Thèses sur le sujet « Treatment of inflammatory disease »

In 117 (male = 48) patients with Crohn's disease (CD) aged 20 to 65 years, bone mineral density (BMD) and biochemical markers of bone turnover were measured. Sex hormone status was assessed in men with CD. Hand skinfold thickness and calcaneal ultrasound were evaluated as screening tools for osteoporosis; and the effect of low impact exercise on BMD was prospectively studied. In patients with colitis, the effects of rectal steroids on bone turnover was investigated. The major determinants of BMD in CD were body weight, corticosteroids and male sex. Hypogonadism was an uncommon cause of flow BMD in men with CD. The bone resorption marker deoxypyridinoline was significantly increased in patients with CD compared to controls (10.97 (9.22) nMDPD/mMCreatinine vs 5.02 (1.03) mMDPH/mMCreatinine, difference in means-=5.95, 95% CI -9.6 to -2.3, p=0.00001) but bone formation markers were not significantly different. Hand skinfold thickness was significantly associated with BMD at the lumbar spine and femoral neck (r=0.43, 95% CI 0.27, 0.57 p<0.0001). However, the relation was inadequate to diagnose osteoporosis in an individual. Similarly, the sensitivity and specificity of calcaneal ultrasound were too low to recommend it as a screening tool for osteoporosis at the hip (sensitivity 66.7%, specificity 85.6%) or spine (sensitivity 75%, specificity 89%). Significant gains in BMD occurred at the trochanter of patients who fully complied with a 12 month exercise programme (+7.7% (8.2) vs +3.1% (5.83), difference in means=4.67%, 95% CI 0.86 to 8.48, p=0.02). Increases in BMD were significantly related to compliance with the exercises and were independent of other potential confounding variables. In patients with colitis, rectally administered corticosteroids had no significant effects on bone turnover. Bone resorption is increased in patients with CD who are at high risk of low BMD. Corticosteroids are an important aetiological factor and rectally administered steroids should be prescribed wherever possible. Low impact exercise could be an effective method of increasing BMD in some patients with CD.

This PhD thesis combines four chapters on different fields of basic research and sets the focus on two circulating viruses of global concern, the orthomyxovirus influenza A virus (IAV) and the alphavirus Ross River virus (RRV). The first three chapters include swine influenza A virus (sIAV) surveillance for the detection and characterisation of IAV subtypes, an in vitro high throughput screening (HTS) on host micro RNAs (miRNAs) for the discovery of novel anti-IAV (H7N9) targets and their underlying mechanisms, and an approach to reduce disease pathogenesis in mice infected with H7N9 by targeting the pro-inflammatory factor CCL2. In a fourth chapter, drug repurposing with the interleukin-1 (IL-1) inhibitor anakinra was investigated to treat RRV-induced bone loss in mice. By combining these four chapters, a broad range of drug discovery is covered in this PhD thesis; Surveillance, HTS target discovery and the application of drug repurposing in animal models of viral diseases.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
Science, Environment, Engineering and Technology
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Current aminosalicylic acid (ASA) preparations are used to induce and control the remission of inflammatory bowel diseases (IBOs); however, only partial success has been achi~ved. Hence, in the absence of a satisfactor~ treatment or a cure, there is a need for the development of further therapeutic strategies to combat IBOs. The objective of this study was to explore new therapeutic avenues by developing novel conjugates of the active ASAs pharmacophore. These conjugates were designed to incorporate the ASA with other moieties which show therapeutically useful properties. This report includes the design, synthesis and characterisation of novel ASA and aliphatic amino acid (AA) conjugates with the antioxidant ethylenediaminetetraacetic acid (EOTA). Diglyceride and model polymeric delivery systems were also investigated, although, these two alternative areas were not highly successful. All the EOTA conjugates· described in this study are readily synthesised in two steps using standard methyl ester and amide-bond formation techniques. A wide range of biochemical properties were studied for the conjugates synthesised in this project. These studies determined the effectiveness of the conjugates in the combat against IBOs. These properties included metal ion chelation, reactive oxygen species (ROS) suppression, proteolytic and pH stabilities, as well as anti-cancer activity. Firstly, the chelation of labile transition metal ions has been shown to remove the ion's pro-oxidant activity. The removal of pro-oxidant activities by chelation could assist in the treatment of IBOs, which has been shown to be linked with oxidative stress. Secondly, due to a link between ROS and IBOs, the discovered ROS suppression would also be advantageous in the combat against IBOs. Thirdly, the gastrointestinal stability of these conjugates is favourable; as, reaching the disease site (Le. the colon and terminal ileum) intact would enable the conjugates to function as designed. And fourthly, due to a link between IBOs and cancer, ethylenediaminetetraacetic acid bis-(5-aminosalicylic acid methyl ester) was tested in the National Cancer Institute's 60 cancer cell-line.· The synthesised conjugates showed an ideal 1: 1 binding ratio with the transition metal ions Cu(lI) and Fe(III). ROS suppression was observed for all the synthesised compounds whe~ chelatcd to redox active transition metal ions. In the stability studies, it was found that the ASA conjuga.tes were· stable to proteolytic attack. This was in contrast to the AA conjugates, which were· readily degraded by protease. Additionally, all the studied conjugates showed excellent pH stabilities. And lastly; the 5-ASA-EDTA conjugate showed promising in vitro anti-cancer activity. This study reports novel anti-inflammatory conjugates, which show promising properties for use in the combat against IBOs. Results from this project have shown that the conjugates are able to chelate toxic, unbound transition metal ions, which in turn forms strong ROS suppressors. The work within this report has also shown excellent protease and pH stabilities of the ASA conjugates, which would enable them to travel through the upper gastrointestinal tract intact. Upon reaching the ~ite of disease, they would be able to either act as novel anti-inflammatory, chelating conjugates, or, as prodrugs which would then release the ASA moiety via bacterial reduction, The anti-cancer activity shown for one of the conjugates would also be beneficial in IBO treatment.

Azathioprine (AZA) and mercaptopurine (MP – also known as 6-mercaptopurine or 6MP) are the first line immunomodulatory treatments for inflammatory bowel disease (IBD) with proven efficacy for multiple clinical outcomes (fistula closure, steroid withdrawal, maintenance of remission etc.). They also have a wide application in the fields of rheumatology, dermatology, haematology and transplant medicine. However these drugs also cause toxicity and may be ineffective. Both of these outcomes can have serious consequences for the individual concerned. A proportion of toxicity caused by these drugs is explained by genetic polymorphism in the enzyme thiopurine methyltransferase (TPMT), however the majority of toxicity remains unexplained and as yet there is no satisfactory explanation for the variable efficacy of these drugs. In this thesis I explore the impact of genetic polymorphism in several novel candidate genes involved in thiopurine metabolism on the success of thiopurine treatment. Single nucleotide polymorphisms (SNPs) in xanthine oxidase/dehydrogenase (XDH) and the final enzymatic step which activates its essential cofactor (molybdenum cofactor sulfurase, MOCOS) are shown to protect against side effects to AZA therapy. Polymorphism in aldehyde oxidase (AOX) and multi-drug resistance protein 5 on the other hand, are shown to predict a lack of response to thiopurine treatment. Sequencing AOX validated the real-time PCR results and suggested that there were no other coding SNPs likely to be contributory. A pharmacogenetic index incorporating these new markers with established predictors of outcome on thiopurines is presented and the clinical utility of such an index discussed. Finally, clinical data supporting the optimisation of azathioprine therapy, both by the measurement of thioguanine metabolite profiles and through co-prescription of allopurinol are presented.

