Liver collagen hydroxylation in murine schistosomiasis.

The activity of procollagen prolyl hydroxylase was measured in fibrotic liver obtained from mice with hepatosplenic schistosomiasis, an animal model of the most prevalent form of human liver fibrosis. Measurable activity of prolyl hydroxylase in fibrotic liver supernatants was 47-fold higher than that of normal liver. The effect of prolyl hydroxylase inhibition on collagen synthesis in fibrotic liver slices was studied, using 8,9-dihydroxy-7-methyl benzo[b]quinolizinium bromide (GPA 1734). This compound was shown in other systems to inhibit prolyl and lysyl hydroxylations by iron chelation at concentrations which did not affect total protein synthesis. The formation of nondialyzable labelled hydroxyproline was inhibited by GPA 1734, 40, 70 and 95% at 30, 50 and 100 micrometer, respectively. Incorporation of proline into total liver protein was unaffected at 30 and 50 micrometer, but was inhibited 20% at 100 micrometer GPA 1734. Underhydroxylated collagen synthesized by liver slices with GPA 1734 was extracted with neutral salt solution and was subsequently hydroxylated with partially-purified prolyl hydroxylase to the same extent as control material synthesized in the absence of GPA 1734.     

Praziquantel pharmacotherapy reduces systemic osteopontin levels and liver collagen content in murine schistosomiasis mansoni

The pathogenesis of schistosomiasis and the mechanism of disease regression after Praziquantel pharmacotherapy are not fully elucidated. Schistosoma mansoni egg antigens directly stimulate the expression of the profibrogenic molecule osteopontin (OPN), and systemic OPN levels strongly correlate with disease severity, suggesting its use as a potential morbidity biomarker. In this study, we investigated the impact of Praziquantel use on systemic OPN levels and on liver collagen deposition in chronic murine schistosomiasis. Praziquantel treatment significantly reduced systemic OPN levels and liver collagen deposition, indicating that OPN could be a reliable tool for monitoring PZQ efficacy and fibrosis regression in murine schistosomiasis.     

Age-related variations in hydroxylation of lysine and proline in collagen

The effect of age on the extent of hydroxylation of lysine and proline both generally and at certain specific sites in collagens from bone, skin and tendon was examined in the chick from the 14-day embryo to the 18-month-old adult. For all collagens there was a marked fall in the overall extent of hydroxylation of lysine with increasing age in both alpha(1) and alpha(2) chains, this fall occurring mostly in a relatively short period immediately after hatching. Hydroxylation of lysine declined to a constant value which, as expected, differed appreciably for each collagen and was considered to be characteristic of the collagen according to its tissue of origin. Hydroxylation of lysine in the N-terminal, non-helical telopeptide region of both alpha(1) and alpha(2) chains, which is important with regard to cross-linking, was relatively high in embryonic collagens. There was, however, a rapid loss of hydroxylation at these sites in skin collagen, occurring both during development of the embryo and in the period immediately after hatching. In contrast some hydroxylation at these sites persisted in bone and tendon collagens and, as judged by examination of peptide alpha(1)-CB1, appeared to reach a constant value in time of about 33% in bone and about 15% in tendon collagen. The actual extent of hydroxylation of lysine in the N-terminal telopeptides and the size of the changes in these values with age appeared to be unrelated to the corresponding whole-chain values, and it is suggested therefore that hydroxylation of telopeptidyl lysine may be under separate enzymic control. The increased hydroxylation of lysine in the embryo was accompanied by only minimal changes in proline hydroxylation, which was very slightly increased in embryonic bone and tendon collagens. Increased hydroxylation of proline in the embryo was, however, readily observed in peptide alpha(1)-CB2 from the helical region of tendon collagen. This hydroxylation was close to the theoretical maximum, in contrast with that observed in post-embryonic tendon, where hydroxylation was incomplete, as in rat tendon (Bornstein, 1967), only four on average, of the six susceptible proline residues being hydroxylated.     

