Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 6 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Primary chick embryo liver cells were used to explore the regulation of δ-aminolevulinic acid synthase and heme oxygenase, the enzymes that catalyze the rate-limiting reactions of heme anabolism and catabolism, respectively. The general focus of the work was the exploration of the novel observation in which glutethimide and iron synergistically induced both δ-aminolevulinic acid synthase and heme oxygenase, a phenomenon that would not be predicted a priori. The course of events appeared to be: first, that heme synthesis was increased after addition of the glutethimide and that iron potentiated heme synthesis; second, the heme induced heme oxygenase five to ten fold; and third, that heme oxygenase degraded the heme permitting an uncontrolled induction of δ-aminolevulinic acid synthase. This induction of δ-aminolevulinic acid synthase could be prevented by the addition of a metalloporphyrin inhibitor of heme oxygenase. Induced δ-aminolevulinic acid synthase activity could be dramatically reduced by the addition of nanomolar concentrations of a metalloporphyrin, inhibitory for heme oxygenase, and heme. Specific observations related to the synergistic induction of heme oxygenase by glutethimide and iron was that the induction of heme oxygenase activity by glutethimide and iron occurred rapidly, with maximal increases occurring four to six hours after original treatment. Induction of heme oxygenase by glutethimide and iron was shown to be dependent on de novoheme synthesis since 4,6-dioxoheptanoic acid, a potent and specific inhibitor of heme biosynthesis, prevented the activity of heme oxygenase from increasing in the presence of glutethimide and iron. Induction of activity was associated with increases in heme oxygenase mRNA and protein; and, when induction was prevented by 4,6-dioxoheptanoic acid, no increase in either mRNA or immunoreactive protein was observed. δ-Aminolevulinic acid synthase activity was also synergistically increased by glutethimide and iron; this increase occurred 4-6 hours after maximal heme oxygenase activity had been attained. The temporal relationship between the induction of δ-aminolevulinic acid synthase and heme oxygenase suggested that the oxygenase depleted a regulatory heme pool that would normally prevent uncontrolled induction of the synthase. When cultures were exposed to tin-mesoporphyrin, a potent inhibitor of heme oxygenase, induction of δ-aminolevulinic acid synthase, normally produced by glutethimide and iron, was prevented. Addition of tin-mesoporphyrin after δ-aminolevulinic acid synthase induction had already been established promptly halted any further induction. When heme or a combination of heme and tin-mesoporphyrin was added after induction of δ-aminolevulinic acid synthase was established, activity of the synthase was rapidly reduced. Finally, experiments in primary chick embryo liver cells with tin-, zinc- and copper- chelated porphyrins were done to assess their effects on activities of δ-aminolevulinic acid synthase, induced by prior treatment of cells with glutethimide and iron. Nanomolar concentrations of zinc- or tin porphyrins reduced δ-aminolevulinic acid synthase activities, while copper-chelated porphyrins did not. When nanomolar concentrations of heme were added with zinc- or tin-porphyrins, δ-aminolevulinic acid synthase activity was further reduced. Effects of the non-heme metalloporphyrins on δ-aminolevulinic acid synthase were closely correlated with their abilities to inhibit heme oxygenase (r=0.78). The largest decrease of δ-aminolevulinic acid synthase (67%) was obtained with zinc-mesoporphyrin and heme. There was a rapid appearance of the cytosolic, precursor form of δ-aminolevulinic acid synthase in the presence of both 10 μM heme or 50 nM zinc-mesoporphyrin and 200 nM heme. Reduction of the half-life of the mRNA from 5.2 hours to 2.2-2.5 hours was observed in the presence of both 10 μM heme or 50 nM zinc-mesoporphyrin and 200 nM heme. In summary, the chick embryo liver cell culture model treated with glutethimide and iron may serve as one experimental model for patients suffering from acute porphyrias, in whom uncontrolled induction of hepatic δ-aminolevulinic acid synthase plays a key role in pathogenesis of disease. The synergistic induction of δ-aminolevulinic acid synthase in the presence of glutethimide and iron may serve as an experimental paradigm for this disease. The reduction of δ-aminolevulinic acid synthase by low doses of zinc-mesoporphyrin and heme may help form the experimental foundation for eventual studies in patients suffering from acute porphyrias.