Background. Mesenchymal stem cells (MSC) based on their immune-modulatory and anti-inflammatory properties have been candidate as therapeutic treatment for several immune disease including inflammatory bowel disease (IBD). Although most of beneficial effects are explained by MSC engrafting at the site of tissue injury with modulation of inflammatory reactions, the mechanisms by which MSC exert their activities needed to be elucidated. Aim of this study was to investigate the mechanisms underlying the therapeutic efficacy of MSC in Dextran Sodium Sulphate (DSS) induced colitis. Methods. Colitis was induced in C57BL6 mice by 3% DSS treatment for 10 days. MSC were isolated from bone marrow of wild type (WT) and GFP-transgenic C57BL6 mice, cultured for 4 weeks and then sorted for Sca-1+, CD31-, cocktail lineage- surface markers. At day 5 of colitic induction the mice were treated intraperitoneally with a single injection of 3x106 GFP-MSC or with 5 daily injections of recombinant murine TNFα-stimulating gene protein 6 (TSG-6) (4 μg). Body weight and disease activity index (DAI) were monitored daily and the damage of murine colonic mucosa was evaluated by endoscopic and histological scores. To follow the migratory activity of the MSC, GFP-MSC were injected intraperitoneally in healthy or colitic mice at day 5 and their presence was assessed by flow cytometry after 24 and 48 hours in the colon and mesenteric lymph nodes. Levels of TSG-6, IL-6 and IFN-γ were measured in serum and in mucosal extracts by ELISA, while MMP activities were quantified by WB. Results. We found that one injected into the peritoneum MSC remain into the peritoneal cavity, where they aggregate along with macrophages and lymphocytes, generating organized structures. Only a small fraction (< 1%) of cells reached the inflamed colon. In vitro and in vivo assays have demonstrated that MSC secrete a multipotent anti-inflammatory protein TSG-6 able to reduce the damage to the colon. In fact, recombinant murine TSG-6 treatment improved survival rate and DSS-induced colitis by reducing markedly both systemic and mucosal levels of IL-6, IFN-γ, neutrophil infiltration, and MMP activities. Conclusions. Overall, these data indicate that the MSC gut-homing is not relevant for exerting their immunomodulatory effects, but MSC dampen the mucosal inflammatory response at distance by releasing a potent anti-inflammatory protein.

Inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), are chronic inflammatory immune-mediated diseases of the gastrointestinal tract. Two-thirds of IBD patients will develop severe disease, with complications that will require frequent surgeries and hospital admissions, and will seriously impair their quality of life. The ultimate clinical challenge of precision medicine in IBD is to find predictive markers to anticipate the development of severe disease and to monitor treatment in these patients.In the first part of my PhD thesis, we have carried out several studies monitoring the biologics used in IBD patients with severe disease. We have evaluated the pharmacokinetics of the following biologics used in IBD patients: infliximab, vedolizumab, and ustekinumab. We have focused on measuring trough levels (TLs) (defined as the serum drug level measured just before the next drug administration) early on after initiating biologic treatment to predict patient outcomes, including long- term responses in patients treated with infliximab and vedolizumab. In addition, we are currently conducting a prospective multicentric study that aims to design a pharmacokinetic model of infliximab at induction in IBD patients (EudraCT: CT 2015- 004618-10) (End of study expected by December 2019 but interim analysis available in the present work). Moreover, we have reported on the efficacy of ustekinumab in a large national cohort of highly refractory CD patients and have also examined the benefit of early measurement of ustekinumab TLs in these patients. Finally, we have reported novel findings on the impact of different wash-out periods (defined as the time frame between the discontinuation of one biologic and the initiation of a second biologic on the pharmacokinetics of the second-line biologic). Altogether, over the past 3 years, our data suggest the importance of measuring TLs early on during induction to predict long-term response to biologics during maintenance therapyIn the second part of my PhD thesis, we have analysed the inter-variability of the immune response in healthy subjects. Inflammation is the obvious key driver and underlying mechanism of disease severity in IBD. Therefore, the magnitude of inflammation must help define the phenotype of mild to severe disease. Delineating the inter-variability of the immune response in a healthy cohort constitutes a fundamental step to uncovering the genetic factors underlying this variability. We have performed whole blood cell cultures in a highly selected population of more than 400 healthy subjects stimulated with several Toll-like receptor (TLR) agonists and a T-cell receptor (TCR) antagonist. We found that the magnitude of the immune response (the high- or low-cytokine producer phenotype) was independent of the cytokine measured and the TLR agonists used. Thus, a donor exhibits a specific immune (cytokine) response or “immunotype” across cytokines released and TLR stimulation. Importantly, the high- or low-cytokine producer phenotype was different and did not overlap between the TLR and TCR stimulation conditions. In other words, a donor who is ahigh-cytokine producer following TLR stimulation will not be a high-cytokine producer following TCR stimulation (or the inverse). Therefore, we have defined TLR- or TCR- related Immunotypes (IT) as “a grading classification of the magnitude of the cytokine immune response” with IT1, IT2, and IT3 as low, intermediate, and high immunotypes. This suggests that two independent TLR and TCR ITs (TLR IT1 and TCR IT3) can co-exist in the same subject. We are now currently evaluating the genetic markers underlying these ITs before validating them in large cohort of IBD patients with mild-to-moderate and severe disease.This PhD thesis provides some data suggesting that the assessment of the pharmacokinetics of biologics early on at the initiation of treatment could help predict how the patient will respond in the long run. In parallel, this PhD thesis provides some advances in the understanding of the inter-variability of the immune response, a fundamental step before the identification of potential genetic markers underlying the inter-variability of inflammation and, hence, the severity of disease in IBD.
Doctorat en Sciences biomédicales et pharmaceutiques (Médecine)
info:eu-repo/semantics/nonPublished

Thiopurines azathioprine and 6-mercaptopurine are effective immune-modulators and cytotoxic agents widely used in the treatment of autoimmune diseases, graft rejection, and cancer. Of concern is the growing epidemiologic evidence that chronic exposure to these agents increases the risk of mesenchymal and solid tumors. Although in vitro and in vivo animal studies suggest that these agents are mutagenic and have the potential to select for mismatch repair deficient mutants, their in vivo mechanism(s) of carcinogenicity in humans remain(s) unclear. We investigated the in vivo mutagenicity of thiopurines in a cross-sectional study of 119 children and adults with inflammatory bowel disease by determining the frequency and spectra of somatic mutations at the HPRT locus using the T-cell cloning assay. We observed a significant increase in the frequency of somatic mutations that was related to total dose (p<0.001) and duration of treatment (p<0.001) among 56 thiopurine treated subjects compared to 63 untreated subjects. By mutation spectra comparison of 1020 mutant isolates, we also observed a significant dose-dependent increase in the proportion of G:C to A:T transitions (p<0.001) that is consistent with the in vitro mutagenic signature of these drugs. Although in vivo selection for HPRT mutants may have confounded the absolute mutation frequency since this is a ratio value, the significant thiopurine dose-dependent correlations with frequency and mutation spectra shift strongly suggest that thiopurine treatment is mutagenic in vivo. We also investigated the prevalence of in vivo clonal proliferation by the combined analyses of HPRT mutations and TCRβ CDR3 regions from mutant isolates and observed a higher prevalence of cell proliferation in association with chronic thiopurine therapy. Thiopurine treated subjects displayed expansions of precursor or committed progenitor cells and post-β rearrangement thymocytes or mature T-cells while untreated adult subjects showed only post-β rearrangement expansions of thymocytes or mature T-cells. Thiopurine treated subjects also uniquely displayed clonal expansions of “hyper-mutable” thymocytes or mature T-cells that gave rise to progenies with independent mutations either through extensive proliferation or from genetic instability. This pattern predominated in children and exhibited the highest occurrences of G:C to A:T mutations. G:C to A:T transitions were also observed in both early precursors or committed progenitors and post-β rearrangement thymocytes or mature T-cells from thiopurine treated subjects compared to untreated subjects who displayed a low frequency of G:C to A:T mutations unrelated to thiopurine therapy in post-β rearrangement thymocytes or mature T-cells. These data provide direct evidence that thiopurine treatment is mutagenic in vivo by mutation induction that may be enhanced through its effects on cell proliferation, and provide the first mechanistic evidence for the carcinogenicity of these therapeutic agents in humans. Our observations also suggest the potential risks to proliferating precursor and mature cells. In children, we may be capturing a subpopulation of “hypermutable” T-cells with a higher propensity to progress to lymphoma, which occur at highest frequency following transplantation.

