Type 1 diabetes (T1D) is a metabolic disorder characterized by destruction of the pancreatic beta-islet cells leading to complete loss of insulin production. Gliclazide is used in Type 2 diabetes (T2D) to stimulate insulin production but it also has beneficial extrapancreatic effects which make it potentially useful in T1D. In fact, some T2D patients continue to use gliclazide even after their diabetes progresses to T1D since it provides better glycemic control than insulin alone. About 30% of a gliclazide dose undergoes enterohepatic recirculation which may contribute to the observed high interindividual variability in its pharmacokinetics. This may limit its efficacy in T1D especially since diabetes can disturb the gut microbiota and give rise to changes in bile composition and enterohepatic recirculation. Improving the absorption of gliclazide through the use of bile acids and probiotics may reduce this variability and improve the efficacy of gliclazide in T1D. The aim of this thesis was to investigate the interaction between the semisynthetic bile acid, 12-monoketocholic acid (MKC) and gliclazide in terms of pharmacokinetics and hypoglycemic effects in a rat model of T1D with and without probiotic pretreatment. A parallel ex vivo (Ussing chamber) study was carried out to investigate the mechanism of the interaction. Sensitive LC-MS and HPLC methods (Chapter 2) were developed to determine the concentrations of gliclazide and MKC in Ringer's solution and rat serum. Diabetes was induced in male Wistar rats by intravenous (i.v.) alloxan (30 mg/kg). Rats with blood glucose concentration > 18 mmol/l and serum insulin concentration < 0.04 [mu]g/l, 2-3 days after alloxan injection were considered diabetic. A total of 280 male Wistar rats (Chapter 3) were randomly allocated into 28 groups (n=10) of which 14 were made diabetic. Then 7 groups of healthy and 7 groups of diabetic rats were gavaged with probiotics (10⁸ CFU/mg, 75 mg/kg) every 12 hours for three days after which single doses of gliclazide (20 mg/kg), MKC (4 mg/kg) or the combination were administered either by tail vein injection (i.v.) or by gavage. The other 14 groups (7 healthy and 7 diabetic) were gavaged with saline every 12 hours for three days and then treated in the same way. Blood samples were collected from the tail vein for 10 hours after the dose and analyzed for blood glucose, serum gliclazide & serum MKC concentrations. Serum concentration-time curves for gliclazide and MKC were used to determine pharmacokinetic parameters. In the parallel ex vivo study (Chapter 4), 88 rats were randomly divided into 22 groups (n=4 rats per group, 8 chambers per rat), of which 11 groups were made diabetic. Of the 22 groups, 8 groups (4 healthy and 4 diabetic) were pretreated with probiotics as described above to study their influence on gliclazide and MKC flux, 8 groups (4 healthy and 4 diabetic) were used to investigate the interaction between gliclazide and MKC during transport, and 6 groups (3 healthy and 3 diabetic) were used to study the influence of selective inhibitors of the drug transporters Mrp2, Mrp3 and Mdr1 on gliclazide flux. 10 cm piece of the ileum was removed from each rat, the underlying muscle layer and connective tissue removed and the epithelial sheets mounted into Ussing chambers. Gliclazide, MKC or a combination were added to either the mucosal or serosal side and samples collected from both sides for 3 h to determine mucosal-to-serosal absorptive flux (Jss[MtoS]) and serosal-to-mucosal secretory flux (Jss[StoM]) of gliclazide and MKC as appropriate. In diabetic rats, gliclazide alone had no effect on blood glucose levels (Ch3, exp2) whereas MKC reduced it from 23 � 3 to 18 � 3 mmol/l (Ch3, exp3) and the combination of gliclazide and MKC reduced it even further from 24 � 4 to 16 � 3 mmol/l (Ch3, exp4). In diabetic rats, probiotic treatment reduced blood glucose by 2-fold (Ch3, exp1) and enhanced the hypoglycemic effect of the combination of gliclazide and MKC (blood glucose decreased from 24 � 3 to 10 � 2 mmol/l). The bioavailability of gliclazide was higher in healthy rats (53.2 � 6.2%) than in diabetic rats (39.9 � 6.0%) (Ch3, exp2). In healthy rats, MKC enhanced gliclazide bioavailability (82.7 � 8.2%) but in diabetic rats MKC had no effect on gliclazide bioavailability (Ch3, exp4). In healthy rats, probiotic pretreatment significantly reduced gliclazide and MKC bioavailabilities (p<0.01) while in diabetic rats, probiotic pretreatment significantly increased the low bioavailability of gliclazide to a level similar to that in healthy rats (Ch3, exp2 & 3). MKC showed clear evidence of enterohepatic recycling and probiotics delayed and reduced its systemic absorption (Ch3, exp3). In ileal tissues from healthy rats, Ussing chamber studies showed gliclazide is most likely a substrate of Mrp2 and Mrp3 (Ch4, exp5) and MKC significantly reduced gliclazide Jss[MtoS] probably through Mrp3 inhibition (Ch4, exp1). In ileal tissue from diabetic rats, MKC had no effect on gliclazide Jss[MtoS] and Jss[StoM] (Ch4, exp2) and none of the inhibitors had any effect of gliclazide flux (Ch4, exp6). This suggests that these transporters are dysfunctional in this model of T1D. Probiotics and MKC have hypoglycemic effects that appear to be enhanced by gliclazide and all appear to interact at the level of ileal drug transporters. The combination of probiotic treatment, gliclazide and MKC exerted the greatest hypoglycemic effect in T1D rats. Accordingly, the application of this combination may have potential in improving the treatment of T1D.

