Action of colchicine on hepatic schistosomal granuloma

Amorphous material and altered collagen fragments within dilated secretory vesicles and cisternae of fibroblast cytoplasm were the main ultrastructural changes seen in hepatic periovular granulomas formed in mice infected with Schistosoma mansoni and treated with colchicine. Despite promoting ultrastructural changes in the fibroblasts found in hepatic periovular granulomas, colchicine administration to infected mice did not significantly change the light microscopic appearance of the hepatic schistosomal lesions, did not diminish the amount of total hepatic collagen, and did not change the collagen isotypes in the granulomas, as observed after a comparative study with non-colchicine treated infected control mice. When administered to mice two weeks after curative treatment of schistosomiasis with praziquantel, colchicine did not seem to increase extracellular collagen degradation or to induce a more rapid resorption of hepatic periovular granulomas, although still promoting ultrastructural alterations in fibroblasts.     

The outcome of acute schistosomiasis infection in adult mice with postnatal exposure to maternal malnutrition

Maternal malnutrition during the lactation period in early development may have long-term programming effects on adult offspring. We evaluated the combined effects of parasitological behaviour and histopathological features and malnutrition during lactation. Lactating mice and their pups were divided into a control group (fed a normal diet of 23% protein), a protein-restricted group (PR) (fed a diet containing 8% protein) and a caloric-restricted group (CR) (fed according to the PR group intake). At the age of 60 days, the offspring were infected with Schistosoma mansoni cercariae and killed at nine weeks post-infection. Food intake, body and liver masses, leptinaemia, corticosteronaemia, collagen morphometry and neogenesis and the cellular composition of liver granulomas were studied. PR offspring showed reduced weight gain and hypophagia, whereas CR offspring became overweight and developed hyperphagia. The pre-patent period was longer (45 days) in both programmed offspring as compared to controls (40 days). The PR-infected group had higher faecal and intestinal egg output and increased liver damage. The CR-infected group showed a lower number of liver granulomas, increased collagen neogenesis and a higher frequency of binucleate hepatocytes, suggesting a better modulation of the inflammatory response and increased liver regeneration. Taken together, our findings suggest that neonatal malnutrition of offspring during lactation affects the outcome of schistosomiasis in mice.     

Changes in collagen and albumin mRNA in liver tissue of mice infected with Schistosoma mansoni as determined by in situ hybridization

We have employed in situ hybridization to evaluate the molecular mechanisms responsible for hypoalbuminemia and increased liver collagen content in murine schistosomiasis. Results were compared using a simplified method of hybridizing isolated hepatocytes from Schistosoma mansoni-infected and normal mouse liver with mouse albumin (pmalb-2) and chick pro-alpha 2(l) collagen (pCg45) probes. Whereas hepatocytes from infected mice showed significantly less albumin mRNA than hepatocytes from control, there were more grains of procollagen mRNA in hepatocytes from infected as compared with control liver. Hybridization of infected liver tissue sections with the collagen probe showed more grains per field in granulomas than in liver regions, whereas with the albumin probe there was more hybridization in liver tissue than in granulomas. These results suggest that in murine schistosomiasis a reduction in albumin mRNA sequence content may be associated with decreased albumin synthesis and ultimately leads to hypoalbuminemia. In addition, although the granuloma seems to be the primary source of type I collagen synthesis, hepatocytes are also capable of synthesizing collagen, especially under fibrogenic stimulation.     

Immunopathology of alkaline phosphataseinduced granulomatous hepatitis in rats

Granulomatous inflammation is a specific type of chronic inflammation in which macrophages and T-cell-mediated immunity to the inciting agent play a pivotal role. In the present study, granulomatous hepatitis was induced in rats by the administration of a single intravenous dose of porcine intestinal alkaline phosphatase. The cellular composition of the hepatic granulomas was analyzed in-situ with a number of recently developed mouse anti-rat monoclonal antibodies to cells of the monocyte-macrophage lineage and lymphocyte subsets. Well-developed granulomas consisted of aggregates of macrophages with central modification into epithelioid cells, a peripheral rim of T- and B-lymphoid cells, including considerable numbers of immunoblasts and plasma cells. In addition, the periphery of the granulomas contained many fat storing cells, a sinusoidal cell type thought to play a central role in hepatic fibrosis. Moreover, intense immunostaining for the extracellular matrix proteins fibronectin and collagen type III was observed at the periphery of the lesions. The granulomas persisted for long periods without eliciting liver cirrhosis. Alkaline phosphatase induced hepatic granulomas in the rat may help to elucidate the contribution of cells of the B-lineage to chronic granulomatous inflammation.     