Youth with inflammatory bowel disease (IBD) have higher rates of depression than healthy youth. A cognitive-behavioral treatment, primary and secondary control enhancement training-physical illness (PASCET-PI), for depressed adolescents with IBD was associated with reductions in depressive symptoms. The purpose of this study was to examine the salience of nonspecific processes (factors inherent in any human relationship) and specific processes (factors related to therapy content) during PASCET-PI sessions and their association with improvements in psychological functioning. Participants included 10 adolescent patients with IBD with mean illness duration of 31.9 months. At intake, eight participants qualified for a diagnosis of major depressive disorder and two for a diagnosis of minor depression. Participants completed measures of depressive symptomatology and clinicians completed the Children's Global Assessment Scale (CGAS). Measures were completed at posttreatment, 6 months posttreatment, and 1 year posttreatment. Independent judges used the Psychotherapy Process Q-sort (PQS) to rate the salience of therapeutic processes for PASCET-PI sessions #2 and #8 for each subject. PQS ratings of PASCET-PI sessions were con-elated with ideal prototypes of cognitive-behavioral treatment (CBT), inteqnrsonal therapy (IPT), and psychodynamic (PD) orientations that were previously developed based on PQS ratings of an ideal session, according to expert therapists. Findings indicate that PASCET-PI sessions most closely resembled the CBT prototype (r = .51, p < .05). Change scores on outcome measures were correlated with PQS-prototype correlates to determine which processes were associated with improved psychological functioning. Findings suggest that reductions in depressive symptomatology were associated with processes characteristic of various orientations. Thus, CBT processes were not exclusive in promoting change. There were strong positive relationships between change scores of the PCS and prototypes of all orientations (CBT, IPT, and PD) at posttreatment and between the CDI and ASQ and all orientations at 6-months follow-up (r = .62 -.72, p < .05). Comparisons of specific process-outcome correlates and nonspecific process-outcome correlates did not reveal significant differences.