Lysine hydroxylation of collagen in a fibroblast cell culture system

The lysine (Lys) hydroxylation pattern of type I collagen produced by human fibroblasts in culture was analyzed and compared. Fibroblasts were cultured from normal human skin (NSF), keloid (KDF), fetal skin (FDF), and skin tissues of Ehlers-Danlos syndrome type VIA and VIB patients (EDS-VIA and -VIB). The type I collagen alpha chains with or without non-helical telopeptides were purified from the insoluble matrix and analyzed. In comparison with NSFs, KDF and FDF showed significantly higher Lys hydroxylation, particularly in the telopeptide domains of both alpha chains. Both EDS-VIA and -VIB showed markedly lower Lys hydroxylation in the helical domains of both alpha chains whereas that in the telopeptides was comparable with those of NSFs. A similar profile was observed in the tissue sample of the EDS-VIB patient. These results demonstrate that the Lys hydroxylation pattern is domain-specific within the collagen molecule and that this method is useful to characterize the cell phenotypes in normal/pathological connective tissues.     

Tachykinin production in granulomas of murine schistosomiasis mansoni

Preprotachykinins, the products of one gene, are the precursor molecules of three mammalian tachykinins called substance P (SP), substance K (SK), and neuropeptide K. An additional mammalian tachykinin, neurokinin B, has also been described. SP and possibly other tachykinins may modulate immunologic responses. Granulomas that form around parasite ova in murine schistosomiasis were examined for tachykinins. Tachykinins were extracted from granulomas by boiling or with detergent. Extracts examined by RIA and HPLC contained only immunoreactive SP. Granulomas were dispersed with collagenase and cultured in vitro for up to 4 h. Only immunoreactive SP appeared in the culture medium. SP immunoreactivity localized solely to granuloma eosinophils as demonstrated by a sensitive immunohistochemical technique. An antiserum that recognized SK, neuropeptide K, and neurokinin B, but which possessed low reactivity to SP, also stained these cells. Only prior absorption of each antiserum with the appropriate synthetic neuropeptide would abrogate the immunostaining. This suggested that tachykinins other than SP were present within these cells. However, results of in situ hybridization experiments intimated that eosinophils produced predominantly preprotachykinin mRNAs which encode SP but are devoid of the SK/neuropeptide K sequence. It is concluded that granuloma eosinophils make predominantly SP in deference to other tachykinins, and that tachykinins other than SP are unlikely to be important in the regulation of the early granulomatous response of murine schistosomiasis.     

Influence of interleukin-2 and interferon-gamma in murine schistosomiasis

Schistosoma mansoni-infected mice were administered at the time of parasite residency in the lung with recombinant murine interleukin (IL)-2 or interferon-gamma (IFN-gamma), to evaluate the impact of cytokines in host responses to primary schistosomiasis. S. mansoni lung-stage schistosomula did not affect plasma lipids levels in BALB/c, while elicited significant (p<0.05) increase in free fatty acids (FA) and decrease in cholesterol plasma levels in C57BL/6 and CD1 mice, and stimulated expression of mRNA for Th2 cytokines in BALB/c and Th1 cytokines in C57BL/6 and CD1 mice. Production of specific antibodies was negligible in the 3 strains. Interleukin-2 treatment elicited significant (p<0.001) decrease in triglycerides (TG) in CD1, and decrease in TG and cholesterol plasma levels and down-regulation of TNF-alpha mRNA expression in C57BL/6 mice. Induction of type 2 cytokines and/or IFN-gamma mRNA expression did not lead to increase in percentage of specific antibody responders in any mouse strain. Exogenous IL-2-related reduction in cholesterol plasma levels and TNF-alpha mRNA expression in C57BL/6 mice was associated with significant (p<0.05) decrease in adult worm recovery and egg count. Treatment with IFN-gamma elicited significant (p<0.05) free FA plasma levels increase in BALB/c and C57BL/6 and decrease in CD1 mice. Expression of type 2 cytokines mRNA was stimulated in BALB/c and CD1 mice, yet was not accompanied with increase in humoral responses. Exogenous IFN-gamma-related reduction in free FA plasma levels and IFN-gamma mRNA response, and up-regulation of TNF-alpha mRNA expression in CD1 mice were associated with significant increase in adult worm burden and egg load. The data were discussed in an attempt to define host factors predictive of resistance to schistosome infection.     

Inhibition of collagen hydroxylation by 2,7,8-trihydroxyanthraquinone in embryonic-chick tendon cells.

