stain. Liver granulomas were measured in hematoxylin-eosin stained sections. Sections from three different depths of the tissue were scanned for granulomas. The total area of the granulomatous/fibrotic lesions was measured in treated or untreated mice. About 25 readings were made for each liver. Granulomas were measured by computerized morphometry using a Scion software program (Scion Corp., Frederick, MD). Granuloma areas of treated or untreated mice are expressed in square micrometres.

Hydroxyproline assay

The hydroxyproline assay was carried out based on previously reported methodology22 using base23 rather than acid hydrolysis for the dissolution of tissue. Liver tissue was weighed and incubated overnight at 37° in 5% KOH (6 ml/100 mg tissue weight). Tubes were occasionally inverted to resuspend the dissolved tissue. Duplicate sets containing 600 µl of tissue sample, 160 µl 10 N NaOH and 40 µl water were hydrolyzed in screw-capped tubes in an autoclave for 20 min. Normal tissues were used as the background for the standards. A volume of 250 µl of samples in duplicate was taken and supplemented with 250 µl chloramine T solution (saturated in 50% n-propanol/H2O). Samples were incubated at room temperature for 3 hr. Then, 500 µl of freshly prepared Ehrlich's reagent (0·15 g/ml in n-propanol/perchloric acid, 2 : 1 v/v) was added to the tubes and incubated in a 65° waterbath for 20 min. Samples were transferred to a 96-well plate and absorbance at 550 nm was recorded. A standard curve of samples with known quantities of hydroxyproline (Sigma) was generated for each assay. The absorbances of unknown samples were compared to the standard curve, and hydroxyproline content per gram of liver tissue was calculated.

RNA and reverse transcription

Total RNA was extracted from the snap-frozen liver tissue of individual mice at three points during the infection. Samples were homogenized in Trizol (Gibco/BRL, Grand Island, NY) and total RNA was isolated according to the manufacturer's suggested protocol. Pure RNA for reverse transcriptase–polymerase chain reaction (RT-PCR) analyses was produced from total nucleic acid mixtures by extensive treatment with RNAse-free DNAse l (Promega Corporation, Madison, WI), and extractions with buffered phenol and then chloroform/isoamylalcohol (24/1). The ethanol-precipitated and recovered RNA was stored in RNAse-free distilled water. The purity of RNA preparations was assessed spectrophotometrically and by PCR in the absence of preceding RT. For RT experiments, 8 µg of RNA was used for cDNA synthesis, which was performed in a sample volume of 80 µl using random primers and Mu-MLV reverse transcriptase (Gibco/BRL) in the reaction as previously described.24

Real-time quantitative analyses

The relative abundance of mRNA species was assessed using the SYBR® Green method on an ABIprism 7700 sequence detector system (Applied Biosystems, Foster City, CA). PCR primers were designed with Gene Runner Software (Hasting Software, Inc., Hasting, NY) from RNA sequences from GenBank (Table 1). All primer sets had a calculated annealing temperature of 60°. Quantitative RT-PCR was performed in duplicate in a 25-µl reaction volume consisting of 2X SYBR Green PCR Master Mix (Applied Biosystems), 900 nm of each primer and 2–3 µl of cDNA. Amplification conditions were 2 min at 50°, 10 min at 95° and 40 cycles of denaturation for 15 s and annealing/extension at 60° for 10 min. Data from real-time assays were calculated using the v1·7 Sequence Detection Software from PE Biosystems (Foster City, CA). Relative expression of MMP, TIMP, and cytokine mRNA was calculated using the comparative Ct method as previously described.25 All values were normalized to the 18S rRNA genes and reported as fold change over background levels detected in untreated control tissues.

Table 1
Sequence of the murine primers used for real-time PCR

Statistical analysis

Data were expressed as mean ± standard error (SE). Groups were compared using Student's two-tailed t-test. Comparisons were deemed statistically significant when P ≤ 0·05.