The overall aim of this thesis was to determine how natural killer (NK) cells regulate virus infections in vivo. Anti-viral mechanisms by which NK cells control murine cytomegalovirus (MCMV) infection in the spleens and livers of adult C57BL/6 mice were first studied, revealing different mechanisms of control in different organs. Three days post-infection, MCMV titers in the spleens of perforin-deficient (perforin 0/0) mice were higher than in wild type controls, but no elevation of liver titers was found in perforin 0/0 mice. NK cell depletion in MCMV-infected perforin 0/0 mice resulted only in an increase in liver viral titers but not in spleen titers. Depletion of IFN-γ in adult C57BL/6 mice by injections with mAbs to IFN-γ resulted in an increase in viral titers in the liver but not in the spleen. Analyses using IFN-γ-receptor-deficient (IFN-γR0/0) mice, rendered chimeric with C57BL/6 bone marrow cells, indicated that even though the donor spleen cells could respond to IFN-γ, the depletion of NK cells in a recipient environment where the host cells could not respond to IFN-γ caused an increase in MCMV titers in the spleens but had little effect in the liver. IFN-γ has the ability to induce a variety of cells to produce nitric oxide (NO), and administrating the nitric oxide synthase (NOS) inhibitor Nω-monomethyl-L-arginine (L-NMA) into MCMV-infected adult C57BL/6 mice resulted in MCMV titer increases in the liver but not in the spleen. These data indicate that in adult C57BL/6 mice, there is a dichotomy in the mechanisms utilized by NK cells in the regulation of MCMV in different organs. In the spleen NK cells exert their effects in a perforin-dependent manner, suggesting a cytotoxic mechanism, whereas in the liver the production of IFN-γ by NK cells may be a predominant mechanism in the regulation of MCMV synthesis. These results may explain why the Cmv-1r (Cmv-1-resistant) locus, which maps closely to genes regulating NK cell cytotoxic function, confers an NK cell-dependent resistance to MCMV infection in the spleen but not in the liver. The ability of adoptively transferred cells to protect suckling mice from MCMV was another model used to study the mechanisms utilized by NK cells in the regulation of MCMV. Adoptive transfers of 129, C57BL/6 and perforin 0/0 spleen cells or lymphokine-activated killer (LAK) cells into 4 - 6 day old MCMV-infected C57BL/6 suckling mice significantly lowered the splenic MCMV titers in these mice compared to the infected controls. Adoptive transfers of C57BL/6 spleen cells into MCMV-infected 129 suckling mice also decreased the amount of MCMV in the 129 suckling mice, but C57BL/6 spleen cells could not regulate MCMV synthesis when adoptively transferred into 129/IFN-γR0/0 suckling mice. These results suggest that, in the suckling mouse model, the regulation of MCMV by the adoptively transferred NK cells is via an IFN-γ-dependent, perforin-independent, Cmv-1-independent mechanism. The Cmv-1 gene locus resides within the NK gene complex, in close proximity to the Ly49 NK cell receptor family. Analyses were carried out to determine if any of the 4 known Ly49 NK cell receptors (Ly49A, C, D and G2) played a role in the control of MCMV synthesis by NK cells. Studies comparing the expression of the different Ly49 NK cell subsets in the spleen and the peritoneal cavity revealed that there were differences in the distribution of the Ly49 receptors on NK1.1+ cells. Three days post-MCMV infection, the percentage of NK1.1+- Ly49+ NK cells in the spleen and the peritoneal cavity were different than in naive controls. Within the splenic NK1.1+ population, increases in NK1.1+ -Ly49A+ and NK1.1+-Ly49G2+ cells but decreases in NK1.1+-Ly49C+ and NK1.1+-Ly49D+ cells were observed. These changes in the spleen were accompanied by a concomitant decrease in NK1.1+ - Ly49A+ cells and increases in NK1.1+-Ly49C+, NK1.1+-Ly49D+ and NK1.1+-Ly49G2+ cells within the NK1.1+ population in the peritoneal cavity. These data suggest that 3 days post-MCMV infection, there may be movement of NK cells between the different organs. The role of Ly49 NK cell receptors in the regulation of MCMV was tested using adult C57BL/6 mice depleted of single or multiple Ly49 NK cell subsets. These in vivo depletions did not affect the ability of the residual NK cells to regulate MCMV synthesis. LAK cells sorted into the different Ly49 NK cell subsets and adoptively transferred into C57BL/6 suckling mice lowered the splenic MCMV titers in these mice. Together, these results indicate that even though there is a redistribution of the Ly49 NK cell subsets during MCMV infection, the presence or absence of anyone of the 4 tested Ly49 NK cell receptors does not affect the regulation of MCMV by NK cells. However, there remain a possibility that one of the undefined Ly49 receptors or an untested NK cell receptor may be important in the control ofMCMV. Most of the cloned NK cell receptors have been shown to bind to MHC class I molecules, and MHC class I antigens have been implicated as modulators of target