Time course of gene expression profiling in the liver of experimental mice infected with Echinococcus multilocularis

Background

Alveolar echinococcosis (AE) is a severe chronic parasitic disease which behaves like a slow-growing liver cancer. Clinical observations suggest that the parasite, Echinococcus multilocularis (E. multilocularis) influences liver homeostasis and hepatic cell metabolism. However, this has never been analyzed during the time course of infection in the common model of secondary echinococcosis in experimental mice.

Methodology/Principal Findings

Gene expression profiles were assessed using DNA microarray analysis, 1, 2, 3 and 6 months after injection of E. multilocularis metacestode in the liver of susceptible mice. Data were collected at different time points to monitor the dynamic behavior of gene expression. 557 differentially expressed genes were identified at one or more time points, including 351 up-regulated and 228 down-regulated genes. Time-course analysis indicated, at the initial stage of E. multilocularis infection (month 1–2), that most of up-regulated pathways were related to immune processes and cell trafficking such as chemokine-, mitogen-activated protein kinase (MAPK) signaling, and down-regulated pathways were related to xenobiotic metabolism; at the middle stage (month 3), MAPK signaling pathway was maintained and peroxisome proliferator-activated receptor (PPAR) signaling pathway emerged; at the late stage (month 6), most of up-regulated pathways were related to PPAR signaling pathway, complement and coagulation cascades, while down-regulated pathways were related to metabolism of xenobiotics by cytochrome P450. Quantitative RT-PCR analysis of a random selection of 19 genes confirmed the reliability of the microarray data. Immunohistochemistry analysis showed that proliferating cell nuclear antigen (PCNA) was increased in the liver of E. multilocularis infected mice from 2 months to 6 months.

Conclusions

E. multilocularis metacestode definitely exerts a deep influence on liver homeostasis, by modifying a number of gene expression and metabolic pathways. It especially promotes hepatic cell proliferation, as evidenced by the increased PCNA constantly found in all the experimental time-points we studied and by an increased gene expression of key metabolic pathways.     

The course of pepsinogens during growth in mice

In the mouse stomach exist two acid proteases separable on the polyacrylamide gel electrophoresis. One of them increases in activity after weaning and is a pepsinogen immunologically related to the pepsinogens of other vertebrates. The other is predominant before weaning and is presumed to be a chymosin-like acid protease.     

Secondary Echinococcus multilocularis infection in severe combined immunodeficient (scid) mice: biphasic growth of the larval cyst mass.

E. multilocularis infection was suppressed in C.B-17 mice after intraperitoneal inoculation of protoscoleces, with larval cysts weighing no more than 1.0 g. In scid mice, which are genetically identical to C.B-17 except for a deficiency in functional lymphocytes, infection progressed and larval cysts reached a mass of 17.5 g at 15 weeks post-infection. The growth of the larval cyst mass in scid mice was similar to that in other susceptible mouse strains, with a biphasic pattern. Histological observations revealed giant cells and granulomatous inflammation in the C.B-17, but not in the scid mice. These results led to the conclusion that suppression of the growth of the larval cyst mass in the initial stage of infection in susceptible mice strains is caused by factors other than the host's lymphocytic immune response.     

Hepatocyte growth factor controls the proliferation of cultured epidermal melanoblasts and melanocytes from newborn mice

Mouse epidermal melanoblasts and melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanocyte-proliferation medium (MDMD) and melanoblast-proliferation medium (MDMDF) supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF). Pure cultured primary melanoblasts and melanocytes were further cultured with MDMD/MDMDF supplemented with hepatocyte growth factor (HGF) from 14 days (keratinocyte depletion). The HGF increased the number of melanoblasts and melanocytes, but not the percentage of differentiated melanocytes in the melanoblast-melanocyte population in the absence of keratinocytes. Flow cytometry analysis showed that melanoblasts and melanocytes in the S and/or G2/M phases of the cell cycle were increased by the treatment with HGF. Moreover, an anti-HGF antibody supplemented to MDMD/MDMDF from the initiation of the primary culture (in the presence of keratinocytes) inhibited the proliferation of melanoblasts and melanocytes, but not the differentiation of melanocytes. These results suggest that HGF is a keratinocyte-derived factor involved in regulating the proliferation of epidermal melanoblasts and melanocytes from newborn mice in cooperation with cAMP elevators and/or bFGF.     

Immune suppression induced by protoscoleces of Echinococcus multilocularis in mice. Evidence for the presence of CD8dull suppressor cells in spleens of mice intraperitoneally infected with E. multilocularis.

