Tumor progression is associated with a significant decrease in the expression of the endostatin precursor collagen XVIII in human hepatocellular carcinomas.

Endostatin inhibits angiogenesis and tumor growth in mice. The role of its endogenous precursor collagen XVIII in human cancer is unknown. In normal tissues, two variants of collagen XVIII, namely, the short and long forms regulate tissue specificity, the long form being almost exclusively expressed by hepatocytes in the liver. We analyzed RNA arrays from 57 hepatocellular carcinomas (HCCs) with common and variant-specific probes and investigated the relationships between collagen XVIII expression and angiogenesis by measuring the CD34-positive microvessel density. Low collagen XVIII expression by tumor hepatocytes was associated with large tumor size (r, -0.63; P < 0.001) and replacement of trabeculae with pseudoglandular-solid architecture (chi2, 28; P < 0.001), which indicate tumor progression. Tumors expressing the highest collagen XVIII levels were smaller and had lower microvessel density (P = 0.01) than those expressing moderate levels; and HCCs with the lowest collagen XVIII levels approached a plateau of microvessel density, which indicated that a decrease in collagen XVIII expression is associated with angiogenesis in primary liver cancer. HCCs recurring within 2 years of resection showed 2.2-fold lower collagen XVIII mRNA than nonrecurring ones (P = 0.02). The findings relied on the hepatocyte-specific long form. Thus, the endogenous expression of the endostatin precursor decreases along with tumor progression in HCCs.

Enhanced myocardial lesions in chronically Trypanosoma cruzi-infected rats subjected to adult thymectomy.

Control animals and rats infected 90 days earlier, by inoculation of 1 x 10(6) trypomastigotes of Trypanosoma cruzi at weaning, were subjected to adult thymectomy (ATx) or sham operation (S-ATx) and assessed 3 months later for the presence of myocardial lesions and levels of lymph node and spleen T-cell populations. Chronic focal myocarditis (CFM) developed in 78% and 84% of S-ATx or ATx infected rats, respectively. While the two groups of infected rats did not differ as to the occurrence of myocardial lesions, large foci of CFM were more prevalent in ATx infected rats. Chronic T. cruzi (Tc) infection resulted in decreased CD4+ and increased CD8+ lymph node and spleen cell, with CD8+ lymphocytes being lowered to normal values in the spleen of the ATx infected group. It is suggested that ATx might act by interfering with a down-regulating immunoregulatory mechanism, leading to an exacerbation of autoimmune reactions believed to be involved in the generation of myocardial damage.

Infection with Trypanosoma cruzi during pregnancy in rats and a decrease in chronic myocardial lesions in their infected offspring.

To ascertain whether maternal infection with Trypanosoma cruzi may influence the course of the parasitic infection in offspring, two groups of female 1 rats were mated with syngeneic sires. One group of females was infected with 10(6) trypomastigotes of T. cruzi three times at weekly intervals. All offspring were nursed by their mothers until weaning and then separated into two groups of young, one to be infected with the same dose of T. cruzi, and the other to remain uninfected. Infection of pregnant rats caused no aggravated disease but resulted in a self-controlled infection that did not cause any deaths or affect their reproductive capacity. The number of young delivered, litter size, fertility coefficient, and offspring weights at weaning were also unaffected by maternal infection; however, the survival coefficient decreased in comparison with values recorded in the offspring of uninfected mothers. The latter finding is likely due to neonatal transmission, since bloodstream forms of T. cruzi were observed in a few offspring of infected mothers. While infected offspring whose mothers had been inoculated with T. cruzi during pregnancy were not protected from acute infection, the occurrence of chronic focal myocarditis was less prevalent when compared with that recorded in chronically infected offspring born to uninfected mothers.

In situ detection of human cytomegalovirus DNA in gastrointestinal biopsies from AIDS patients by means of various PCR-derived methods.