cell sensitivity to NK cell-mediated lysis. The regulation of virus infections and the fate of NK cells and their natural targets was examined in β2-microglobulin-deficient mice [β2m (-/-)], which have defective MHC class I expression. Infections with either the NK cell-sensitive MCMV or the NK cell-resistant lymphocytic choriomeningitis virus (LCMV) significantly augmented NK cell activity in either C57BL/6 or β2m (-/-) mice. Depletion of NK cells in vivo with antiserum to asialo GM1 markedly enhanced the synthesis of MCMV but had no effect on the synthesis of LCMV in either strain of mouse. Adoptively transferred β2m (-/-) spleen cells lowered splenic MCMV titers in C57BL/6 suckling mice, not unlike adoptively transferred C57BL/6 spleen cells. Analysis of naturally NK cell-sensitive thymocyte targets from these virus-infected β2m (-/-) mice revealed no cell surface expression of class I MHC detectable by conformation-dependent or -independent antibodies, but the virus infections enhanced class I expression on thymocytes from C57BL/6 mice. The sensitivity of C57BL/6 thymocytes to NK cell-mediated lysis was markedly reduced after in vivo poly inosinic:cytidylic (poly I:C) treatment or viral infection; in contrast, the sensitivity of the β2m (-/-) thymocytes was significantly less affected by poly I:C or viral infection. These data indicate that the normal expression of MHC class I antigens on NK cells or their targets is not required for the anti-viral functions of NK cells against an NK-sensitive virus (MCMV) nor do they protect an NK-resistant virus (LCMV) from the anti-viral activity of NK cells. Together, the data presented in this thesis help to further our understanding of the mechanisms utilized by NK cells in the control ofMCMV in both adult and suckling mice, and also help clarify the roles played by Ly49 NK cell receptors and MHC class I molecules in the regulation of MCMV.

Bile acid metabolism is integrally involved in cholesterol homeostasis in mammals because it is the major means by which cholesterol is eliminated from the body. We have undertaken an effort to study the molecular mechanisms underlying the regulation of bile acid metabolism by isolating and characterizing the cDNA and gene for an enzyme that hydroxylates lithocholic acid (LCA) at position 6β, lithocholic acid 6β-hydroxylase; the first bile acid-induced gene reported. LCA is a very hydrophobic, toxic bile acid formed from chenodeoxycholic acid in the gut lumen upon reduction of the 7α-hydroxy group by microbial enzymes. The proper elimination of LCA is essential for maintenance of the bile acid pool and for prevention of cholestasis which results from LCA precipitating in the cannaculi of the liver when its concentration is high. The LCA 6β-hydroxylase cDNA was isolated by differential hybridization of hamster liver libraries prepared from animals fed either a cholic acid enriched diet or a cholestipol-rich chow and was named CYP3A10 based on its homology with other cytochrome P450s (P450) in family 3A. We found that CYP3A10 was essentially expressed only in males. A statistical analysis of RNA from young males fed with cholic acid and normal chow showed that the cholic acid induction was about 50% at the RNA level. We determined the biological nature of the protein encoded by CYP3A10 by expression of the cDNA in COS cells. Microsomes prepared from transfected cells were assayed with LCA as a substrate and found to hydroxylate LCA predominantly at position 6β. We examined whether CYP3A10 could hydroxylate other steroid compounds by assays with testosterone, progesterone and androstenedione and found that, although 6β-hydroxylase (as well as others) activity was observed with all three, LCA was the preferred substrate based on kinetic analysis. A developmental time course of CYP3A10 expression in males showed little expression before puberty, a striking induction of expression at puberty and a fourfold induction thereafter through adulthood. We then examined the male-specific expression of CYP3A10 in hamster liver. We disrupted the pattern of GH secretion in male hamsters by hypophysectomy, neonatal glutamate treatment and by continuous infusion of GH via osmotic minipumps (to mimic the female pattern of GH secretion) and found no significant effect on CYP3A10 expression. Conversely, in females, hypophysectomy and neonatal glutamate treatment significantly induced CYP3A10 expression 5- to 10-fold. Additionally, when females treated neonatally with glutamate were injected twice daily with GH as adults (to mimic the male pattern of GH secretion), the levels of CYP3A10 expression were not significantly different from those of normal males. These results led us to conclude that the pattern of GH secretion in males does not control the male-specific expression of CYP3A10 but that in females expression can be induced by altering the tonic secretion of GH. No significant effect on CYP3A10 expression was observed by castration of adult males, indicating that circulating androgens were not required for expression. We