ABSTRACT 1. Background. Paediatric neuroimmunology is a rapidly evolving field both as regards clinical-radiological phenotyping, biomarker development, and therapeutic possibilities. In this latter aspect, while a growing armamentarium of treating agents is becoming available, this is not mirrored by quality evidence and definite recommendations on treatment strategies, drugs’ efficacy and tolerability. This is especially true in paediatric age, where most data is derived from adult studies. Objective. To investigate clinical and therapeutic aspects of decision making in paediatric autoimmune and immune-mediated inflammatory conditions. In particular, the aims of this work include: exploring the available immune therapeutic agents and their mechanisms of action; investigating the use of immune therapy in autoimmune encephalitis; investigating the use, efficacy and tolerability of individual immune therapeutic agents in different clinical situations in paediatric neurology (intravenous immunoglobulin in Sydenham’s chorea; intravenous immunoglobulin in paediatric neurology; therapeutic plasma exchange in anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis; rituximab in neuromyelitis optica spectrum disorders; mycophenolate mofetil, azathioprine and methotrexate in anti-NMDAR encephalitis; mycophenolate mofetil in paediatric autoimmune and immune-mediated central nervous system (CNS) conditions). 2. Methods. The present PhD thesis is articulated into sub-projects carried out at the Department of Women's and Children's Health in Padua, Italy, and at the Children's Hospital at Westmead, Sydney, Australia. Study designs include six literature reviews (available immune therapeutic agents and their mechanisms of action; immune therapy in autoimmune encephalitis; immune therapy in herpes simplex virus-induced anti-NMDAR encephalitis; intravenous immunoglobulin in Sydenham’s chorea; therapeutic plasma exchange in paediatric anti-NMDAR encephalitis; mycophenolate mofetil, azathioprine and methotrexate in paediatric anti-NMDAR encephalitis) and four original studies with observational retrospective design (immune therapy in paediatric anti-NMDAR encephalitis; intravenous immunoglobulin in paediatric neurology; rituximab in neuromyelitis optica spectrum disorders; mycophenolate mofetil in paediatric autoimmune and immune-mediated CNS conditions). Of these latter original studies, one includes an Italian population (immune therapy in paediatric anti-NMDAR encephalitis), one a single-center Australian population (intravenous immunoglobulin in paediatric neurology), and two include an international cohort of paediatric patients (rituximab in neuromyelitis optica spectrum disorders; mycophenolate mofetil in paediatric autoimmune and immune-mediated CNS conditions). Most of the projects have been concluded, whereas two are in their final phases (mycophenolate mofetil, azathioprine and methotrexate in paediatric anti-NMDAR encephalitis; mycophenolate mofetil in paediatric autoimmune and immune-mediated CNS conditions). 3. Results. Key findings are presented for the main study objectives. 3.1 Immune therapeutic agents and their mechanisms of action. 3.1.1 Immune therapeutic agents and their mechanisms of action (literature review). First-line treatments typically include corticosteroids, intravenous immunoglobulin, and plasmapheresis, while for severe disease second-line ‘induction’ agents such as rituximab or cyclophosphamide are used. Steroid-sparing agents such as mycophenolate mofetil, azathioprine or methotrexate are often used in potentially relapsing or corticosteroid-dependent diseases. Lessons from adult neuroimmunology and rheumatology could be translated into pediatric autoimmune CNS disease in the future, including the potential utility of monoclonal antibodies targeting lymphocytes, adhesion molecules for lymphocytic migration, cytokines or their receptors, or complement. Finally, many agents used in other fields have multiple mechanisms of action, including immunomodulation, with potential utility in neuroimmunology, such as antibiotics, psychotropic drugs, probiotics, gut health, and ketogenic diet. 3.2 Immune therapy in autoimmune encephalitis. 3.2.1 Autoimmune encephalitis with antibodies targeting neuronal surface antigens (systematic literature review). Most studies on immune therapy in autoimmune encephalitis associated with antibodies to cell surface antigens are retrospective cohorts, and there are no randomised controlled trials. Most clinicians use first-line therapy (steroids, intravenous immunoglobulin, plasma exchange), and if severe or refractory, second-line therapy (rituximab, cyclophosphamide). When present, tumours should be removed. There are common therapeutic themes emerging. Firstly, patients given immune therapy do better and relapse less than patients given no treatment. Secondly, patients given early treatment do better. And thirdly, when patients fail first-line therapy, second-line therapy improves outcomes and reduces relapses. Given the retrospective uncontrolled data, the literature has inherent bias, including severity and reporting bias. 3.2.2 Clinical and therapeutic aspects of the Italian cohort of paediatric anti-NMDAR encephalitis (national retrospective observational study). We described a new case series of 20 children (50% females), with anti-NMDAR encephalitis referred by 13 Italian centers (mean age at onset 8 years, range 3-17). Onset was with neurological symptoms in 70%, and with behavioral/psychiatric disturbances in 30%. Most patients developed a severe clinical picture (90%), and 41% experienced medical complications; children 12-18 years old seemed to be more severe and symptomatic than younger patients. All children received first-line immune therapy; second-line treatment was administered to 45%. Relapses occurred in 15%. At last follow-up (mean 23.9 months, range 5-82), 85% patients had modified Rankin Scale (mRS) 0-1; this rate was higher among older patients, and in those receiving first immune therapy within 1 month. 3.2.3 Herpes simplex virus-induced anti-NMDAR encephalitis (systematic literature review). 43 patients with herpes simplex encephalitis (HSE) followed by anti-NMDAR encephalitis were identified in the literature (31 children). Latency between HSE and anti-NMDAR encephalitis was significantly shorter in children than adults (median 24 vs. 40.5 days; p=0.0057). Compared to the HSE phase, anti-NMDAR encephalitis was characterized by significantly higher frequency of movement disorder (2.5% in HSE, vs. 75% in anti-NMDAR encephalitis; p<.0001), and by significantly lower rate of seizures (70% in HSE, vs. 30% in anti-NMDAR encephalitis; p=0.0011). Compared to adults, during anti-NMDAR encephalitis children had significantly more movement disorder (86.7% in children, vs. 40% in adults; p=0.0064) and less psychiatric symptoms (41.9% in children, vs. 90% in adults; p=0.0251). Children also had a slightly higher median mRS than adults during the acute phase of anti-NMDAR encephalitis (5 vs. 4; p=0.0146). During anti-NMDAR encephalitis, 84.6% patients received acyclovir (for ≤7 days in 22.7%; long-term antivirals in 18% only), and 92.7% immune therapy, but none had recurrence of HSE clinically or using CSF HSV-PCR (median follow-up 7 months). 3.3 Modes of use, efficacy and tolerability of individual immune therapeutic agents in different clinical situations in paediatric neurology. 3.3.1 Intravenous immunoglobulin in Sydenham’s chorea (systematic literature review). The studies reviewed on intravenous immunoglobulin in Sydenham’s chorea demonstrate a short-term benefit in symptomatic improvement. However, they do not clarify an optimum timing and duration for use of intravenous immunoglobulin, and do not provide data on the effect on long-term neurological and psychiatric complications. 3.3.2 Intravenous immunoglobulin in paediatric neurology (single-center retrospective observational study). 196 children received intravenous immunoglobulin for neuroimmunological indications at the Children’s Hospital at Westmead, Australia, between 2000 and 2014 (28.1% had Guillain-Barré syndrome) (15.5% of all hospital indications). In total, 1669 intravenous immunoglobulin courses were administered (total 57221 g, median 78 g/patient, range 12-5748 g). Highest median number of courses was in chronic inflammatory demyelinating polyneuropathies, opsoclonus-myoclonus-ataxia, suspected immune-mediated epilepsies and Rasmussen’s encephalitis. Adverse reactions occurred in 25.5%, mostly minor. Outcome at follow-up was best in anti-NMDAR encephalitis, Guillain-Barré syndrome and myasthenia gravis, and worst in Rasmussen’s encephalitis and epilepsies. The total cost for intravenous immunoglobulin was 2,595,907 American dollars (median $3,538/patient, range $544-260,766). 45.4%-57.1% patients received intravenous immunoglobulin for ‘weak’ indications or ‘not listed’ in international guidelines. Some entities frequently treated with intravenous immunoglobulin in current practice, such as anti-NMDAR encephalitis and transverse myelitis, are not listed in most guidelines. 3.3.3 Therapeutic plasma exchange in anti-NMDAR encephalitis (systematic literature review). 71 articles were identified (mostly retrospective), reporting a total of 242 children treated with therapeutic plasma exchange for anti-NMDAR encephalitis (73.2%, 93/127 females; median age at onset 12 years, range 1-18). Median time to immunotherapy was 21 days (range 0-190). In most cases, therapeutic plasma exchange was given with steroids and intravenous immunoglobulin (69.5%, 89/128), or steroids only (18%, 23/128); in a minority, it was associated with intravenous immunoglobulin only (7%, 9/128), or was the only first-line treatment (5.5%, 7/128). In 54.5% (65/119), therapeutic plasma exchange was the third treatment after steroids and intravenous immunoglobulin, in 31.1% (37/119) the second after steroids or intravenous immunoglobulin; only in 14.3% (17/119) was it the first treatment. Second-line immunotherapies were administered in 71.9% (100/139). Higher rates of full/substantial recovery at follow-up were observed with immunotherapy given ≤30 days from onset (69.4%, 25/36) compared to later (59.2%, 16/27), and when therapeutic plasma exchange was associated with steroids (66.7%, 70/105) rather than not (46.7%, 7/15). Significant adverse reactions to therapeutic plasma exchange were reported in 6 patients. 3.3.4 Rituximab in paediatric neuromyelitis optica spectrum disorders (international retrospective observational study). 16 patients treated with at least two courses of rituximab for neuromyelitis optica were included (14 females; mean age 9.6 years, range 1.8-15.3). The patients had a mean of 6.1 events (range 1-11) during a mean follow-up of 6.1 years (range 1.6-13.6), and received a total of 76 rituximab courses (mean 4.7, range 2-9) in 42.6-year cohort treatment. Before rituximab, 62.5% received azathioprine, mycophenolate mofetil or cyclophosphamide. Mean time from rituximab to last documented B cell depletion and first repopulation was 4.5 and 6.8 months respectively, with large inter-patient variability. Earliest repopulations (2.7 and 2.9 months) occurred with the lowest rituximab doses. Significant reduction between pre and post rituximab annualized relapse rate (ARR) was observed (p=0.003). During rituximab, 6 patients were relapse-free, although 21 relapses occurred in 10 patients, including 13 ‘repopulation’, 3 ‘depletion’, and 4 ‘depletion failure-related relapses’. Of the 13 ‘repopulation relapses’, 4 had CD19 10-50x106cells/L, 10 inadequate monitoring (≤1 CD19 in the 4 months before relapses), and 5 delayed re-dosing ≥10 days after repopulation detection. 3.3.5 Mycophenolate mofetil, azathioprine and methotrexate in anti-NMDAR encephalitis (systematic literature review). 76 patients treated with mycophenolate mofetil/azathioprine/methotrexate for paediatric-onset anti-NMDAR encephalitis were included (age range at onset 0.8-18 years; 69.7% females; 49.1% had ≥1 relapse), reported in 37 articles. Mycophenolate mofetil was used in 53.9%, azathioprine in 25%, methotrexate in 15.8%; an additional 5.3% received two among mycophenolate mofetil/azathioprine/methotrexate. Mycophenolate mofetil/azathioprine/methotrexate were not preceded by any second-line therapy (rituximab/cyclophosphamide) in 47.7%, and were administered only after relapses in 46.8%. Among the subgroup treated with mycophenolate mofetil/azathioprine/methotrexate after the first event, relapses occurred in 8.3% only. Time on mycophenolate mofetil/azathioprine/methotrexate was median 9 months (range 1-48). Median annualised relapse rate was 0.45 (mean 1, range 0-6.67) before mycophenolate mofetil/azathioprine/methotrexate (excluding onset), and 0 (mean 0.06, range 0-1.3) during/after mycophenolate mofetil/azathioprine/methotrexate. Adverse reactions were reported only for mycophenolate mofetil (cytomegalovirus colitis and respiratory infection; grade 3 Common Terminology Criteria for Adverse Events v4.0). Relapse rate was significantly higher in patients started on first immune therapy (any) >30 days after onset (85.7%) compared to those treated early (31.2% (p=0.0272). 3.3.6 Mycophenolate mofetil in paediatric autoimmune and immune-mediated central nervous system conditions (international retrospective observational study). 44 children were included (30/44, 68.2% females). 43.2% (19/44) had proven or suspected autoimmune encephalitis, 31.8% (14/44) autoimmune inflammatory demyelinating CNS diseases, and 25% (11/44) other autoimmune/immune-mediated CNS conditions. Worst mRS was median 4 (range 2-6). Disease course was relapsing in 52.3% (23/44), monophasic in 38.6% (17/44), and chronic/chronic-progressive in 9.1% (4/44). Before mycophenolate mofetil, all patients received first-line (steroids: 44/44, 100%; intravenous immunoglobulin: 23/44, 52.3%; plasma exchange: 14/44, 31.8%) and 38.6% (17/44) second-line immune therapies (cyclophosphamide: 12/44, 27.3%; rituximab: 6/44, 13.6%). Median age at mycophenolate mofetil commencement was 9.3 years (range 1.4-16.4). Mycophenolate mofetil was started at median 9.5 months from onset (range 1-127; ≤6 months in 31.8%, 14/44). In 55% (22/40) of patients, mycophenolate mofetil was started only after ≥2 events had occurred. Median duration of treatment with mycophenolate mofetil was 18 months (mean 23.2, range 0.3-73). Median annualised relapse rate (excluding patients with chronic/chronic-progressive disease) was 0.52 (mean 0.86, range 0-3) before mycophenolate mofetil (excluding first events), and 0 (mean 0.36, range 0-4.64) during mycophenolate mofetil. 20.5% (8/39) patients relapsed during mycophenolate mofetil; compared to patients who did not relapse (31/49, 79.5%), these patients were younger (median age at onset 4.2 years versus 7.6), were more frequently females (8/8, 100% versus 21/31, 67.7%), had lower rate of second-line treatments before mycophenolate mofetil (1/8, 12.5% versus 15/31, 48.4%), a later commencement of mycophenolate mofetil (>6 months after onset in 7/8, 87.5% versus 22/35, 58.1%), and more frequently they were started on mycophenolate mofetil only after ≥2 events had occurred (7/8, 87.5% versus 14/35, 45.2%). Adverse reactions to mycophenolate mofetil occurred in 18.2% (8/44) of cases (6/8: grade 2, 2/8: grade 3 Common Terminology Criteria for Adverse Events v4.0). 4. Conclusion. The present thesis is a collection of ten works exploring several aspects of the clinical and therapeutic decision-making in paediatric autoimmune and immune-mediated inflammatory conditions. A growing array of immune therapies are becoming available in paediatric neurology, also derived from the experience in immune modulation from other fields of paediatrics and in adult patients. While quality data and definite recommendations are generally lacking, there are common themes emerging, such as the utility of early and aggressive immune therapy in certain clinical situations, such as in autoimmune encephalitis. The use of immune therapy is still characterised by a great heterogeneity between physicians in many neurological conditions, for examples as regards therapeutic plasma exchange and steroid sparers in anti-NMDAR encephalitis, reflecting not only the lack of definite recommendations, but also different treating habits and potential practical difficulties, such as with therapeutic plasma exchange in children and uncooperative patients. Even when recommendations do exist, such as for the use of intravenous immunoglobulin in neurology, current practice is not always adherent to the guidelines, suggesting both the need for greater adherence to existing recommendations and the need for recommendations to be updated to accommodate emerging indications. In other cases, finally, the utility and safety of treatments such as steroid sparing agents warrants further investigations in several fields of paediatric neurology, such as in anti-NMDAR encephalitis. In all cases, both currently accepted and future potential agents have adverse effects, which can be severe. A comprehensive understanding of the therapeutic aspects should not go without the ability to understand each clinical situation in all its facets, taking into considerations not only potential effects, adverse reactions and mechanisms of treatment agents, but also the pathophysiology, the severity of the acute disease, the risk of relapses and of permanent disability, in a complex ‘risk-versus-benefit’ determination and a tailored approach.