The development of new in situ assessment of HCMV disease on endoscopical gastrointestinal biopsies from AIDS patients is described and compared with the viral load measured by semiquantitative solution-phase PCR (SQ-PCR). Ten biopsies were examined by viral isolation, standard histology, in situ hybridization (ISH), in situ PCR-hybridization (PCR-ISH) and SQ-PCR, using the same target sequence. The methods developed for in situ HCMV detection were HCMV primers, the plasmid pCMV 406-S, a vector-free-digoxigenin-labelled HCMV-362 probe and the pSK + MCS nonsense probe. Paraffin-embedded MRC5 cells, either HCMV-infected or uninfected served as controls of specificity for ISH. beta-Actin primers were designed as markers of DNA integrity. Computerized models of the PCR, solution-phase and in situ PCR on formalin-fixed DNA indicated that HCMV and beta-actin primers were efficient and specific. Nine biopsies were negative for HCMV by histology and virus isolation. SQ-PCR revealed 80,000; 80 and < 80 HCMV genomic equivalents in 6, 2 and 2 biopsies, respectively. In 8 biopsies, both ISH and PCR-ISH identified positive nuclei in the intestinal epithelium, with sparing of the lamina propria. This indicates that an improvement in in situ methods can help the timely diagnosis of HCMV infection. Direct in situ PCR with beta-actin primers showed a positive signal in all the nuclei in the tissue sections, whereas omission of Taq polymerase resulted in an absence of signal, implying optimal in situ PCR. The data suggest an early-stage reactivation of HCMV, possibly harboured in the intestinal epithelium.

Assessing matrix metalloproteinase expression and activity in hepatocellular carcinomas.

The matrix metalloproteinases (MMPs) constitute a large family of zincand calcium-dependent endopeptidases that cleave extracellular matrix components (1). Hence, MMPs are classified according to their substrate specificities: interstitial collagenases, stromelysins, gelatinases, membrane-type matrix metalloproteinases (MT-MMPs), and elastase (Table 1). The regulation of MMP activity involves gene expression, proteolytic processing of the propeptides to active forms, and inhibition by specific tissue inhibitors of matrix metalloproteinase (TIMPs). MMPs are involved in situations that require extracellular matrix remodeling, including wound healing, development, inflammation, fibrosis angiogenesis, and tumor invasion (2-5). Several complementary methods have provided an insightful description of the expression levels of MMPs and their pathological correlates. These include immunohistochemistry and Northern, Western, and dot blots. Additionally, the activity of MMPs is evaluated by gel substrate analysis. This approach has demonstrated that an increase in the expression of MMP2 (6,7), MT1-MMP (7), TIMP1, and TIMP2 (8-10) is associated with liver fibrosis. Similarly, in hepatocellular carcinomas, a high expression of MMP2, MMP9, MT1-MMP, and matrilysin is related to tumor aggressiveness (11-14). Consistently, by gel substrate analysis, MMP2 activity is increased in primary and secondary liver cancers (13,15,16). By in situ hybridization, the sources of.

Blocking Wnt signaling by SFRP-like molecules inhibits in vivo cell proliferation and tumor growth in cells carrying active ß-catenin.

Constitutive activation of Wnt/ß-catenin signaling in cancer results from mutations in pathway components, which frequently coexist with autocrine Wnt signaling or epigenetic silencing of extracellular Wnt antagonists. Among the extracellular Wnt inhibitors, the secreted frizzled-related proteins (SFRPs) are decoy receptors that contain soluble Wnt-binding frizzled domains. In addition to SFRPs, other endogenous molecules harboring frizzled motifs bind to and inhibit Wnt signaling. One of such molecules is V3Nter, a soluble SFRP-like frizzled polypeptide that binds to Wnt3a and inhibits Wnt signaling and expression of the ß-catenin target genes cyclin D1 and c-myc. V3Nter is derived from the cell surface extracellular matrix component collagen XVIII. Here, we used HCT116 human colon cancer cells carrying the ΔS45 activating mutation in one of the alleles of ß-catenin to show that V3Nter and SFRP-1 decrease baseline and Wnt3a-induced ß-catenin stabilization. Consequently, V3Nter reduces the growth of human colorectal cancer xenografts by specifically controlling cell proliferation and cell cycle progression, without affecting angiogenesis or apoptosis, as shown by decreased [(3)H]-thymidine (in vitro) or BrdU (in vivo) incorporation, clonogenesis assays, cell cycle analysis and magnetic resonance imaging in living mice. Additionally, V3Nter switches off the ß-catenin target gene expression signature in vivo. Moreover, experiments with ß-catenin allele-targeted cells showed that the ΔS45 ß-catenin allele hampers, but does not abrogate, inhibition of Wnt signaling by SFRP-1 or by the SFRP-like frizzled domain. Finally, neither SFRP-1 nor V3Nter affect ß-catenin signaling in SW480 cells carrying nonfunctional Adenomatous polyposis coli. Thus, SFRP-1 and the SFRP-like molecule V3Nter can inhibit tumor growth of ß-catenin-activated tumor cells in vivo.