found that gonadal hormones (e.g. estrogen and progesterone) do not have a suppressive effect on CYP3A10 expression in females since ovariectomy did not induce expression. Many genes are "imprinted" neonatally by exposure to a given effector for developmental-, tissue- or sexually regulated expression. We investigated whether neonatal androgen exposure was required for male-specific expression of CYP3A10 by castrating hamsters neonatally and determining the level of CYP3A10 expression in adulthood. Our results indicate that androgens are required neonatally for CYP3A10 expression since no expression was observed in neonatally castrated hamsters. We were unable to induce expression in neonatally castrated hamsters by either GH or testosterone injections. These results suggest several notable points 1) that CYP3A10 expression is programmed neonatally by androgen exposure; 2) that androgens exert their effect directly on the liver and not via the hypothalamus; 3) that neither testosterone nor GH can restore CYP3A10 expression when males have not been exposed to androgens neonatally; and 4) that in experimental conditions, females can be induced to express CYP3A10, which indicates that there are two modes for regulating expression: by "imprinting" in males and by GH and testosterone in females. We are now studying the molecular mechanisms involved in the bile acid-mediated induction and the male-specific expression of CYP3A10. We have cloned approximately 8 kb of 5' flanking DNA from a hamster genomic library and sequenced about 1 kb of proximal DNA. Primer extension and S1 digestion analyses indicate that the mRNA for CYP3A10 has multiple transcription initiation sites clustered about 90 bp from the initiator methionine codon. We have also prepared CYP3A10 promoter/lacZ chimeric constructs to begin delineating the cis-acting elements controlling CYP3A10 expression and regulation. We used H2.35 cells as recipients because they are a mouse hepatocyte cell line that has been transformed with a temperature sensitive SV40. These cells can be grown at the permissive temperature and can be induced to behave like liver cells, the differentiated condition, by switching to a nonpermissive temperature. We have found that the construct with 1 kb of proximal CYP3A10 5' flanking DNA was able to express the reporter gene at higher levels under differentiated conditions, which were consistent with higher expression of an albumin promoter/lacZconstruct, upon switching the cells to the more liver phenotype. The system characterized and described here is ideally suited for dissecting the molecular details governing bile acid-mediated regulation and sexually dimorphic expression of liver genes. Very little is known about both these very important biological phenomena. Much could be learned about transcriptional regulation of liver genes by investigating the cis-elements and trans-acting factors mediating regulation of CYP3A10 expression.

No trato digestório a colite ulcerativa apresenta necrose no intestino como processos patofisiológicos. Este projeto visou estudar neurônios com códigos químicos do sistema nervoso entérico (SNE) e a morfologia estrutural do intestino grosso de animais com colite ulcerativa. Grupos: a) Colite: injetados com TNBS, b) PBS: injetados com PBS e c) controle. Os tecidos foram preparados por métodos imunohistoquímicos de duplas marcações do receptor P2X7 com NOS, ChAT, Calb, Calr, anti-HuC/D (pan-neuronal) e S100 (células glias). No grupo Colite, no plexo mioentérico, o receptor P2X7 estava diminuído. No tecido lesado apresentou aumento de neutrófilos e da lâmina própria, alteração de colágeno e destruição do epitélio e células caliciformes. Reduziram colocalizações de neurônios com receptor no plexo mioentérico e aumento no plexo submucoso. Houve reduções nas densidades e áreas dos neurônios no SNE. Conclui-se que a colite afetou os neurônios entéricos e células gliais, causou alterações morfológicas, sendo assim, pode afetar motilidade intestinal. / In the digestive tract ulcerative colitis have a bowel necrosis as pathophysiological processes. This project aimed to study neurons with their respective chemicals codes the enteric nervous system (ENS) as well as structural morphology of the distal colon of animals with ulcerative colitis. Groups: a) colitis: TNBS injected, b) PBS: PBS injected and c) control. The tissues were prepared by immunohistochemical methods for double marking with P2X7 receptor, ChAT, Calb, Calr, anti-HUC / D (pan-neuronal) and S100 (glial cells). In the colitis group, the myenteric plexus, the P2X7 receptor decreased. In the injured tissue showed increased neutrophils, alteration and destruction of collagen and epithelial goblet cells. There were reduced colocalizations of neurons with receptor in myenteric plexus and increase in submucosal plexus. There were reductions in the densities and areas neurons of ENS. Concluded that colitis affected enteric neurons and glial cells, causing morphological changes and could be affect intestinal motility.