Prolyl 4-hydroxylase (EC 1.14.11.2) is an essential enzyme in the post-translational modification of collagen. Inhibitors of this enzyme are of potential interest for the treatment of diseases involving excessive deposition of collagen. 2,7,8-Trihydroxyanthraquinone (THA) is an effective inhibitor of prolyl 4-hydroxylase by virtue of its ability to compete with the co-substrate 2-oxoglutarate (Ki = 40.3 microM). Using a simple and reproducible assay for collagen hydroxylation, we show that THA inhibits the hydroxylation of collagen in embryonic-chick tendon cells in short-term culture, with an IC50 value (concn. giving 50% inhibition) of 32 microM. In comparison, the ethyl ester of 3,4-dihydroxybenzoic acid has an IC50 value of 0.1 mM against collagen hydroxylation by chick tendon cells, whereas its Ki versus isolated prolyl 4-hydroxylase is 49 microM.     

Eosinophils from granulomas in murine schistosomiasis mansoni produce substance P

Granulomas are chronic, usually focal, tissue-destructive inflammatory reactions that usually form around slowly degradable, poorly soluble substances. They are dynamic lesions, regulated by complex immune mechanisms. Tachykinins are a family of neuropeptides characterized by the common C-terminal amino acid sequence -Phe-X-Gly-Leu-Met-NH2. One such tachykinin, substance P, has been reported to modulate immunologic responses. In this investigation, granulomas were examined for substance P. Granulomas were isolated from the livers of mice infected with murine schistosomiasis, and substance P was extracted. Immunoreactive substance P was detected by RIA. The authenticity of the molecule was confirmed by elution profile on HPLC. Immunoreactive substance P, identified by immunostaining, localized to eosinophils derived from collagenase-dispersed granulomas. Granulomas were then probed for expression of the gene for substance P (preprotachykinin). Preprotachykinin mRNA was localized to granuloma eosinophils by in situ oligonucleotide hybridization. It is concluded that substance P is present within the granuloma as a result of preprotachykinin production by eosinophils.     

Macrophages as effector cells of protective immunity in murine schistosomiasis

Mice infected with Schistosoma mansoni develop a dramatic (five- to eightfold) increase in numbers of peritoneal leukocytes, and approximately 65% of these cells are macrophages. By several biochemical and cytochemical criteria, these cells were comparable to resident peritoneal macrophages of normal mice. However, macrophages from schistosome-infected mice exhibited significant nonspecific tumoricidal activity in vitro, a function associated with immunologically activated cells. The time course for development of activated macrophages in the peritoneal cavity was dependent upon the route of infection. Cytotoxic cells were present in the peritoneal cavity by 3 weeks after intraperitoneal infection, but were not evident until several weeks later in animals infected percutaneously, subcutaneously, or intravenously. However, by 3 weeks after subcutaneous infection, tumoricidal macrophages appeared in the peritoneal cavity after intraperitoneal challenge with soluble schistosome antigens. Macrophage activation was independent of the development of egg granulomas, since tumoricidal cells could be found prior to the onset of egg production and were also present in mice infected with only male worms. Development of activated macrophages in these instances is thus consistent with previous observations on induction of T lymphocyte reactivity toward schistosomula. Since other manipulations known to activate macrophages have been shown to induce partial resistance to schistosome infection, the finding that macrophage activation results from primary S. mansoni infection itself suggests that these cells may play a major role in acquired immunity to this parasite.     

Splenic suppressor cells and cell-mediated cytotoxicity in murine schistosomiasis.

An impairment of the capacity to generate alloantigen-specific cytotoxic T lymphocytes (CTL) was observed in mixed lymphocyte cultures (MLC) established with spleen cells from mice infected with Schistosoma mansoni. This impairment, which was observed as early as the eighth week of infection, could be abrogated by the fractionation of spleen cell suspensions by the carbonyl iron/magnet method prior to the establishment of MLC. Cocultivation of normal spleen cells with increasing numbers of splenocytes from S. mansoni-infected syngeneic mice resulted in a dosage-dependent suppression of CTL generation. This "infectious suppression" was not sensitive to antiserum against mouse thymic lymphocyte antigen (MTLA). The present studies suggest the role of a macrophage rather than a T cell as the suppressor cell in this model of cell-mediated immunity in schistosome-infected mice.