Results

Effect of praziquantel treatment on survival rate, body weight, organ weight and spleen cell count in infected mice

Praziquantel treatment increased the survival rate of the infected mice. Three months after treatment, around 45% of untreated mice survived while none of the praziquantel-treated mice died (Table 2). Indeed, no mortality occurred even 12 months after treatment. Whereas no significant difference in body weight was observed between the control and experimental groups, a significant decrease in liver and spleen weights between 3 and 12 months of drug treatment was noted. Spleen cell count was also lower at 3 months and onward after treatment. This reflected the diminished immune/inflammatory response of the treated animals (Table 3).

Table 2
Survival rates of CBA/J mice infected with Schistosoma mansoni and treated with praziquantel
Table 3
Body weights, organ weights and spleen cell counts of CBA/J mice infected with Schistosoma mansoni and treated with praziquantel

Effect of praziquantel treatment on liver pathology of infected mice

Figure 1 shows photomicrographs of typical liver granulomas. The granuloma of an untreated mouse at 3 months contains a live egg surrounded by the predominantly eosinophilic cellular reaction. Deposited matrix surrounds the cells (Fig. 1a). The granuloma of a treated animal contains the remnants of an egg, is less cellular and less condensed, and shows heavy concentric bands of fibrous tissue (Fig. 1b). At 6 months, the granuloma of an untreated mouse is less cellular and more fibrous, whereas that of a treated animal is mostly fibrous, without the previously seen concentric collagen bands (Figs 1c and d). Twelve months after treatment, the resorbing lesion contains a few scattered cells and thin disorganized bands of collagenous fibres (Fig. 1e).

Figure 1
Photomicrographs of florid and involuting liver granulomas at different stages of fibrous tissue resorption. Infected mice were treated orally with 300 mg/kg praziquantel on two successive days. At 3, 6 and 12 months after treatment, control and treated ...

Effect of praziquantel treatment on liver granuloma size

In addition to liver pathology, we also monitored granulomatous responsiveness in the livers of praziquantel-treated and untreated infected mice. As Table 4 shows, relative to untreated controls drug-treated mice showed significant gradual reduction in granuloma size at 3 and 6 months after treatment. Compared with untreated controls 6 months post-infection, the fibrous foci at 12 months post-treatment were significantly reduced in size.

Table 4
Effect of praziquantel treatment on the involution of granulomatous response in Schistosoma mansoni-infected mice

Effect of praziquantel treatment on liver collagen gene expression and hydroxyproline content

A major thrust of this study was to examine whether drug treatment induced diminished collagen gene expression and the resorption of deposited fibrous tissue. As Fig. 2 shows, at 3 months, livers of treated mice showed significant diminution only in collagen IV gene expression. In contrast, significant reduction in collagen I, III and IV gene expression was observed at 6 and 12 months after praziquantel treatment. Changes in the liver content of collagen, a major extracellular matrix component of fibrosis, were also evaluated by measuring hepatic hydroxyproline levels. As shown in Table 5, livers of age-matched uninfected mice had low hydroxyproline levels. By the eighth week of the infection, liver collagen content increased about 3-fold. At 3 months this increased 2·5-fold compared to the 8-week post-infection values. At 3 months post-treatment, the collagen content in the liver has not yet significantly decreased compared with untreated controls. Whereas at 6 months the hydroxyproline content in the livers of untreated mice showed a further increase, that of drug-treated animals significantly decreased. At 12 months after drug treatment, hydroxyproline values further decreased. Compared with untreated controls 6 months post-infection, the total amount of deposited fibrous tissue diminished by about 71%.