MMP2 activation by collagen I and concanavalin A in cultured human hepatic stellate cells.

Fibrosis occurs in most chronic liver injuries and results from changes in the balance between synthesis and degradation of extracellular matrix components. In fibrotic livers, there is a markedly increased activity of matrix metalloproteinase 2 (MMP2), a major enzyme involved in extracellular matrix remodeling. We have previously shown that hepatic stellate cells secrete latent MMP2 and that MMP2 activation occurs in coculture of hepatic stellate cells and hepatocytes concomitantly with matrix deposition. In the present work we investigated the effects of various extracellular matrix components and concanavalin A, an inducer of immune-mediated liver injuries, on MMP2 activation in cultured human hepatic stellate cells. Collagen I induced a dose-dependent MMP2 activation, which was not blocked by both actinomycin and cycloheximide. Collagen VI, laminin, and a reconstituted basement membrane (matrigel) were ineffective in inducing activation. Specific antibodies against the subunits of alpha2beta1 integrins, the major collagen I receptor, induced partial inhibition of MMP2 activation. Treatment of cells with concanavalin A resulted in a marked activation of MMP2 that correlated with the proteolytic processing of MT1-MMP, the MMP2 activator, from a Mr=60 kd toward a Mr=43 kd polypeptide. Actinomycin and cycloheximide inhibited the MMP2 activation induced by concanavalin A. Recombinant tissue inhibitor of metalloproteinase-2 and the MMP inhibitor BB-3103, but not PMSF, blocked MMP2 activation induced by collagen I or concanavalin A, and MT1-MMP processing to its Mr-43 kd form. These results suggest that the accumulation of collagen I may specifically contribute to the remodeling of extracellular matrix in fibrotic livers by inducing MMP2 activation.

Activation of matrix metalloproteinase-2 from hepatic stellate cells requires interactions with hepatocytes.

Activation of matrix metalloproteinase (MMP)-2, the 72-kd collagenase IV/gelatinase A, is involved in extracellular matrix remodeling. It has been suggested that a membrane-type MMP (MT-MMP-1) and the tissue inhibitor of metalloproteinase (TIMP)-2 are involved in MMP-2 processing, but the exact mechanism(s) of its activation remains unclear. We have investigated the role of cell-cell cooperation in the activation of pro-MMP-2 in the liver, using pure cultures and co-cultures of hepatocytes and hepatic stellate cells (HSCs). Northern blot analysis and in situ hybridization showed that, in both pure and co-cultures, HSCs, but not hepatocytes, expressed MMP-2, TIMP-2, and MT-MMP-1 mRNA. Zymography analyses revealed the latent form of MMP-2 in medium from 2-day-old pure HSC cultures with higher amounts in medium from hepatocyte/HSC co-cultures. When hepatocytes were added to 10-day-old HSC cultures, the activated form of MMP-2 was detected, concomitantly with the deposition of an abundant extracellular matrix. Incubation of plasma membrane-enriched fractions from hepatocytes with conditioned medium from pure HSC cultures generated the activated species of MMP-2 (62 and 59 kd). Activation of pro-MMP-2 by hepatocyte membranes was inhibited by EDTA, heat, and trypsin but not by serine proteinase inhibitors. These data show that the co-expression of TIMP-2, MMP-2, and MT-MMP-1 by HSCs does not lead to secretion of the activated form of MMP-2. Hepatocytes, which do not express MMP-2, TIMP-2, or MT-MMP-1, induce MMP-2 activation through a plasma membrane-dependent mechanism(s), thus suggesting that cell-cell interactions are involved in this process in vivo.