Enterohepatic Helicobacter species (EHS) are emerging infectious disease agents. Infection of the enterohepatobiliary tract of several mammals by this group of bacteria results in various pathological disorders. The availability of the Helicobacter hepaticus sequenced and annotated genome, allowed molecular characterisation of the responses of H. hepaticus to host factors such as bile. The adaptation/responses of the bacterium to bovine, porcine and human bile were investigated using proteomics and transcriptomics. Ninety-one different proteins were identified in the responses of H. hepaticus response to the three types of bile. These proteins participate in several key cellular processes including DNA replication; protein transcription, translation and folding; oxidative stress response; motility; virulence; and metabolism. In particular, the bacteria deployed several strategies such as inhibition of the TCA cycle and the electron transport chain as well as iron sequestration to ensure control of the levels of hydroxyl radicals. The results of this study revealed also the modulation by bile of the expression of H. hepaticus genes involved in response to oxidative stress and virulence. The responses of human HEp-2 and Huh7-derived cell-lines to H. hepaticus and Helicobacter bilis, respectively, were investigated employing proteomics and transcriptomics. One-hundred and twenty different proteins were differentially expressed in the responses of the human cells to the presence of Helicobacter spp. in the cell cultures. These proteins are involved in regulation of cell proliferation and structure; metabolism; protein transcription, translation and modification; stress response; and tumour induction. For example, in co-cultures of Huh7-derived cells and H. bilis, the activation of several mitochondrial and endoplasmic reticulum stress-related proteins and the dysregulation of several apoptosis effectors were suggested as mechanisms that could result in the death of the liver cells. Importantly, the differential expression of several tumour-related proteins by the Huh7 cells supported a possible role for Helicobacter spp. in liver cancer.

Cholangiocarcinoma (CCA), or cancer of the bile ducts, is extremely prevalent in people from Laos and Thailand whose staple diet is uncooked fish which harbour the liver fluke, Opisthorchis viverrini. There is no stronger link between a parasite and cancer than that between O. viverrini and CCA. Indeed WHO data suggests that one in six infected people contract liver cancer derived from the fluke. In vitro and in vivo studies have indicated that the fluke’s excretory/secretory (ES) proteins are mitogenic and likely make significant contributions to the initiation of CCA. To identify these carcinogenic components I undertook two distinct yet related approaches - (1) traditional protein purification methods to separate ES products, specifically targeting mitogenic proteins, and, (2) bioinformatic screening of 5,000 expressed sequences tags (ESTs) and ES proteins characterized by shotgun proteomic approaches, searching for homologues of molecules that are associated with human cancers. The protein purification approach utilized a cell proliferation assay that I developed for measuring cell replication rates in NIH-3T3 fibroblast and human CCA (KKU-100) cell lines stimulated with ES products. ES products were separated by a combination of ion exchange, hydrophobic interaction, size exclusion and a final ion exchange polishing chromatography steps. ES products and chromatographically separated ES proteins were added to cultured cells to observe mitogenic activity. A four-step purification process resulted in the isolation of 23 and 31 kDa proteins that stimulated cell proliferation at just picomolar quantities. These proteins account for a very small proportion of the total protein biomass (6 ppm and 39 ppm respectively) secreted by the parasite. Their identities are currently being explored using alternate proteomic approaches. Some growth factors bind to heparin, so an alternative purification process was developed using a heparin affinity column to purify ES mitogens. In combination with ion exchange chromatography a 20 kDa heparin-binding protein was identified using tandem mass spectrometry as a member of the sperm-coating protein 65 (SCP)-like extracellular proteins, also called SCP/Tpx-1/Ag5/PR-1/Sc766 (SCP/TAPS; Pfam accession number no. PF00188). The O. viverrini heparin-binding SCP/TAPs protein shared similarity with secreted proteins from other parasitic helminths including the hookworm activation-associated protein family, some of which are known to bind to host cells. In silico screening of the O. viverrini ESTs and ES peptides generated by mass spectrometry for proteins associated with cell proliferation and cancer revealed numerous secreted proteins of interest. One of these proteins shared identity with granulin, a vertebrate growth factor. The cDNA corresponding to this protein was termed Ov-grn-1. The predicted molecular characteristics of Ov-GRN-1 (isoelectric point and molecular weight) corresponded with the biochemical properties of the semi-purified mitogen that was chromatographically purified from ES products. Recombinant Ov-GRN-1 was expressed in E. coli in inclusion bodies and the purified denatured protein was refolded to produce a soluble protein. Refolded Ov-GRN-1 stimulated proliferation of NIH-3T3 fibroblasts at nanomolar concentrations and induced shape changes in affected cells. Antibodies raised to recombinant Ov-GRN-1 inhibited the ability of O. viverrini ES products to induce proliferation of fibroblasts and the KKU-100 CCA cell line in vitro, indicating that Ov-GRN-1 is the major growth factor present in O. viverrini ES products. This is the first report of a secreted growth factor from a parasitic worm that induces proliferation of host cells, and supports a role for this fluke protein in establishment of a tumourigenic environment that may ultimately manifest as CCA.