Figure 2
Relative collagen gene expression levels in the livers of infected, praziquantel-treated mice. Real-time RT-PCR assays were performed for the livers of mice at (a) 3 months, (b) 6 months and (c) 12 months after treatment. Data represent means ± ...
Table 5
Hydroxyproline content in liver tissue of CBA/J mice infected with Schistosoma mansoni and treated with praziquantel

Effect of praziquantel treatment on gene expression of MMPs, TIMPs and cytokines

Matrix metalloproteinases are key enzymes in the degradation of collagens present in the matrix of deposited fibrous tissue.26 In order to establish a role for MMP gene expression in the resorption of deposited fibrous matrix, we followed, over 12 months post-treatment, the expression of several metalloproteinase genes that code for gelatinase (MMP-2 and -9), stromelysin (MMP-3, and -10), collagenase (MMP-8 and -13) and membrane-bound matrix-active enzymes (MMP-14 and -16) active in the liver. The results presented in Fig. 3 show that at 3 months after treatment levels of most of the MMP gene messages diminished compared with controls. The increase in MMP-9 levels was statistically not significant (Fig. 3a). By 6 months, a highly significant increase in MMP-10 expression was noted, whereas expression of the other MMP genes was either unchanged or diminished significantly compared with values of the untreated group (Fig. 3b). At 12 months after treatment, levels of messages for all MMP genes were significantly decreased, but the expression of the MMP-10 gene continued to be somewhat elevated (Fig. 3c).

Figure 3
Relative MMP gene expression levels in the livers of infected, praziquantel-treated mice. Experimental conditions and statistical evaluations are as in Fig. 2.*, P < 0·05.

According to our working hypothesis, during the process of fibrous tissue resorption MMP gene expression should be enhanced, and that of the inhibitors (TIMPs) should be curtailed. Examination at intervals after treatment of TIMPs 1–4 showed that already at 3 months TIMP-1 gene expression drastically decreased to one-tenth of the level of untreated controls, whereas mRNA for TIMP-2, -3 and -4 showed no or little change (Fig. 4a). By 6 months post-treatment, in addition to the decreased TIMP-1, messenger levels for TIMP-2 and -3 were also diminished, while for TIMP-4 levels were comparable to those of untreated controls (Fig. 4b). At 12 months, TIMP-1 gene message levels were still diminished, whereas expression of the three TIMP genes did not significantly differ from the 6-month control values (Fig. 4c).

Figure 4
Relative TIMP gene expression levels in the livers of infected, praziquantel-treated mice. Experimental conditions and statistical evaluations are as in Fig. 2.

Cytokines such as TNF-α27 and TGF-β,28,29 as well as released nitric oxide (NO),30 are known to influence MMP and TIMP expression. Therefore, we intended to correlate cytokine and iNOS message levels with those of MMP and TIMP. As seen in Fig. 5, compared with untreated control livers, TNF-α messenger showed no change at 3 months, whereas a drastic drop was seen by 6 and 12 months in the treated animals. TGF-β gene expression decreased by 70% at 3 months post-treatment and remained at this level until the conclusion of the study. Expression of the iNOS gene significantly diminished by 3 months and onwards compared with untreated controls. As a corollary, type 1 and type 2 cytokine production by SEA-stimulated splenocytes was also examined. Compared with controls at the post-treatment times, a conspicuous and significant increase in IL-4 and IL-5 production was observed in the treated group between 3 and 12 months post-treatment. By 6 and 12 months, an increase in IL-13, and to a lesser degree IL-10 secretion was noticed in cell cultures of drug-treated mice.

Figure 5
Relative TNF-α, TGF-β and iNOS gene expression levels in the livers of infected, praziquantel-treated mice. Experimental conditions and statistical evaluations are as in Figs 2 and and33.

However, compared with the 3-month production level, IL-10 secretion at 6 and 12 months post-treatment greatly decreased. IFN-γ production at 3 and 6 months exceeded that of the untreated group, but by 12 months levels decreased and were equal to the 6-month control values. The overall levels of secreted TGF-β were very low throughout the period of the examination, with minimal or no difference between treated and control groups (Fig. 6).

Figure 6
Lymphokine levels in splenic culture supernatants of infected untreated and praziquantel-treated mice. Spleen cells were tested at 3, 6 and 12 months after treatment. A concentration of 5 × 106 cells/ml was stimulated with 10 µg/ml SEA ...