The promoter of the long variant of collagen XVIII, the precursor of endostatin, contains liver-specific regulatory elements.

The endostatin precursor collagen XVIII is expressed at high levels in human livers, the main source being hepatocytes. We have studied the regulatory elements in the promoter 2 of the Col18a1 gene that directs the transcription of the NC1-517 variant of collagen alpha1(XVIII), which is the main form expressed in the liver. The 5'-flanking region of Col18a1 gene was cloned, and a series of 5'-deletions from -3286 bp to +285 bp were linked to the luciferase reporter gene. Transfection experiments in HepG2 cells allowed to identify a silencer-like element containing putative HNF1 and HNF3 sites and activator elements containing stretches of GC-rich sequences. Another putative HNF3 site in close apposition to a NF1/CTF site was localized upstream of the silencer-like element. Cotransfection experiments showed that the Col18a1 promoter 2 was transactivated by Sp1 and HNF3alpha. Gel-shift analyses showed that HNF3, NF1/CTF, and Sp1-like sites specifically recognized nuclear factors. Super-shift experiments indicated that HNF3beta was the major form of HNF3 interacting with the HNF3/NF1 site. The well-differentiated hepatoma cell line mhATFS315 transfected with a truncated form of HNF3beta, which competitively blocks HNF3 transactivating activity, expressed the Col18a1gene at a very low level. Taken together, these data strongly suggest that Col18a1 is a liver-specific gene. Furthermore, gel-shift analyses performed with nuclear factors prepared from well-differentiated hepatocellular carcinomas showed increased HNF3/NF1 binding activity compared with normal livers. Consequently, the precursor of endostatin might be differently expressed according to the differentiated and/or transformed state of hepatocytes.

Differential expression and origin of membrane-type 1 and 2 matrix metalloproteinases (MT-MMPs) in association with MMP2 activation in injured human livers.

Matrix metalloproteinase-2 (MMP2) activation is associated with basement membrane remodeling that occurs in injured tissues and during tumor invasion. The newly described membrane-type MMPs (MT-MMPs) form a family of potential MMP2 activators. We investigated the localization and steady-state levels of MT1-MMP and MT2-MMP mRNA, compared with those of MMP2 and tissue inhibitor of MMP-2 in 22 hepatocellular carcinomas, 12 liver metastases from colonic adenocarcinomas, 13 nontumoral samples from livers with metastases, 10 benign tumors, and 6 normal livers. MMP2 activation was analyzed by zymography in the same series. The expression of MT1-MMP mRNA and the activation of MMP-2 were increased in hepatocellular carcinomas, metastases, and cholestatic nontumoral samples. MT2-MMP mRNA was rather stable in the different groups. MT1-MMP mRNA levels, but not MT2-MMP mRNA, correlated with MMP-2 and tissue inhibitor of MMP-2 mRNA levels and with MMP2 activation. In situ hybridization showed that MT1-MMP mRNA was expressed in stromal cells, and MT2-MMP mRNA was principally located in both hepatocytes and biliary epithelial cells. Consistently, freshly isolated hepatocytes expressed only MT2-MMP mRNA, and culture-activated hepatic stellate cells showed high levels of MT1-MMP mRNA. These results indicate that in injured livers, MMP2 activation is related to a coordinated high expression of MMP2, tissue inhibitor of MMP-2, and MT1-MMP. Furthermore, the finding of a preferential expression of MT2-MMP in hepatocytes, together with our previous demonstration that the activation of stellate cell-derived MMP2 in co-culture requires interactions with hepatocytes (Am J Pathol 1997, 150:51-58), suggests that parenchymal cells might play a pivotal role in the MMP2 activation process.