Significance of schistosomal granuloma modulation

Hepatic Schistosoma mansoni periovular granulomas undergo changes in size, cellular composition and appearance with time. This phenomenon, known as "immunological modulation", has been thought to reflect host immunological status. However, as modulation has not been observed outside the liver, participation of local factors, hitherto little considered, seems crucial. Components of the extracellular matrix of periovular granulomas of the mouse were particularly studied in three different organs (liver, lung and intestine) and during three periods of infection time (acute, intermediate and chronic) by means of histological, biochemical and immunofluorescence techniques, while quantitative data were evaluated by computerized morphometry, in order to investigate participation of local factors in granuloma modulation. Results confirmed modulation as a exclusively hepatic phenomenon, since pulmonary and intestinal granulomas, formed around mature eggs, did not change size and appearance with time. The matricial components which were investigated (Type I, III and IV collagens, fibronectin, laminin, proteoglycans and elastin) were found in all granulomas and in all organs examined. However, their presence was much more prominent in the liver. Elastin was only found in hepatic granulomas of chronic infection. The large amount of extracellular matrix components found in hepatic granulomas was the main change responsible for the morphological aspects of modulation. Therefore, the peculiar environment of the liver ultimately determines the changes identified in schistosomal granuloma as "modulation".     

Bioresorption of Porous 3D Matrices Based on Collagen in Liver and Muscular Tissue

Highly porous cylinder-shaped 3D matrices with diameters of 1.3 and 3 mm were obtained by lyophilization of collagen solution. A study in vivo of the mechanism and rate of resorption of the resulting material showed that complete resorption of the matrix occurred 6 weeks after their implantation into liver tissue and 3 weeks after implantation into muscle tissue of animals. Surrounding tissues were not altered or damaged. Histological analysis revealed that, simultaneously with the resorption of matrix collagen, connective tissue and blood vessels were formed. This allows us to recommend the developed porous material based on collagen for use as matrices for tissue engineering and cellular transplantation.     

Detection of vasoactive intestinal peptide and localization of its mRNA within granulomas of murine schistosomiasis

Schistosomiasis mansoni is a parasitic disease resulting in the deposition of ova predominantly in the liver and intestines. These ova secrete antigens which induce host sensitization and evoke focal granulomas. The granulomas are intricate delayed-hypersensitivity reactions governed by numerous cellular and humoral interactions. They displace or destroy normal tissue. Vasoactive intestinal peptide (VIP) is one of several neuropeptides which exert a broad range of biologic actions that may include modulation of immune responses. In this study, VIP was sought within liver granulomas isolated from Schistosoma mansoni-infected, CBA/J mice. Granuloma extracts contained appreciable amounts of immunoreactive VIP as detected by radioimmunoassay. Immunoreactive VIP was shown, by each of two chromatographic methods, to elute as a single peak coinciding with that of synthetic VIP. In situ hybridization was performed with an oligonucleotide probe complementary to a portion of the nucleotide sequence encoding VIP on preproVIP mRNA (antisense probe). Radiolabeled VIP probe adhered exclusively to granuloma eosinophils and to eosinophils within a peritonitis induced in normal mice by proteose peptone. Hybridization of radiolabeled VIP probe in the presence of unlabeled probe substantially attenuated binding. A sense probe failed to bind. These data suggest that the granulomas contain authentic VIP and that eosinophils express the gene for this molecule.     

Organ-dependent differences in composition and function observed in hepatic and intestinal granulomas isolated from mice with Schistosomiasis mansoni

In murine schistosomiasis mansoni, eggs deposited in the liver and intestines induce a cell-mediated granulomatous reaction. Previous studies have shown that maximal granuloma size differs in the liver and various segments of the gastrointestinal tract. The objective of this investigation was to isolate intestinal granulomas and to determine whether organ-dependent differences in cell composition and granuloma function exist. Intestinal granulomas representative of those in tissue were isolated by a combination of chemical and mechanical techniques. When dissociated by collagenase, these lesions yielded a viable heterogeneous population of inflammatory cells. Granulomas from the liver, colon, and ileum showed differences in cellular composition. Liver lesions contained the largest number of T and B lymphocytes, eosinophils, and mast cells whereas ileal granulomas comprised mostly macrophages. Immunofluorescence studies on frozen tissue sections revealed that T and B lymphocytes also displayed different patterns of distribution within granulomas from different tissues. In contrast to isolated cultured liver granulomas that produced MIF-like substances, isolated colonic and ileal granulomas had weak or no MIF activity. It thus appears that granuloma formation in various organs is influenced by local factors that could affect the ultimate resolution of the lesions.     