The project concerns the development of a drug delivery system for the treatment of inflammatory bowel disease. This condition, which affects more than 2 million people in Europe alone, is characterised by inflammatory lesions which may be confined to the large bowel (ulcerative colitis) or extend through the intestinal tract (Crohn's disease). The components of disease activation lend themselves to treatment with immunomodulators such as the folate analogue methotrexate; although systemic sideeffects are significant. :. To o overcome this limitation, a local delivery system was investigated. In this thesis, folate has been used as a safer surrogate in the development of a silicone ribbon-based oral dosage form.

Pharmaceutical Sciences
Ph.D.
The 5-HT7¬ receptor is the most recently discovered 5-HT receptor subtype. 5-HT7 is a GPCR that exhibits a regulatory role in many biological functions in both the central nervous system (CNS) and the periphery. Recent literature has demonstrated a connection between the 5-HT7 receptor and Inflammatory Bowel Disease (IBD) progression. IBD is a devastating disease that affects 1.4 million Americans. Patients suffer from life altering symptoms as a result of severe, chronic inflammation of the gastrointestinal tract. Current treatments mitigate symptoms with no effect on disease progression. Targeting the 5-HT7 receptor as a novel treatment option is a viable medicinal chemistry project that could result in a therapy capable of providing relief to IBD patients. A novel series of butyrolactones were discovered during a prior thesis project completed by Dr. Rong Gao at Temple University’s School of Pharmacy. Broad screening indicated that many of the compounds within this series were potent binders of the 5-HT7 receptor. These results led to the initiation of a medicinal chemistry program aimed at the development of this series with the intent to identify novel 5-HT7 receptor antagonists that are suitable for pre-clinical and clinical evaluation for the treatment of IBD. Medicinal chemistry strategies were utilized in order to optimize each structural aspect of the butyrolactone pharmacophore. This required the preparation of several small series of compounds wherein one structural feature was systematically changed while the remaining features were held constant. The particular properties that were studied for optimization included 5-HT7 affinity, subtype selectivity, liver microsomes stability (mouse and human), and the topological polar surface area (to minimize CNS penetration). Implementing these strategies led to the identification of potent 5-HT7¬ antagonists, some of which exhibited excellent subtype selectivity and improved mouse liver microsome stability. Two analogs, 170073 and 230168, were chosen for further study. Both analogs exhibited adequate in vivo pharmacokinetic profiles capable of supporting efficacy in an in vivo setting. 170073 distributed rapidly and extensively into brain tissue, while 230168 moderately distributed into brain tissue. Moving forward, reducing CNS penetration will become a top priority. These two compounds were examined in the DSS induced mouse model of IBD and both exhibited efficacy. Specifically in the acute DSS model of colitis, 170073 and 230168 significantly lowered the disease activity index, mitigated histological damage and reduced the production of proinflammatory cytokines. In addition, 170073 demonstrated efficacy in the chronic DSS model of colitis. 230168 has yet to be tested in the chronic model. The results of this dissertation support the validity of this project and the use of 5-HT7¬ ¬¬antagonists as a potential novel treatment option for IBD.
Temple University--Theses

Pelvic Inflammatory Disease (PID) is one of the most common causes of morbidity in women. PID is a polymicrobial infection of the female reproductive tract, and is associated with pelvic pain, abnormal uterine bleeding, and tubal damage that can lead to ectopic pregnancies and infertility. It is curable but the effects of PID can be permanent if not properly diagnosed and treated. PID presents as a spectrum of disease and is often missed at early stages; even acute PID can be difficult to diagnose, as there is no single conclusive diagnostic test. Currently, PID is identified and treated syndromically because pelvic pain is the only consistent clinical finding. The Center for Disease Control and Prevention (CDC) recommends doxycycline, a broad-spectrum antibiotic, for treatment but doxycycline can cause gastrointestinal irritation and local inflammation leading to an incomplete treatment. Most cases of PID are polymicrobial infections of the tubes and endometrium, which are not accessible to culture due to the difficulty of procuring samples above the naturally contaminated vagina and distal cervix. Given the difficulty of properly diagnosing PID and the limitations and side effects of the current treatments, there is an urgent need for new approaches for improving the accuracy for diagnosis and treatment of PID. We propose a new and practical approach to collect sterile specimen samples from the endometrium for more accurate PID diagnosis, and to treat the reproductive tract locally using doxycycline-loaded nanoparticles. The proposed research presents a novel sterile uterine sampler cover (SUSC) device that can safely and effectively collect uncontaminated specimen samples from the uterus, and also deliver nano-encapsulated drugs directly to the site of infection. The analysis of uncontaminated endometrium samples is expected to provide an understanding of uterine flora in symptomatic and asymptomatic women, and will lead to the identification of infective microbes in symptomatic women for pathogen-specific treatment. The use of nano-encapsulated doxycycline will enable localized drug delivery to lower drug dosage and minimize side effects for the patient. The doxycycline-loaded nanoparticles are characterized and evaluated based on their drug release properties, size distribution, and tissue response in vitro. This research will lead towards a more effective approach for the diagnosis and treatment of PID while freeing women from prolonged systemic treatments and their adverse effects. Moreover, this research will increase our understanding of the uterine biome under various hormonal and pathologic conditions, in symptomatic and asymptomatic women.