Increased extracellular matrix remodeling is associated with tumor progression in human hepatocellular carcinomas.

Matrix metalloproteinase-2 (MMP2) is a key enzyme in the process of extracellular matrix remodeling involved in tumor invasion and metastasis. The activation of MMP2 involves interplay with the membrane type-matrix metalloproteinase-1 (MT1-MMP) and the tissue inhibitor of metalloproteinase-2 (TIMP2). In vitro, activated hepatic stellate cells are a main source of MMP2 and collagen I induces MMP2 activation. The steady-state mRNA levels of MMP2, MT1-MMP, TIMP2, collagen I, collagen IV, and laminin gamma1 were compared with MMP2 activity in 55 hepatocellular carcinomas, 47 matching nontumor biopsies and 19 histologically normal livers. In hepatocellular carcinomas, increased collagen I mRNA levels were strongly associated with those of MMP2 (Spearman R =.74, P <.001), MT1-MMP (R =.65, P <.001) and TIMP2 (R = 0.61, P <.001). MMP2 activity was correlated with the mRNA expression of collagen I (R =.45 P <.01), collagen IV (R =.40, P <.01) and laminin gamma1 (R =.33, P <.05). Unlike collagen IV and laminin gamma1 mRNAs, MMP2, MT1-MMP, TIMP2, collagen I mRNA levels were increased in nonencapsulated compared with encapsulated tumors (P <.05). In addition, MMP2 activity was fourfold higher (P <.01) in tumors arising in cirrhotic livers than in those arising in noncirrhotic livers. Moreover, tumor recurrence was associated with 4.6- and 2.8-fold (P <.05) higher collagen I and MMP2 mRNA levels, respectively, in hepatocellular carcinomas arising in cirrhotic livers. Thus, a high extracellular matrix remodeling favors tumor progression in hepatocellular carcinomas.

In situ detection of matrix metalloproteinase-2 (MMP2) and the metalloproteinase inhibitor TIMP2 transcripts in human primary hepatocellular carcinoma and in liver metastasis.

BACKGROUND/AIMS: Metalloproteinase (MMP)-2 and the metalloproteinase inhibitor TIMP2, play a critical role in tumor invasion. We have investigated the cellular sources of MMP2 and TIMP2 in primary and secondary human liver cancers. METHODS: Using in situ hybridization and zymography, we analyzed surgical biopsies from matching pairs of tumoral and non-tumoral liver from six hepatocellular carcinomas and seven liver metastases and from four liver donors. The cellular sources of MMP2 and TIMP2 were further characterized using an anti-alpha-smooth muscle actin antibody on contiguous sections. RESULTS: In hepatocellular carcinoma and liver metastases, in situ hybridization showed that MMP2 and TIMP2 mRNA were expressed by anti-alpha-smooth muscle actin-positive cells at the invasive front. Slender fibroblasts embedded in a denser matrix were MMP2(+)/TIMP2(+)/anti-alpha-smooth muscle actin(+). Intratumor microvessels showed a strong labeling for MMP2 but weak for TIMP2 mRNA. In contrast, the endothelial lining of the central veins was MMP2(+)/TIMP2(+) in non-tumoral areas with signs of blood-flow obstruction. In control livers, MMP2 and TIMP2 mRNA distribution was restricted to fibroblasts and endothelial cells within portal tracts and scattered sinusoidal cells. Direct zymography of samples comprising the invasive front revealed variable amounts of both proMMP2 and its active form in hepatocellular carcinoma, whereas strong bands corresponding to both active and latent forms of MMP2 were detected in liver metastases. CONCLUSIONS: The striking density of MMP2(+)/TIMP2(+)/anti-alphaSM(+) stellate-shaped cells in the perisinusoidal space adjacent to liver tumors suggests that hepatic stellate cells, upon differentiation to myofibroblasts, may contribute to the dissemination of liver metastases through the sinusoidal network.