CD4+CD25+ regulatory cells contribute to the regulation of colonic Th2 granulomatous pathology caused by schistosome infection

Eggs of the helminth Schistosoma mansoni accumulate in the colon following infection and generate Th2-biassed inflammatory granulomas which become down- modulated in size as the infection proceeds to chronicity. However, although CD4+CD25+FoxP3+ regulatory T cells (T(regs)) are known to suppress Th1-mediated colitis, it is not clear whether they control Th2-associated pathologies of the large intestine which characterise several helminth infections. Here we used a novel 3D-multiphoton confocal microscopy approach to visualise and quantify changes in the size and composition of colonic granulomas at the acute and chronic phases of S. mansoni infection. We observed decreased granuloma size, as well as reductions in the abundance of DsRed+ T cells and collagen deposition at 14 weeks (chronic) compared to 8 weeks (acute) post-infection. Th2 cytokine production (i.e. IL-4, IL-5) in the colonic tissue and draining mesenteric lymph node (mLN) decreased during the chronic phase of infection, whilst levels of TGF-β1 increased, co-incident with reduced mLN proliferative responses, granuloma size and fibrosis. The proportion of CD4+CD25+FoxP3+T(regs): CD4+ cells in the mLN increased during chronic disease, while within colonic granulomas there was an approximate 4-fold increase. The proportion of CD4+CD25+FoxP3+T(regs) in the mLN that were CD103+ and CCR5+ also increased indicating an enhanced potential to home to intestinal sites. CD4+CD25+ cells suppressed antigen-specific Th2 mLN cell proliferation in vitro, while their removal during chronic disease resulted in significantly larger granulomas, partial reversal of Th2 hypo-responsiveness and an increase in the number of eosinophils in colonic granulomas. Finally, transfer of schistosome infection-expanded CD4+CD25+T(regs) down-modulated the development of colonic granulomas, including collagen deposition. Therefore, CD4+CD25+FoxP3+T(regs) appear to control Th2 colonic granulomas during chronic infection, and are likely to play a role in containing pathology during intestinal schistosomiasis.     

Lysosome participation in collagen resorption by hepatocytes during regression of liver cirrhosis

Electron microscopy and electron histochemistry employed in experiments on mice with liver cirrhosis regression have demonstrated the presence of vacuoles with collagen in the cytoplasm of hepatocytes as well as the presence in these vacuoles of a reaction product to acid phosphatase. Based on the data obtained it is concluded that during liver cirrhosis regression, there takes place an intracellular resorption of collagen by hepatocytes via phagocytosis with an active involvement in this process of lysosomal enzymes.     

Host nutritional status as a contributory factor to the remodeling of schistosomal hepatic fibrosis

Weaning Swiss mice were percutaneously infected with 30 cercariae of Schistosoma mansoni and submitted to a shifting either from a deficient to a balanced diet or vice-versa, for 24 weeks. The nutritional status was weekly evaluated by measurements of growth curves and food intake. Hepatic fibrosis and periovular granulomas were studied by histological, morphometric and biochemical methods. All mice fed on a deficient diet failed to develop periportal "pipestem" fibrosis after chronic infection. An unexpected finding was the absence of pipestem fibrosis in mice on normal diet, probably related to the sample size. The lower values for nutritional parameters were mainly due to the deficient diet, rather than to infection. Liver/body weight ratio was higher in "early undernutrition" group, after shifting to the balanced diet. Volume density and numerical density of egg granulomas reached lowest values in undernourished animals. The amount of collagen was reduced in undernourished mice, attaining higher concentrations in well-fed controls and in "late undernutrition" (balanced diet shifted to a deficient one), where collagen deposition appeared increased in granulomas. That finding suggested interference with collagen degradation and resorption in "late" undernourished animals. Thus, host nutritional status plays a role in connective tissue changes of hepatic schistosomiasis in mice.     

Dynamics of acid phosphatase activity in the liver in the process of involution of cirrhosis

Changes in the total activity of acid phosphatase in the liver as well as changes in the enzyme activity in hepatocytes and connective tissue cells of fibrosis layers were investigated, using quantitative histochemical method, in the process of mouse cirrhosis involution. After discontinuation of CCl4 injection, the animals with cirrhosis were divided into two groups. In the first group the resection of the left lobe of the liver was performed. The animals of the second group were not subject to operation. The results demonstrate that there is a close correlation between lysosomal hydrolase activity of hepatocytes and connective tissue cells of the liver and collagen resorption during cirrhosis involution. The most intensive lysis of collagen takes place within the first three weeks of cirrhosis involution in both experimental groups. Partial resection in cirrhosis has no significant effect on the changes and level of total activity of lysosomal hydrolase enzymes in the liver during cirrhosis involution.     