At least 1.4 million of Americans suffer from Inflammatory Bowel Diseases (IBD). IBD (Crohn’s disease and ulcerative colitis) is a spontaneously relapsing, immunologically mediated disorder of the gastrointestinal tract. Complete medical cure remains a challenge and the probability of relapse is over 70%. Curcumin has been shown to have a protective role in mouse models of inflammatory bowel diseases (IBD) and to reduce the relapse rate in human ulcerative colitis (UC), thus making it a potentially viable supportive treatment option. The objective of this research project was to provide a preclinical evaluation of curcumin’s efficacy in relevant models of human IBD, and to investigate the molecular mechanisms of its protective mechanism of action. (1) We investigated the effect of dietary curcumin in trinitrobenzene sulfonic acid (TNBS)-induced colitis in SJL/J mice (Th-1/Th-17 response) and in BALB/c mice (Th-1/Th-2 response). We demonstrated that the efficacy of dietary curcumin varies in the two strains. Although the exact mechanism underlying these differences remains unclear, our observations suggest that the therapeutic value of dietary curcumin may vary depending on the nature of immune dysregulation. (2) We further confirmed those findings and we investigated the effects of curcumin on the development of colitis, immune activation, and in vivo NF-κB activity in germ-free IL-10^(–/–) colonized with specific pathogen-free microflora. In this model resembling CD, we demonstrated that IL-10 and curcumin act synergistically to downregulate inflammation. (3) Neutrophil aberrant accumulation at the intestinal mucosa is a characteristic hallmark of inflammatory conditions such as ulcerative colitis. Neutrophil transepithelial migration leads to an impaired epithelial barrier function, perpetuation of inflammation and tissue destruction. Therefore, we investigated the effect of curcumin on neutrophil polarization and motility. Our results indicated that curcumin interferes with colonic inflammation partly through chemokine expression inhibition and neutrophil chemotaxis and chemokinesis inhibition. We also demonstrated that curcumin significantly reduced epithelial tissue injury generated by neutrophil transepithelial migration and protease release. Those findings significantly add to our understanding of the mechanism by which curcumin affects the innate and adaptive immune response in IBD and may help develop innovative therapeutic strategy for IBD.

Dissertation presented to obtain a Ph.D. degree (Doutoramento) in Chemistry at the Instituto de Tecnologia Quimica e Biol6gica da Universidade Nova de Lisboa
Carbon Monoxide, CO, has been recognized as an endogenously produced, potent biological mediator involved in many defense mechanisms both in physiologic and pathologic situations. As a result of these signaling processes, CO possesses a strong therapeutic potential on a wide range of disease indications. However, the hardly avoidable safety and practical problems associated with therapeutic inhalation of toxic CO gas, led to the search for molecules capable of delivering CO to tissues in a living organism in a controlled and therapeutically useful manner. From all the areas of the chemical space where such CO-Releasing Molecules (CO-RMs) can be found, Metal Carbonyls Complexes (MCCs) seems to be the most versatile. It is the purpose of this Thesis to provide an extensive characterization of the behavior of MCCs in the presence of biological molecules and media, in order to identify the chemical and structural parameters that are more relevant to define the profile of a therapeutically effective metal-based CO-RM drug.(...)
This Thesis was financially supported by Fundacao para a Ciencia e Tecnologia, European Social Fund grant number SFRH/BDE/15501/2004 and ALFAMAResearch and Development of Pharmaceutical Drugs Ltd

Inflammatory bowel disease (IBD) is a group of diseases in the gastrointestinal field that is becoming more commonly diagnosed among patients. IBD is usually characterized as a group of chronic diseases affecting the digestive tract that are caused by a multitude of factors including genetic, environmental, mucosal, and immune contributors. One of the subgroups of IBD is very early onset IBD (VEO-IBD), which is diagnosed in children under the age of 6. VEO-IBD is a rare yet unique case of IBD, which reports poor response to conventional adult-onset IBD treatments. Nutrition is an alternative treatment that can decrease inflammation and allow IBD patients to achieve remission. This proposed study explores whether formula-based diets, which have been strongly correlated with reduced IBD inflammation and symptoms, will impact VEO-IBD patients. A mouse model will be set up with one control group of healthy mice and two variable groups of VEO-IBD characteristic mice, with 60 mice in each group. The mice will be fed three formula-based dietary regiments including camel’s milk, Pediasure, and liquid vitamin D3 twice daily for 90 days. All three of these dietary treatments have been proven to decrease inflammation in adult-onset IBD patients. The inflammation and severity of symptoms will be monitored every two days through Western blotting protein levels of IL10 (a genetic marker for VEO-IBD) and physiological tests. If nutrition has a positive effect on the VEO-IBD induced mice, then a decrease in inflammation and VEO-IBD symptoms should be observed. This study is vital to future treatment plans by determining the influence of formula-based diets in alleviating symptoms of VEO-IBD patients.

Ischemic stroke is a leading cause of death and long-term disability in the world. Although many pathological aspects of mechanisms are considered to be involved in the stroke, accumulating evidences implicated that inflammation accounts for its progression and complications. Tumor necrosis factor-alpha (TNF-α) and nitric oxide are considered as key mediators produced by cells like microglia in the pathogenesis of the disease. Therefore, the development of therapies targeting at the suppression of nitric oxide and TNF-α productions may ameliorate the severity of ischemic stroke. Gastrodia and Uncaria Decoction (GUD) is a traditional herbal decoction that is commonly used in the therapy of post-ischemic stroke in China. Although it shows great efficacy in clinical treatment, few studies have been conducted to investigate the mechanisms of action of GUD. Furthermore, GUD contains a complex mixture of constituents and the effects of these compounds are unknown. In this study, individual herbs from GUD were extracted and the bioactive fractions were further separated using liquid-liquid partition, silica gel chromatography and high performance liquid chromatography (HPLC). The inhibitory effect of the extracts on lipopolysaccharide (LPS)-stimulated nitric oxide production in BV-2 microglial cells was utilized as the biological marker for the screening. After several rounds of purification, a purified bioactive compound was isolated. After spectroscopic analysis by nuclear magnetic resonance and gas chromatography-mass spectrometry, the compound was identified as genipin (1R,4aS,5,7aS-tetrahydro-1-hydroxy-7-(hydroxymethyl)-cyclopenta[c]pyran-4-carboxylic acid, methyl ester). Mechanisms of the suppressive action on signaling pathways were investigated in the LPS-activated BV-2 cells. Our results demonstrated that genipin can dose-dependently inhibit LPS-stimulated nitric oxide overproduction. It can also suppress mRNA levels and protein expressions of inducible nitric oxide synthase (iNOS) and TNF-αupon LPS-induction. In addition, the phosphorylations of phosphoinositide-3 kinase (PI3K) and protein kinase B (Akt) were suppressed. In contrast, the phosphorylations of mitogen-activated protein kinases (MAPKs), nuclear translocation of nuclear factor-κB (NF-κB) p65 and degradation of inhibitory κB-α (IκB-α) were not affected by genipin. Finally, genipin protected murine Neuro-2a neuroblast against neurotoxicity stimulated by the conditioned media transferred from LPS-challenged BV-2 cells. In conclusion, the anti-inflammatory effects of genipin are via the modulation of PI3K/Akt signaling pathway. Genipin and its synthetic analogues may have great potential for developing into new drugs in treating ischemic stroke. In addition, genipin can be used as the chemical marker to standardize the extract of Eucommia ulmoides Oliver as anti-inflammatory agents for treating inflammatory conditions associated with ischemic stroke.
published_or_final_version
Paediatrics and Adolescent Medicine
Master
Master of Philosophy