Sp1-mediated transactivation of LamC1 promoter and coordinated expression of laminin-gamma1 and Sp1 in human hepatocellular carcinomas.

The laminin-gamma1 chain is present in most basement membranes and is involved in various physiological and pathological processes, including carcinogenesis in the liver. We have investigated the role of the transcription factor Sp1 in the activation of the LamC1 gene, which encodes laminin-gamma1, both in hepatocytes and in human hepatocellular carcinomas. DNAse I hypersensitive sites were mapped in the murine LamC1 promoter using early hepatocyte primary cultures in which LamC1 becomes activated. Three hypersensitive sites were found in enhancer-like elements that contain GC-rich regions. Gel-shift analyses showed that specific complexes were resolved using GC-containing oligonucleotides and Faza 567 hepatoma cells, which constitutively express laminin-gamma1 at a high level. Increased GC-binding activity was observed using nuclear extracts from early hepatocyte cultures versus normal liver. Sp1 overexpression in normal hepatocytes transfected with an Sp1 expression vector induced a marked increased of laminin-gamma1 mRNA content and co-transfection of promoter fragments in Drosophila melanogaster SL2 cells demonstrated that Sp1 transactivates LamC1. In human hepatocellular carcinomas, Sp1 and laminin-gamma1 mRNA were simultaneously expressed at high levels, and gel-shift experiments demonstrated a higher GC-binding activity to Sp1 compared with control livers. In situ hybridization indicated that cells exhibiting a high content of laminin-gamma1 mRNA were also strongly positive for Sp1 mRNA, including both cancer cells at the invasion front and stromal cells. These results show that Sp1 is involved in the activation of LamC1 that occurs in human hepatocellular carcinomas.

Overexpression of matrix metalloproteinase-2 and tissue inhibitor of matrix metalloproteinase-2 in liver from patients with gastrointestinal adenocarcinoma and no detectable metastasis.

Degradation of basement membranes is a key step in tumoral invasion, mainly mediated by matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). Since the liver is a main target for metastases from gastrointestinal adenocarcinoma, we have investigated MMP2 and TIMP2 expression by RT-PCR, in situ hybridization and zymography in the liver of patients with gastrointestinal adenocarcinomas and no detectable hepatic metastasis (n = 12), in tumoral and nontumoral liver from patients with hepatic metastasis (n = 9) and in control liver (n = 4). MMP2 and TIMP2 mRNA levels were increased in liver from patients with gastrointestinal adenocarcinomas and no detectable metastasis, compared with those of either control liver (5-fold and 3.2-fold, respectively) or nontumoral areas of liver from patients with metastasis (7.8-fold and 3-fold, respectively). MMP2 and TIMP2 transcripts were located in mesenchymal cells of portal tracts and sinusoids. MMP2 was mainly in its latent form. In liver from patients with hepatic metastasis, the tumoral/nontumoral ratios for MMP2 and TIMP2 mRNA were 6.2 +/- 4 and 1.5 +/- 0.4, respectively. Both transcripts were localized in the stromal cells of liver metastases, and the active form of MMP2 was found only in the tumoral areas. In the matching nontumoral areas the signals for MMP2 and TIMP2 mRNA were restricted to mesenchymal cells in portal tracts and sinusoidal cells. Our data show that liver stromal cells express high levels of MMP2 and TIMP2 in patients with colonic carcinoma without liver metastasis, suggesting the distant induction of these transcripts by the primary tumor.

Tumor hepatocytes and basement membrane-Producing cells specifically express two different forms of the endostatin precursor, collagen XVIII, in human liver cancers.