Immunoregulatory states induced by i.p. inoculation with the metazoan parasite Echinococcus multilocularis in the murine system were investigated. Proliferative responses and IL-2 production induced by Con A in spleen cells from BALB/c mice were significantly depressed at an early stage after infection with E. multilocularis protoscoleces (PSC). Addition of plastic-adherent cells from normal syngeneic mice to the nonadherent spleen cells from infected mice did not restore the depressed Con A responsiveness. On the other hand, exogenous IL-2 reconstituted completely the proliferative responses to Con A. Flow cytometry analysis revealed that CD4- CD8+ cells with a low density of CD8 Ag (CD8dull cells) increased in spleens from infected mice 2 weeks after inoculation. Addition of the spleen cell subpopulation containing the CD8dull cells, but not that depleted of the CD8dull cells, to normal spleen cells resulted in marked suppression of the Con A responses. These findings suggest that the CD8dull cells detected in spleens of mice inoculated with E. multilocularis PSC may play a key role in the suppressive regulation of immune responses. The relevance of the immune suppression seen in the early stages of experimental infection with E. multilocularis PSC to the eventual establishment of a host-parasite relationship is discussed.     

Gastrointestinal uptake of trace elements are changed during the course of a common human viral (Coxsackievirus B3) infection in mice.

Most infectious diseases are accompanied by a change in levels of several trace elements in the blood. However, it is not known whether changes in the gastrointestinal uptake of trace elements contribute to this event. Coxsackievirus B3 (CVB3), adapted to Balb/c mice, was used to study whether infection induces gene expression of metallothionein (MT1) and divalent-metal transporter 1 (DMT1) in the intestine and liver and hepcidin in the liver, as well as whether trace elements in these tissues are changed accordingly. Quantitative expression of CVB3, MT1, DMT1 and hepcidin was measured by real-time RT-PCR and six trace elements by ICP-MS on days 3, 6 and 9 of the infection. The copper/zinc (Cu/Zn) ratio in serum increased as a response to the infection. High concentrations of virus were found in the intestine and liver on day 3 and in the intestine on day 6. MT1 in the intestine and liver increased on days 3 and 6. The increase of MT1 in the liver correlated positively with Cu and Zn. Hepcidin in the liver showed a non-significant increase on days 3 and 6 of the infection, whereas DMT1 in the intestine decreased on day 9. Accordingly, iron (Fe) in the liver increased progressively during the disease, whereas in the intestine DMT1 was negatively correlated to Fe. Arsenic (As), cadmium (Cd) and mercury (Hg) were found to decrease to various degrees in the intestine, serum and liver. Thus, enteroviral infections, and possibly many other infections, may cause a change in the gastrointestinal uptake of both non-essential and essential trace elements.     

Suppression of humoral response during the course of Candida albicans infection in mice

This paper aims at demonstrating the non-specific immunosuppression as regards thyme-dependent antigens sheep erythrocytes (SRBC) during the course of Candida albicans systemic infection.Three lots of syngeneic /BALB/c mice, 8–12 weeks of age, were used. The first normal lot was inoculated via the intraperitoneal route with a (SRBC) suspension (4×10⁸ cells ml) in a Hank's balanced saline solution. The primary response of antibodies formed by splenic cells was measured from 4 to 8 days after inoculation using the direct plaque forming cells technique. The second lot was infected by the same route with a suspension of Candida albicans (1×10⁷ cells). Positive retrocultures from the blood and kidneys of these infected mice were obtained. These yeasts cultivated in a Sabouraud medium were harvested after 20 h at 37 °C. Following the same methodology the immune response to SRBC was determined. The serum obtained from infected mice was transferred to a third lot of mice at different intervals during the course of the infection. The immune response to SRBC was done by the direct plaque-forming cells technique. Controls were carried out using normal donors and recipients.A suppression of the immune response was obtained as from the 2nd day of inoculation up to the 28th day. It was not possible to transfer such suppression passively by means of the serum.These results suggest that the systemic infection by Candida albicans induce a non-specific immunosuppression in the organism, already demonstrated in viral infections, bacteria, protozoaria and metazoaria in mammals.In some way, this will contribute to explain the mechanisms of immune response to Candida albicans.     

Changes in the balance between mitogenic and antimitogenic lipid second messengers during proliferation, cell arrest, and apoptosis in T-lymphocytes.

Control of lymphocyte cell survival and proliferation is critical for both the immune response and for the prevention of autoimmune and infectious diseases. The actions of interleukin-2, the major T-cell regulatory cytokine, are mediated by the complex network of divergent signalling pathways controlled by its high-affinity receptor. Various studies have indicated that the generation of certain lipid second messengers is an important mechanism in the control of proliferation and cell death. We have examined the relationship between diacylglycerol and ceramide and the levels of the lipids phosphatidylcholine and sphingomyelin, their potential precursors, in the human T-cell line Kit 225 cultured in three distinct conditions to favor apoptosis, cell arrest, and proliferation. Our data show that, in proliferating cells, the ratios of diacylglycerol/ceramide and phosphatidylcholine/sphingomyelin are higher than those found in arrested cells and increase with time in culture. These ratios are rapidly reversed in apoptotic cells. Further experiments reveal that de novo synthesis of both diacylglycerol and phosphatidylcholine is greatest in proliferating cells, whereas sphingomyelin synthase activity is increased in cells undergoing apoptosis. In summary, our results demonstrate for the first time that the ratio of mitogenic/antimitogenic lipids changes dramatically during T-cell proliferation and cell death. These results indicate that lipid second messengers and the enzymes that are responsible for their generation may provide targets for novel therapeutic interventions in the clinical management of immunosuppression and autoimmune disease.     