Fibroblast stimulation in schistosomiasis. IX. Schistosomal egg granulomas from congenitally athymic mice are deficient in production of fibrogenic factors

Schistosomal egg granulomas spontaneously secrete fibrogenic factors, suggesting that there exists a molecular link between granulomatous inflammation and hepatic fibrosis in schistosomiasis. To further assess this possibility, we compared elaboration of fibrogenic factors by egg granulomas isolated from Schistosoma mansoni-infected euthymic mice that develop substantial liver fibrosis, with those elaborated by similarly infected congenitally athymic mice that develop minimal fibrosis. Conditioned medium from cultures of granulomas from euthymic mice stimulated fibroblast proliferation, chemotaxis, and synthesis of collagen, collagenase, tissue inhibitor of metalloproteinases, and hyaluronate, whereas those prepared from cultures of granulomas isolated from athymic mice were relatively or absolutely deficient in such activities. These observations provide a correlation between the presence of fibrosis in vivo and the production of fibrogenic factors and reinforce our hypothesis that granuloma-derived fibrogenic factors play a role in the pathogenesis of liver fibrosis in schistosomiasis. Furthermore, the results of this study suggest a central role of T lymphocytes in the fibrogenic process.     

The electron-histochemical detection of the activity of thiol proteinases in normal and cirrhotically altered liver]

The distribution co-activity of thiol proteinases (cathepsin B and cathepsin H) was investigated in normal and cirrhotic liver by the electron-cytochemical method. The reaction product was localized on lysosomes of Kupffer's cells, hepatocytes, endotheliocytes and fibroblasts. Extracellular activity of thiol proteinases was revealed in normal as well as in cirrhotic liver. The reaction product was situated on collagen fibrils near hepatocytes and connective tissue cells. The results evidence that besides participation in intracellular degradation of different proteins, thiol proteinases are secreted by hepatocytes and connective tissue cells of the liver to the intercellular space and can take part in extracellular collagen resorption.     

Inhibitory effects of glucocorticoids on collagen synthesis by mouse sponge granulomas and granuloma fibroblasts in culture.

The basis for the glucocorticoid-mediated decrease in tissue collagen was studied in mouse granulomas and in primary granuloma fibroblast cultures. Injection of mice for 12 days with dexamethasone (0.35 mg/kg body weight) resulted in a 50--70% inhibition of collagen synthesis and accumulation in polyvinyl sponge-induced granulomas whereas total protein synthesis was inhibited by only about 25%. The decreased collagen content of the granuloma was accounted for by both a reduced fibroblast number and diminished synthesis per cell. Growth rates, total protein synthesis and collagen synthesis were the same in granuloma fibroblast cultures derived from control or steroid-treated mice. However, addition of 3.10(-7) M hydrocortisone to the culture medium caused a 30--50% inhibition of both collagen and non-collagen protein synthesis in firbroblasts from either source. These inhibitory effects were dose- and time-dependent with a lag time of 12--24 h. Prolyl hydroxylase activity was reduced both in sponge granulomas from glucocorticoid-treated mice and in hydrocortisone-treated fibroblast cultures. However, protein synthesis was inhibited to the same extent as the inhibition of prolyl hydroxylase activity and there was no effect on peptidyl prolyl hydroxylation. These results indicate that the glucocorticoid-induced reduction of collagen synthesis and accumulation observed in mouse granulomas and primary granuloma fibroblast cultures is not specific for this protein. Furthermore, glucocorticoid-induced inhibition of collagen synthesis cannot be attributed to underhydroxylation of collagen prolyl residues.     

Alterations of the intestinal innervation in mice infected with Schistosoma mansoni.