The Dissertation treats the exacting wards of the zoological gardens - Colobus guereza, Colobus angolensis and Colobus polykomos. The first part summarizes the knowledge of breeding especially in our zoological gardens. Special attention is payed to feeding which is considered as a limiting factor of breeding. The following part compares morphology of the digestive tract of the Colobinae with the other herbivores and with the other monkeys, especially from the point of view of morphology. In the end of the Disseration, an informative experimental observation of cellulose (roughage) digestion is described. Key words: Multiple-compartment stomach, Angolan colobus, Artiodactyls, Digestive system, Cellulose digestion

The Dissertation treats the exacting wards of the zoological gardens - Colobus guereza, Colobus angolensis and Colobus polykomos. The first part summarizes the knowledge of breeding especially in our zoological gardens. Special attention is payed to feeding which is considered as a limiting factor of breeding. The following part compares morphology of the digestive tract of the Colobinae with the other herbivores and with the other monkeys, especially from the point of view of morphology. In the end of the Disseration, an informative experimental observation of cellulose (roughage) digestion is described. Key words: multiple-compartment stomach, Angolan colobus, artiodactyls, digestive system, cellulose digestion

Objective: Treatment compliance in children with Crohn’s disease is associated with higher levels of symptom remission. We hypothesized that the management, comorbidities, and complications for children with Crohn’s disease would differ based on a diagnosis of noncompliance. Methods: Using the Kids’ Inpatient Database for 2006-2012, we identified young patients (<21 >years) with a diagnosis of Crohn’s disease. Diagnoses and procedures were analyzed according to a recorded diagnosis of noncompliance. Multivariable logistic regression analysis was performed to examine the association between noncompliance and the outcomes of interest. Results: There were 28,337 pediatric Crohn’s disease hospitalizations identified with 1,028 (3.6%) hospitalizations having a diagnosis of both Crohn’s disease and noncompliance. The mean age of the study population was 15.9 years and 48.9% were girls. Black patients ( multivariable adjusted odds ratio, aOR,2.27; 95% CI:1.84-2.79) and those in the lowest income quartile (aOR 1.57; 95% CI:1.20-2.05) had an increased likelihood of a noncompliance diagnosis than respective comparison groups. Noncompliant patients had an increased likelihood of concurrent depression, nutritional deficiency, and anemia. Patients with a diagnosis of noncompliance had lower rates of intestinal obstruction (4.0% vs 6.3%), intraabdominal abscesses (2.0% vs 4.2%,), and underwent fewer major surgical procedures (aOR 0.40; 95% CI:0.31-0.53) and large bowel resections (aOR 0.44; 95% CI:0.31-0.64) than patients without this diagnosis. Conclusions: We found significant differences in socioeconomic status and race among hospitalized children with Crohn’s disease with, as compared to those without, a diagnosis of noncompliance. Children with noncompliance have different comorbidities, disease-related complications, and are managed differently. Possible explanations for observed treatment differences include a reluctance to offer surgery to those with a diagnosis of noncompliance, a refusal of intervention by noncompliant patients, or implicit bias. Further investigation is warranted to better define noncompliance in this population and to determine the implications of this diagnosis.

Nonalcoholic steatohepatitis (NASH) is a severe liver disorder characterized by triglyceride accumulation, severe inflammation, and fibrosis. With the recent increase in prevalence, NASH is now the leading cause of liver transplantation, with no approved therapeutics available. Despite years of research, the exact molecular mechanism of NASH progression is not well understood, but fat accumulation is believed to be the primary driver of the disease. Therefore, diacylglycerol O-acyltransferase 2 (DGAT2), a key enzyme in triglyceride synthesis, has been explored as a NASH target. RNAi-based therapeutics is revolutionizing the treatment of liver diseases, with recent chemical advances supporting long term gene silencing with single subcutaneous administration. Here we identified a hyper-functional, fully chemically stabilized GalNAc conjugated siRNA targeting DGAT2 (Dgat2-1473) that upon injection elicits up to three months of DGAT2 silencing (>80-90%, p<0.0001) in wild-type and NSG-PiZ “humanized” mice. Using an obesity-driven mouse model of NASH (ob/ob-GAN), Dgat2-1473 administration prevents and reverses triglyceride accumulation (> 50%, p:0.0008), resulting in significant improvement of the fatty liver phenotype. However, surprisingly, the reduction in liver fat didn’t translate into a similar impact on inflammation and fibrosis. Thus, while Dgat2-1473 is a practical, long-lasting silencing agent for potential therapeutic attenuation of liver steatosis, combinatorial targeting of a second pathway may be necessary for therapeutic efficacy against NASH.