The thiopurines, mercaptopurine (MP) and its pro-drug azathioprine (AZA), are the first line immunomodulators used in the management of inflammatory bowel disease (IBD). Unfortunately, 30 - 40% of patients are unable to derive benefit from these medicines as a result of drug toxicity or treatment non-response. The main active metabolites of these drugs are the phosphorylated thioguanine nucleotides (TGNs) and methylated derivatives of mercaptopurine (MeMP). Recent experience suggests that measurement of these metabolites can be used to explain inter-individual variation in response to treatment and identify opportunities to optimize therapy. In particular, some patients with IBD display sub-optimal TGN levels and unexpectedly high MeMP concentrations, a phenotype which is known as thiopurine hypermethylation. Importantly this skewed drug metabolism can be circumvented using a low dose of AZA/MP in combination with allopurinol. This thesis provides an original contribution to knowledge by firstly exploring and characterizing thiopurine hypermethylation in patients with IBD to determine its impact on clinical response. Secondly, it investigates the mechanism of thiopurine hypermethylation and other thiopurine-induced drug toxicities using both candidate gene and genome-wide approaches, with the goal of identifying biomarkers that can be used to predict outcomes prior to the start of treatment. Finally, it resolves the biochemical interaction between thiopurines and allopurinol, which has remained unexplained for over half a century.

The prevalence of obesity in both the developed and developing world have increased, which leads to diverse health outcomes and is placing a heavy burden on the economy. Abdominal obesity proved to be one of the main features in predicting metabolic and cardiovascular disease (CVD) risk and may be the link that unifies the metabolic syndrome (MS) through pro-inflammatory pathways. While the pathogenesis of the MS and each of its components are complex and not well understood, abdominal obesity remains the mechanism that relates to increased lipolysis causing the liver to increase blood glucose and very low lipoprotein output. This in turns leads to raised blood glucose, triglycerides, low-density lipoprotein cholesterol (LDL-C), blood pressure and inflammatory markers (C-reactive protein, interleukin-6 and tumor necrosis factor-a) and decreased high-density lipoprotein cholesterol (HDL-C). Prevention of the metabolic syndrome and treatment of its main characteristics are now considered of utmost importance in order to combat the increased CVD risk and all-cause mortality. Decreasing sedentary behaviour through regular physical activity is a key element in successful treatment of obesity through an increase in energy expenditure, but the ability to decrease low-grade systemic inflammation may be an even greater outcome. Aims The aims of this study was firstly, to determine by means of a literature review, how obesity could be related to a state of chronic systemic inflammation (increased CRP and IL-6). Secondly to determine whether physical activity could serve as a suitable method to decrease inflammation associated with obesity and related disorders. Thirdly to determine if abdominal obesity is a predictor of the metabolic syndrome and CVD and finally, to determine if measures of obesity can predict risk for the metabolic syndrome and CVD risk. Methods For this review study, a computer-assisted literature search were utilized to identify research published between 1990 and 2005. the following databases were utilized for the search: NEXUS, Science Direct, PubMed and Medline. Keywords related to obesity (abdominal obesity, overweight), metabolic syndrome (insulin resistance syndrome, dysmetabolic syndrome, syndrome X), cardiovascular disease (coronary heart disease, coronary artery disease), cardiovascular risk factors (hypertension, dyslipidemia, diabetes mellitus, physical activity), inflammatory markers (CRP, IL-6, chronic low-grade inflammation) and physical activity (fitness, exercise and training) were included as part of the search, including the references identified by previous reviewers (not identified as part of the computerized literature search). Results and conclusions Several research studies concluded that obesity could be an inflammatory disorder due to low-grade systemic inflammation. Adipose tissue is known to be a sectretory organ producing cytokines, acute phase reactants and other circulating factors. The synthesis of adipose tissue TNF-a could induce the production of IL-6, CRP and other acute phase reactants. CRP is a acute phase reactant, synthesized primarily in hepatocytes and secreted by the liver in response to a variety of inflammatory cytokines of which IL-6 and TNF-a are mainly involved. CRP increases rapidly in response to trauma, inflammation and infection. Thus, enhanced levels of CRP can be used as a marker of inflammation. Several studies of large population cohorts provide evidence for an inverse, independent dose-response relation between plasma CRP concentration and level of physical activity in both men and women. Trends for decreased IL-6, TNF-a and CRP concentrations were linear with increasing amounts of reported exercise in most of the research studies, physical activity proved effective in lowering measures of adiposity (BMI, WHR, WC and percentage body fat) and obesity related inflammatory markers (CRP & IL-6). Thereby indicating a potential anti-inflammatory effect. In the studies reviewed in this article abdominal obesity is identified as a predictor and independent risk factor for CVD in both men and women. High levels of deep abdominal fat have also been correlated with components of the metabolic syndrome, glucose intolerance, hyperinsulinemia, hypertension, diabetes, increases in plasma triglyceride levels and a decrease in HDL-C levels (dyslipidemia) in many of the studies. Prospective epidemiological studies have revealed that abdominal obesity (determined by WC and WHR) conveys an independent prediction of CVD risk and is more relevant compared to general obesity (determined by BMI). Abdominal fat has been linked to metabolic risk factors like high systolic blood pressure, atherogenic dyslipidemia, with increased serum TG and decreased HDL-C, and glucose intolerance. Although magnetic resonance imaging (MRI) and computerized tomography (CT) have been used successfully in many studies to measure adipose compartments of the abdomen (subcutaneous and visceral fat), anthropometrical measures like WHR and WC have been proven to be an effective measure in predicting the metabolic syndrome. WC has also been included in the metabolic syndrome definitions of the WHO, ATP Ill and new IDF.
The prevalence of obesity in both the developed and developing world have increased, which leads to diverse health outcomes and is placing a heavy burden on the economy. Abdominal obesity proved to be one of the main features in predicting metabolic and cardiovascular disease (CVD) risk and may be the link that unifies the metabolic syndrome (MS) through pro-inflammatory pathways. While the pathogenesis of the MS and each of its components are complex and not well understood, abdominal obesity remains the mechanism that relates to increased lipolysis causing the liver to increase blood glucose and very low lipoprotein output. This in turns leads to raised blood glucose, triglycerides, low-density lipoprotein cholesterol (LDL-C), blood pressure and inflammatory markers (C-reactive protein, interleukin-6 and tumor necrosis factor-a) and decreased high-density lipoprotein cholesterol (HDL-C). Prevention of the metabolic syndrome and treatment of its main characteristics are now considered of utmost importance in order to combat the increased CVD risk and all-cause mortality. Decreasing sedentary behaviour through regular physical activity is a key element in successful treatment of obesity through an increase in energy expenditure, but the ability to decrease low-grade systemic inflammation may be an even greater outcome. Aims The aims of this study was firstly, to determine by means of a literature review, how obesity could be related to a state of chronic systemic inflammation (increased CRP and IL-6). Secondly to determine whether physical activity could serve as a suitable method to decrease inflammation associated with obesity and related disorders. Thirdly to determine if abdominal obesity is a predictor of the metabolic syndrome and CVD and finally, to determine if measures of obesity can predict risk for the metabolic syndrome and CVD risk. Methods For this review study, a computer-assisted literature search were utilized to identify research published between 1990 and 2005. the following databases were utilized for the search: NEXUS, Science Direct, PubMed and Medline. Keywords related to obesity (abdominal obesity, overweight), metabolic syndrome (insulin resistance syndrome, dysmetabolic syndrome, syndrome X), cardiovascular disease (coronary heart disease, coronary artery disease), cardiovascular risk factors (hypertension, dyslipidemia, diabetes mellitus, physical activity), inflammatory markers (CRP, IL-6, chronic low-grade inflammation) and physical activity (fitness, exercise and training) were included as part of the search, including the references identified by previous reviewers (not identified as part of the computerized literature search). Results and conclusions Several research studies concluded that obesity could be an inflammatory disorder due to low-grade systemic inflammation. Adipose tissue is known to be a sectretory organ producing cytokines, acute phase reactants and other circulating factors. The synthesis of adipose tissue TNF-a could induce the production of IL-6, CRP and other acute phase reactants. CRP is a acute phase reactant, synthesized primarily in hepatocytes and secreted by the liver in response to a variety of inflammatory cytokines of which IL-6 and TNF-a are mainly involved. CRP increases rapidly in response to trauma, inflammation and infection. Thus, enhanced levels of CRP can be used as a marker of inflammation. Several studies of large population cohorts provide evidence for an inverse, independent dose-response relation between plasma CRP concentration and level of physical activity in both men and women. Trends for decreased IL-6, TNF-a and CRP concentrations were linear with increasing amounts of reported exercise in most of the research studies, physical activity proved effective in lowering measures of adiposity (BMI, WHR, WC and percentage body fat) and obesity related inflammatory markers (CRP & IL-6). Thereby indicating a potential anti-inflammatory effect. In the studies reviewed in this article abdominal obesity is identified as a predictor and independent risk factor for CVD in both men and women. High levels of deep abdominal fat have also been correlated with components of the metabolic syndrome, glucose intolerance, hyperinsulinemia, hypertension, diabetes, increases in plasma triglyceride levels and a decrease in HDL-C levels (dyslipidemia) in many of the studies. Prospective epidemiological studies have revealed that abdominal obesity (determined by WC and WHR) conveys an independent prediction of CVD risk and is more relevant compared to general obesity (determined by BMI). Abdominal fat has been linked to metabolic risk factors like high systolic blood pressure, atherogenic dyslipidemia, with increased serum TG and decreased HDL-C, and glucose intolerance. Although magnetic resonance imaging (MRI) and computerized tomography (CT) have been used successfully in many studies to measure adipose compartments of the abdomen (subcutaneous and visceral fat), anthropometrical measures like WHR and WC have been proven to be an effective measure in predicting the metabolic syndrome. WC has also been included in the metabolic syndrome definitions of the WHO, ATP Ill and new IDF.
Thesis (M.A. (Human Movement Science))--North-West University, Potchefstroom Campus, 2006.