Endostatin is an endogenous inhibitor of angiogenesis and tumor growth in mice, which may be generated by proteolytic cleavage of collagen XVIII. In normal tissues, 2 variants of the endostatin precursor, namely the SHORT and LONG forms, regulate tissue specificity. We analyzed 53 human liver biopsies (18 hepatocellular carcinomas, 16 metastases of colorectal cancer, 3 cholangiocarcinomas, and 16 controls) by RNA dot blots, double-labeling immunohistochemistry, and in situ hybridization, using common and variant-specific probes. Tumor hepatocytes expressed the LONG form, whereas cholangiocarcinoma cells expressed the SHORT form, which was deposited in tumor basement membranes. Metastatic colorectal carcinoma cells did not express collagen XVIII. In the stromal compartment of primary and metastatic cancers, myofibroblasts and vascular endothelial cells expressed the SHORT form. Both basement membrane components, collagen IV and the SHORT collagen XVIII form, were codistributed and their mRNA levels strongly correlated (R =.75, P <.001). In addition, freshly isolated human hepatocytes expressed the LONG form and culture-activated stellate cells the SHORT form. Moreover, the full-length LONG form is a plasma protein. Thus, the LONG form is a hepatocyte-specific variant, and the SHORT form is a major component of the tumor extracellular matrix in primary and metastatic liver cancers. In the clinical context, the global expression of the endogenous endostatin precursor, collagen XVIII, in liver cancer results from the combined expression profiles of tumor cells, stromal cells, and nontumor hepatocytes at the advancing edge of the tumor, particular to each type of cancer.

Collagen XVIII is localized in sinusoids and basement membrane zones and expressed by hepatocytes and activated stellate cells in fibrotic human liver.

Type XVIII collagen is a recently discovered nonfibrillar collagen associated with basement membranes in mice and expressed at high levels in human liver. We studied the origin, distribution, and RNA levels of type XVIII collagen in normal and fibrotic human livers by in situ hybridization, immunohistochemistry, and Northern and dot blots and compared procollagen alpha1(XVIII) RNA levels with those of procollagen alpha1(IV) and laminin gamma1, the two major components of liver basement membranes. In normal liver, type XVIII collagen was heavily deposited in perisinusoidal spaces and basement membrane zones. The major source of type XVIII collagen was hepatocytes and, to a lesser extent, endothelial, biliary epithelial, and vascular smooth muscle cells and peripheral nerves. In cirrhosis, type XVIII collagen formed a thick deposit along capillarized sinusoids. Grain counts after in situ hybridization showed myofibroblasts to increase their expression 13-fold in active and twofold in quiescent fibrosis, whereas hepatocytes increased their expression only twofold in both active and quiescent fibrosis. Activated stellate cells in vitro expressed type XVIII collagen at high levels. These data indicate that type XVIII collagen is a component of the perisinusoidal space and is associated with basement membrane remodeling. Hepatocytes and activated stellate cells are important sources of type XVIII collagen in normal and fibrotic liver respectively, which suggests tissue-specific regulation of its expression.

Acute and chronic experimental Trypanosoma cruzi infection in the rat. Response to systemic treatment with recombinant rat interferon-gamma.

We examined the effects of recombinant rat interferon-gamma (IFN-gamma) injections on the parasitologic, serologic, immunologic and histopathologic features of acute and chronic experimental Trypanosoma cruzi (T. cruzi) infections in "l" rats. Upon infection at weaning, two rat groups were allocated to receive a 20-day cycle of IFN-gamma injections, 20,000 IU/rat each, which started at 1, and 7 days post-infection (pi). Treatment with IFN-gamma, initiated at either 1 or 7 days pi, resulted in comparatively lower peak parasitemias (P < 0.02) but in similar levels of anti-T. cruzi circulating antibodies and serum IFN-gamma activities. The latter appeared significantly increased during acute infection whereas biologically active tumor necrosis factor was virtually undetectable in serum from infected rats regardless of whether they had been given IFN-gamma or not. The prevalence of chronic focal myocarditis in IFN-gamma-treated infected rats showed no differences with respect to the one recorded in control-infected counterparts. The inverse CD4/CD8 ratio of spleen and lymph node T cells that usually accompanies chronic infection was reversed by IFN-gamma. Mononuclear cells carrying class II I-A and I-E molecules, that were found to have increased at both compartments, appeared also modified upon IFN-gamma treatment with an overincrease of I-A-positive cells, and a normalization of I-E-bearing cells.