Detection of the Echinococcus granulosus diagnostic arc 5 in sera from patients with surgically-confirmed E. multilocularis infection.

The Echinococcus granulosus diagnostic arc 5 was revealed by sera from an Alaskan and a Swiss patient with surgically confirmed E. multilocularis infections. The possibility of each patient harboring a concurrent infection with both parasites may be disregarded on the basis of radiologic, scintillographic and surgical data. This conclusion is compatible with ecologic and epidemiologic considerations. These observations suggest that the immunoelectrophoresis test based on arc 5 positivity is not E. granulosus-specific as originally described.     

Humoral response to sheep red blood cells in C57L/J mice during early and chronic stages of infection with Echinococcus multilocularis cysts.

C57L/J male mice infected with Echinococcus multilocularis cysts were challenged intraperitoneally at 4, 8, and 12 weeks postinfection (p.i.) with 3 x 10(9) sheep erythrocytes. The direct plaque forming cells, 2-mercaptoethanol (2-ME) sensitive (4-day sera) and 2-ME-resistant (8-day sera) haemagglutinin responses were significantly elevated at eight and 12 weeks p.i. as compared to controls or the fourweeks postinfected mice. Possible mechanisms of immunopotentiation in the chronically infected mice is discussed in relation to the larval cyst mass, splenomegaly, and B cell hyperplasia in the lymphoid tissues during the course of infection.     

Hepatocyte growth factor modulates migration and proliferation of human microvascular endothelial cells in culture.

Epidermal growth factor (EGF) induces tubular formation of cultured human omental microvascular endothelial (HOME) cells and EGF also stimulates cell migration as well as expression of tissue type plasminogen activator (t-PA). Here we studied the effects of hepatocyte growth factor (HGF) on cell proliferation, cell migration and expression of t-PA and other related genes. Migration of confluent HOME cells into the denuded space was stimulated by HGF after being wounded with razor blade, but at a reduced rate in comparison with EGF. HOME cells could be proliferated in response to exogenous 100 ng/ml of HGF at rates comparable to that of 20 ng/ml EGF. The chemotactic activity of HOME cells was significantly stimulated by HGF in a dose-dependent manner when assayed by Boyden chamber. HGF did not efficiently enhance expression of both the t-PA gene and a tissue inhibitor of metalloproteinase gene whereas it stimulated expression of plasminogen activator inhibitor-1. Our present study provides a new evidence that some of the biological effects of HGF on HOME cells in culture are similar to those of EGF.     

Hepatocyte growth factor/scatter factor binds to small heparin-derived oligosaccharides and stimulates the proliferation of human HaCaT keratinocytes.

Hepatocyte growth factor/scatter factor (HGF/SF) acts via a dual receptor system consisting of the MET tyrosine kinase receptor and heparan sulfate or dermatan sulfate proteoglycans. In optical biosensor binding assays, competition by oligosaccharides for binding of HGF/SF to immobilized heparin showed that disaccharides failed to compete, whereas tetrasaccharides inhibited HGF/SF binding (IC(50) 8 microg/ml). The inhibitory potency of the oligosaccharides increased as their length increased by successive disaccharide units, to reach a maximum (IC(50) 1 microg/ml) at degree of polymerization (dp) 10. In binding assays, HGF/SF was found to bind directly to oligosaccharides as small as dp 4, and the binding parameters were similar for oligosaccharides of dp 4-14 (k(a) 2.2-45.3 x 10(6) m(-1) s(-1), k(d) 0.033-0.039 s(-1), and K(d) 9-16 nm). In human keratinocytes, HGF/SF stimulated DNA synthesis, and this was dependent on a sustained phosphorylation of p42/44(MAPK). In chlorate-treated and hence sulfated glycosaminoglycan-deficient HaCaT cells, the stimulation of DNA synthesis by HGF/SF was almost abolished. Heparin-derived oligosaccharides from dp 2 to dp 24 were added together with HGF/SF to chlorate-treated cells to determine the minimum size of oligosaccharides able to restore HGF/SF activity. At restricted concentrations of oligosaccharides (4 ng/ml), HGF/SF required decasaccharides, whereas at higher concentrations (100 ng/ml) even tetrasaccharides were able to partly restore DNA synthesis. The results suggest that HGF/SF binds to a tetrasaccharide and that although this is sufficient to enable the stimulation of DNA synthesis, longer oligosaccharides are more efficient, perhaps by virtue of their ability to bind more easily other molecules.     