Schistosomiasis mansoni is a parasitic disease in which granulomas form around schistosome eggs in the liver and intestines. The purpose of this study was to determine the alterations in the intrinsic innervation of the distal ileum and proximal colon resulting from schistosomiasis. Using murine schistosomiasis mansoni, we examined light microscopic preparations stained with osmium-zinc iodide or the dihydronicotinamide adenine dinucleotide: nitro BT oxidoreductase (NADH) method. We also examined specific populations of peptidergic nerves (vasoactive intestinal polypeptide and substance P) using an avidin-biotin complex (ABC) immunohistochemical technique. We found that granulomas focally destroyed the enteric nerves. Occasionally nerves were found within granulomas, particularly at the periphery of the lesions. Nerve cell bodies close to granulomas had altered staining, which included increased staining for vasoactive intestinal polypeptide. The distribution of nerve injury varied between the 2 enteric segments studied. In the distal ileum, the principal injury was to the myenteric plexus; whereas, the submucous and mucosal plexuses were predominantly damaged in the proximal colon. The physiologic significance of this injury to the enteric nerves requires elucidation.     

Potential synergies between matrix proteins and soluble factors on resorption and proteinase activities of rabbit bone cells

Human growth hormone (GH) has recently been found to stimulate osteoclastic resorption, cysteine-proteinase and metalloproteinase activities (MMP-2 and MMP-9) in vitro via insulin-like growth factor-I (IGF-I) produced by stromal cells. The present study investigated the effects of two extracellular matrix components (vitronectin and type-I collagen) on hGH- and hIGF-1-stimulated osteoclastic resorption and proteinase activities in a rabbit bone cell model. After 4 days of rabbit bone cell culture on dentin slices with vitronectin coating, hGH and hIGF-1 stimulated bone resorption and hIGF-1 upmodulated cysteine-proteinase activities. MMP-2 expression (but not resorption, cathepsin or MMP-9 activities) was upmodulated by hGH and hIGF-1 on dentin slices coated with type I collagen as compared to those without coating. Then, vitronectin was synergistic with hIGF-1 in the regulation of cysteine-proteinase production whereas collagen showed synergy with hGH and hIGF-1 in the regulation of MMP-2 production. Anti-alphavbeta3 totally abolished the effects of hGH and hIGF-1 on metalloproteinase release, but had no influence on cathepsin release. The results suggest that cysteine-proteinase modulation is not mediated by alphavbeta3 integrin (strongly expressed on osteoclastic surface) whereas the resorption process and metalloproteinase modulation are clearly mediated by this integrin. Our finding about the collagen coating also suggests that hGH- and hIGF-1-stimulated MMP-2 activity are mediated, along with alphavbeta3 integrin, by another adhesion molecule.     

Spontaneous lesions in cynomolgus monkeys used in toxicity studies.

Spontaneous lesions in wild-caught, laboratory-maintained cynomolgus monkeys used in drug-toxicity studies were examined histopathologically in an effort to better distinguish toxic changes from spontaneous lesions and assess the toxicity of drugs more exactly. In the liver and kidney, where toxic changes are observed frequently, many spontaneous lesions were observed. Infiltration of mononuclear cells, vacuolization of the hepatocytes, dilatation of the renal tubules, and vacuolization of the renal epithelia were observed at a relatively high frequency. It is considered important to examine these changes carefully, because they closely resemble the changes recognized as toxic. Deposition of brownish pigment was observed in various organs such as the liver, kidney, spleen, intestinal tract, lung and brain, however the type of pigment differed among the organs, and histochemical examination revealed anthracosis or accumulation of hemosiderin, or melanin. Since the monkeys were caught in the wild, many parasitic lesions were observed especially in the large intestine and liver. Helminthous worms were frequently observed in the granulomas in the large intestine, however, no parasites were observed in the granulomas in the liver. Such lesions in the liver may be misinterpreted as toxic changes, when only scars or inflammatory lesions are observed.     

Entamoeba histolytica: acute granulomatous intestinal lesions in normal and neutrophil-depleted mice

To study the role of neutrophils in the innate resistance to Entamoeba histolytica intestinal infection in mice, animals were treated with anti-neutrophil monoclonal antibodies prior to intracecal parasite inoculation and the resulting lesions were compared with normal mice that had been equally infected. In contrast to our previous finding that neutrophils are critical in eliminating E. histolytica infection in the liver, we show here that neutrophils are not absolutely required to eliminate E. histolytica infection from the intestine. Although the neutrophils are not critical for resolution of the E. histolytica infection, neutrophils do appear to provide some measure of protection as the intestinal amoeba burden was higher at early timepoints after infection in the neutropenic animals. In addition, we found that while both the normal and the neutrophil-depleted mice developed ulcerative lesions in the colon, the neutropenic mice had an increased frequency of granulomas that formed around the amoeba. Thus, our findings appear to be the first evidence showing that granulomatous inflammation can occur after intestinal infection in mice using axenically cultured amoeba.