Discussion

In the present study, we analysed MMP and TIMP gene expression and its relationship to fibrous tissue resorption in praziquantel-treated, S. mansoni-infected mice. In agreement with previous observations,911 chemotherapy of mice significantly ameliorated both morbidity and mortality. Within the first 6 months of worm eradication, hepato-splenomegaly significantly diminished. Moreover, the 50% mortality observed in the untreated infected group was completely eliminated. This was very conspicuous at 1 year post-treatment, by which time all the untreated mice were dead, whereas all the treated ones survived. High survival in our experiments compared with mortality reported previously in drug-treated mice31 is attributed to the light worm load, the different drug dosage and the murine strain used. Three months after chemotherapy, granulomas decreased in size, became less cellular, and contained thick concentric fibrous bands. By 1 year granulomas were replaced by small residual fibrous foci that contained mostly thin, disorganized fibrils and amorphic hyalinized pink-staining tissue. At that time, total liver collagen content significantly decreased to about one-third of the 6-month post-infection control values. These observations are in agreement with previous publications.915

Gene expression analysis revealed two major factors that could influence total liver collagen content and the process of fibrous tissue resorption: (1) gradually diminishing collagen gene expression and (2) a change in the balance of MMP:TIMP gene expression in favour of the former. Compared with infected control mice, within 1 year, a gradual decrease in collagen I, III and IV gene expression was seen in the treated animals. With the elimination of eggs and the subsidence of the inflammatory response, collagen gene expression was minimal, indicating the termination of the fibrotic process.

MMPs are the major enzymes that degrade the various types of collagen.26 After drug therapy, expression of several MMP genes showed dynamic changes. At 3 months, with the exception of MMP-9, all the examined MMP genes showed diminished expression compared with the livers of untreated mice. Previous observations established that collagen production and collagenase activity peaked at the height of the granulomatous response.7,8 Here, we showed that collagen and MMP gene expression diminished in a linked fashion in the treated animals. It appears that, with the weakening of the inflammatory signal(s) and in the face of decreased collagen production and deposition, expression of MMP genes also decreased. With the further involution of the granulomatous response at 6 months, expression of collagen I, III and IV genes continued to diminish. In contrast, MMP-10 (stromelysin-2) gene expression was greatly enhanced. Because the gene codes for an enzyme that has broad substrate specificity and degrades collagens I, III and IV as well as gelatin,32 its heightened expression is consistent with the ongoing resorption of fibrous liver tissue. At 12 months post-treatment, with the exception of MMP-10 message, all other MMP gene expression diminished in comparison with the values seen at 6 months in the untreated group. Because by that time the bulk of the deposited fibrous tissue had been resorbed, the curtailed MMP gene expression is appropriate.

Collagenases are regulated by TIMPs that block their action.20,33 Therefore, it is noteworthy that within 3 months post-treatment TIMP-1 expression diminished 10-fold compared with livers of untreated mice. This strong decrease, which persisted until 12 months, is highly significant because TIMP-1 is the predominant gene expressed in the granulomatous livers (K. P. Singh et al., manuscript in preparation). Because TIMP-1 blocks the enzymatic activity of several different MMPs,20,33 its greatly diminished production should enhance collagenase activity and fibrous tissue degradation. In addition to TIMP-1, by 6 months after treatment, TIMP-2 and -3 gene expression also decreased. Thus, the balance in MMP:TIMP expression is shifted towards MMP, even in the face of lowered MMP gene expression. This shift constituted the basis of scar tissue resorption. This is supported by the difference in the collagen content of control livers (6-month post-infection) and 12-month post-treatment livers, which shows a resorption of over 70% of the deposited fibrous tissue. These observations are in agreement with those on carbon tetrachloride-induced reversible rat liver fibrosis, where resolution of the fibrosis was associated with decreased TIMP expression.34 Whereas using TIMP-1- and TIMP-2-deficient C57BL/6 mice those investigators found no role for the inhibitors in schistosome egg-induced fibrogenesis,35 here we showed an association between greatly diminished TIMP-1 expression and enhanced MMP-mediated resorption of liver fibrous tissue.