Cholangiocarcinoma (CCA), or cancer of the bile ducts, is extremely prevalent in people from Laos and Thailand whose staple diet is uncooked fish which harbour the liver fluke, Opisthorchis viverrini. There is no stronger link between a parasite and cancer than that between O. viverrini and CCA. Indeed WHO data suggests that one in six infected people contract liver cancer derived from the fluke. In vitro and in vivo studies have indicated that the fluke’s excretory/secretory (ES) proteins are mitogenic and likely make significant contributions to the initiation of CCA. To identify these carcinogenic components I undertook two distinct yet related approaches - (1) traditional protein purification methods to separate ES products, specifically targeting mitogenic proteins, and, (2) bioinformatic screening of 5,000 expressed sequences tags (ESTs) and ES proteins characterized by shotgun proteomic approaches, searching for homologues of molecules that are associated with human cancers. The protein purification approach utilized a cell proliferation assay that I developed for measuring cell replication rates in NIH-3T3 fibroblast and human CCA (KKU-100) cell lines stimulated with ES products. ES products were separated by a combination of ion exchange, hydrophobic interaction, size exclusion and a final ion exchange polishing chromatography steps. ES products and chromatographically separated ES proteins were added to cultured cells to observe mitogenic activity. A four-step purification process resulted in the isolation of 23 and 31 kDa proteins that stimulated cell proliferation at just picomolar quantities. These proteins account for a very small proportion of the total protein biomass (6 ppm and 39 ppm respectively) secreted by the parasite. Their identities are currently being explored using alternate proteomic approaches. Some growth factors bind to heparin, so an alternative purification process was developed using a heparin affinity column to purify ES mitogens. In combination with ion exchange chromatography a 20 kDa heparin-binding protein was identified using tandem mass spectrometry as a member of the sperm-coating protein 65 (SCP)-like extracellular proteins, also called SCP/Tpx-1/Ag5/PR-1/Sc766 (SCP/TAPS; Pfam accession number no. PF00188). The O. viverrini heparin-binding SCP/TAPs protein shared similarity with secreted proteins from other parasitic helminths including the hookworm activation-associated protein family, some of which are known to bind to host cells. In silico screening of the O. viverrini ESTs and ES peptides generated by mass spectrometry for proteins associated with cell proliferation and cancer revealed numerous secreted proteins of interest. One of these proteins shared identity with granulin, a vertebrate growth factor. The cDNA corresponding to this protein was termed Ov-grn-1. The predicted molecular characteristics of Ov-GRN-1 (isoelectric point and molecular weight) corresponded with the biochemical properties of the semi-purified mitogen that was chromatographically purified from ES products. Recombinant Ov-GRN-1 was expressed in E. coli in inclusion bodies and the purified denatured protein was refolded to produce a soluble protein. Refolded Ov-GRN-1 stimulated proliferation of NIH-3T3 fibroblasts at nanomolar concentrations and induced shape changes in affected cells. Antibodies raised to recombinant Ov-GRN-1 inhibited the ability of O. viverrini ES products to induce proliferation of fibroblasts and the KKU-100 CCA cell line in vitro, indicating that Ov-GRN-1 is the major growth factor present in O. viverrini ES products. This is the first report of a secreted growth factor from a parasitic worm that induces proliferation of host cells, and supports a role for this fluke protein in establishment of a tumourigenic environment that may ultimately manifest as CCA.

Diss. (sammanfattning) Stockholm : Karol. inst., 2007. / Härtill 4 uppsatser.

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

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [92]-106).

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2008. / Vita. Bibliography: p.157-158