Changes in humoral responses to Trypanosoma cruzi during the course of infection in mice held at elevated temperature.

A parasite-specific, enzyme-linked immunosorbent assay and immunoblot analysis were used to examine the development of humoral immunity in Trypanosoma cruzi-infected C3H mice that survive acute infection when held at elevated environmental temperature. Both parasite-specific antibody levels and numbers of antigens identified increased during infection in mice held at 36 C, with the greatest reactivity measured in sera from mice that had resolved parasitemias. Heat shock of culture forms of T. cruzi resulted in production of different antigens, but there was no strong difference in the antigens recognized by sera from mice held at room temperature and those recognized by sera from mice held at 36 C. Immunoblot analysis using blood-form trypomastigote antigens identified a band of approximately 61 kDa produced by trypomastigotes in mice held at 36 C that was strongly detected by sera from mice held at 36 C. Little if any reactivity to this antigen was observed using sera from mice held at room temperature.     

Infertility due to growth arrest of ovarian follicles in Sl/Slt mice

Sl, Sld, and Slt are mutant alleles at the steel locus. All Sl/Sld and most Sl/Slt female mice are infertile, but the cause of the infertility is different. Germ cells are absent in Sl/Sld ovaries but present in Sl/Slt ovaries. The infertility of Sl/Slt female mice was attributed to the growth arrest of ovarian follicles, and the mechanism was analyzed by producing aggregation chimeras between Sl/Slt and +/+ embryos. Sl/Slt oocytes were ovulated and fertilized in Sl/Slt----+/+ chimeras. We investigated the origin of granulosa cells in the growing follicles and that of granulosa-derived luteal cells in the chimeras by using the electrophoretic pattern of phosphoglycerate kinase-1 and the histochemical activity of beta-glucuronidase as markers. Granulosa cells of Sl/Slt genotype developed and constituted pregnant corpora lutea in Sl/Slt----+/+ chimeras. Therefore, the growth arrest of Sl/Slt ovarian follicles may not be due to an intrinsic defect in granulosa cells but may instead be due to an intrinsic defect in ovarian stromal cells. This suggests that normal stromal cells are essential for the development of ovarian follicles.     

Demonstration of the lymphokine mechanism of the reinforcement of lymphoid proliferation during liver regeneration in mice

Changes in the ability of mouse splenocytes to proliferate early after partial hepatectomy have been studied. Proliferative activity of mouse splenocytes in mixed lymphocyte culture and in spontaneous proliferation in vitro test increased within 4, 17 and 24 hours after operation as compared with pseudo-operated control. After 4 hours of cultivation splenocytes of partially hepatectomized mice excreted into culture medium factor(s) that enhanced proliferative activity of intact mouse lymphocytes both in vitro and in vivo. The factor is thermolabile, operates nonspecifically, and is produced by T-lymphocytes.     

Granulomatous reactivation during the course of a leprosy infection: reaction or relapse

Background

Leprosy is a chronic granulomatous infectious disease and is still endemic in many parts of the world. It causes disabilities which are the consequence of nerve damage. This damage is in most cases the result of immunological reactions.

Objectives

To investigate the differences between a type 1 leprosy (reversal) reaction and relapse on using histopathology.

Methods

The histopathological changes in 167 biopsies from 66 leprosy patients were studied. The patients were selected when their sequential biopsies demonstrated either different patterns or maintained the same pattern of granulomatous reaction over more than two years during or after the treatment of leprosy.

Results

In 57 of the patients studied, a reactivation was seen which coincided with a decrease in the bacteriological index (BI), suggesting that this reactivation (reversal reaction or type 1 leprosy reaction) coincides with an effective capacity for bacteriological clearance. In nine patients, an increase of the bacteriologic index (IB) or persistence of solid bacilli occurred during the reactivation, indicating proliferative activity, suggestive of a relapse. The histopathological aspects of the granulomas were similar in both groups.

Conclusion

Bacterioscopy provided the only means to differentiate a reversal reaction from a relapse in patients with granulomatous reactivation. The type 1 leprosy reaction may be considered as a part effective immune reconstitution (reversal, upgrading reaction) or as a mere hypersensitivity reaction (downgrading reaction) in a relapse.     

Hepatocyte growth factor incorporated chitosan nanoparticles augment the differentiation of stem cell into hepatocytes for the recovery of liver cirrhosis in mice

Background  

Short half-life and low levels of growth factors in the niche of injured microenvironment necessitates the exogenous and sustainable delivery of growth factors along with stem cells to augment the regeneration of injured tissues.