The cellular source(s) of collagenases still needs clarification. Previously, we have shown that both macrophages and eosinophils isolated from the vigorous, florid granulomas secrete collagenases.36 Moreover, ongoing studies showed MMP-10 expression in granuloma myofibroblasts isolated from acute infection lesions. With the involution of the granulomas in drug-treated mice, the cellular content of the lesions gradually diminished. Because at 6 and 12 months post-treatment MMP gene expression is elevated, even though many of the granulomas disappeared, leaving behind small acellular or sparsely cellular fibrotic spots, it is plausible that the source of the secreted collagenases is hepatic myofibroblasts, Kupffer cells, and hepatocytes.29

During the egg granulomatous inflammatory response, both pro, IL-13-mediated37,38 and antifibrogenic (IFN-γ, IL-12) cytokine signals are activated.39,40 However, in general very little data exist on signals that induce enhanced collagenase expression. At 6 and 12 months, the livers of drug-treated mice showed diminished TNF-α (a potential MMP inducer)27 and TGF-β (a TIMP inducer)41 gene expression. The significantly diminished gene expression of the latter is supportive of reduced TIMP-1 induction. A similar drop in the TNF-α signal may also explain the observed diminished expression of the majority of MMPs. Because such enzymes are also regulated at the gene level by IL-1, platelet derived growth factor (PDGF), epidermal growth factor (EGF) and β-fibroblast growth factor (FGF) cytokines,20 at present no conclusion can be drawn about the putative cytokine that enhanced MMP-10 gene expression. The diminished iNOS expression in drug-treated mice is in agreement with previous observations that associated iNOS expression with the evolving granulomatous response42 and antifibrotic effect.43

A comparison at 3, 6 and 12 months of memory T-cell responses of infected untreated versus treated mice revealed a shift from the balanced Th0 to enhanced IL-4, IL-5 and, to a lesser degree, IL-13 production. A similarly enhanced cytokine expression in drug-treated, schistosome-infected humans was also observed.44,45 Elimination of worm-induced immunosuppression may be one factor causing this phenomenon. A similar explanation in our experiments cannot be excluded. It is noteworthy that, with the cessation of SEA stimulus at 6 and 12 months post-treatment, IL-10 production greatly decreased. This decrease may be associated with increased IL-5 production observed at 12 months, as shown previously in infected anti-IL-10 mAb-treated animals.46 It is of interest that antigen-stimulated memory splenocytes produced elevated levels of IL-13 but not TGF-β. Whether the strong anamnestic response of IL-5 and profibrogenic IL-13 on re-infection will produce enhanced tissue eosinophilia and fibrotic reaction will be examined in a subsequent study. The possible role of the sustained antifibrotic IFN-γ signal at 3 and 6 months after treatment in stimulated memory cells also deserves future attention.

In mice, the slow resorption of fibrous tissue following treatment has been linked to the timing of the treatment. Early treatment, started at 8 weeks post-infection, as was the case in our study, has yielded better resorption of the fibrous tissue compared with later treatment.47 This is probably related to the degree of cross-linkage of the deposited collagen, the extent of which is directly related to resistance to enzymatic degradation.48

Previous publications demonstrated that after antihelminthic drug treatment liver fibrosis undergoes a slow process of resorption.1116 The present study is in agreement with previous studies. Here, we provide for the first time the molecular basis for the dynamics of the MMP:TIMP gene expression that regulates the resorption process. With the cessation of inflammation and reparative fibrous tissue production, TIMP-1 expression greatly diminished. Messages for most of the MMPs remained at similar levels or decreased compared with controls. Thus, removal of the inhibitory effect of TIMPs and the unhindered action of MMPs might have constituted the basis for the process of resorption. Future studies aimed at correlating gene expression with protein function and regulation of MMPs and TIMPs should be useful in the elucidation of the resorption process.

Acknowledgments

The work was supported by Public Health Service grants AI12913 from the National Institute of Allergy and Infectious Diseases (DLB) and AR-42541 (APH). Schistosome life stages for this work were supplied through NIH NIAD contract NO1-AI-55270.

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