The cardiovascular diseases (CVDs), resulting from complications of atherosclerosis, remain the leading cause of morbidity and death worldwide. Atherosclerosis as a chronic inflammatory disease, involves both innate and adaptive arms of immunity in which macrophages play the orchestral role in modulating lesion initiation, progression, and potentially devastating thrombotic complications. Available evidences support the notion of a central role of oxidative stress, due mainly to the imbalance between antioxidants and reactive oxygen species (ROS) in CVDs. Furthermore, the pathology is frequently associated with dynamic changes in macrophage activation, with classically activated M1 cells implicated in initiating and sustaining inflammation and M2 or M2-like cells associated with resolution or smoldering chronic inflammation. Among endogenous antioxidants, the thiordoxine-1 (Trx1) plays a central role in several diseases including CVD. Thus, the ubiquitous Trx1 has been reported to exert a myriad of beneficial roles. Indeed, it regulates not only cellular redox homeostasis and acts as a principal antioxidant defense system, but it also affects energy metabolism, modulates the immunological and inflammatory responses, and controls cell growth and survival. In contrast, its truncated form (Trx-80), exerts an opposite effects. However, several studies reported the beneficial role of Trx system in CVDs but the detailed molecular mechanism is not addressed yet. Therefore, the present study aims to investigate the role of both Trx1 and Trx80 in the biology of atherosclerosis through the modulation of macrophage polarization and the implicated signaling pathways as well. Our in vitro major findings, using human macrophages and murine peritoneal macrophages, revealed that Trx1 on one hand promoted the polarization of anti-inflammatory M2 macrophages through downregulation of p16INK4a and suppressing nuclear translocation of activator protein-1 (AP-1) and Ref-1 as evidenced by the expression of the CD206 and IL-10 markers. On the other hand Trx1 also reduced the lipopolysaccharide (LPS)-induced differentiation of inflammatory M1 macrophages, as indicated by the decreased expression of the M1 cytokines, tumor necrosis factor-α (TNF-) and monocyte chemoattractant protein-1 (MCP¨-1). By contrast, Trx80 treatment attenuated the polarization of anti-inflammatory M2 macrophages induced by IL-4 or IL-4/IL-13 even it potentiated LPS-induced M1 activation. To validate our obtained in vitro results, hyperlipoproteinemic C57Bl/6.ApoE2.ki mice and human atherosclerotic vessel specimens from patients undergoing vascular surgery were used. Consistently, Trx1 and Trx80 affected macrophage phenotype in thymus, liver and atherosclerotic lesions. As a consequence, Trx1 reduced whereas Trx80 increased the aortic lesion area in mice. Plasma levels of cholesterol and triglycerides did not changed by the treatment. To further explore our results, the implicated signaling pathways has been studied and it was found that both Trx1 and Trx80 activated Akt. Furthermore, Trx80 uses mTOR signaling pathway to exert its effect in polarizing macrophages toward M1 phenotype since it activated mTOR in a dose-dependent manner as demonstrated by the increased phosphorylation of P70S6K. Based on our results, Trx1 antagonizes whereas Trx80 potentiates atherosclerosis through changing M1/M2 phenotypes. Therefore, Trx1 represents a promising target for therapeutic interventions.

A Doença de Crohn (CD) e a Colite Ulcerativa (UC) são as principais enfermidades componentes das Doenças Inflamatórias Intestinais (DII). Embora existam vários medicamentos atualmente empregados para atenuar a inflamação descontrolada no intestino, tratar as complicações ou prolongar os períodos de remissão clínica, não há, ainda, uma terapia que seja totalmente efetiva para estas doenças. Os glicocorticoides (GCs), anti-inflamatórios comumente usados nas DII, possuem eficácia limitada e mais da metade dos pacientes se tornam refratários ou dependentes da medicação. Por outro lado, as estatinas são conhecidas por possuírem propriedades pleiotrópicas e seu uso concomitante com os GCs tem gerado boas perspectivas em várias doenças autoimunes e inflamatórias, inclusive nas DII. Apesar de já existirem indicativos de melhora de pacientes com DII pela utilização combinada destas drogas, ainda há escassez de dados que mostrem as alterações causadas no sistema imunológico. Assim, o objetivo desse trabalho foi avaliar os efeitos imunomoduladores do uso concomitante de GCs e estatinas na colite experimental induzida por dextran sulfato de sódio (DSS) em camundongos C57BL/6. Os resultados mostraram que o uso contínuo de GCs (dexametasona - DX), associados ou não a estatinas (atorvastatina - ATO), não alterou o curso da doença e antecipou a morte dos camundongos, enquanto que o oposto foi observado com o uso isolado de ATO. Tratamentos em curto prazo (3 doses) contendo ATO (isolada ou associada à DX) causaram melhora clínica e histológica dos animais doentes, diminuíram o número de leucócitos circulantes (principalmente monócitos) e de células mononucleares na lâmina própria (LP), a frequência de células CD11b+ na LP, a frequência de células dendríticas (DCs) CD11b+CD11c+ e CD11b-CD11c+ no baço e a frequência de células CD4+ produtoras de IFN- nos linfonodos mesentéricos (LNM). Entretanto, ambos os esquemas terapêuticos aumentaram a frequência de linfócitos T CD8+ no baço e LNM. Ainda, as terapias inibiram a proliferação de esplenócitos tratados in vitro, diminuíram a síntese de IL-6 e, quando em baixas concentrações, aumentaram a produção de IL-10. Diferencialmente, o tratamento combinado pareceu exercer os efeitos acima descritos de modo mais pronunciado do que o uso isolado de estatina. Adicionalmente, diminuiu os níveis de expressão de RNAm das citocinas IL-1, IL-17 e IFN- no local da inflamação, reduziu o número de linfócitos circulantes, de leucócitos no baço e LNM e de linfócitos T CD4+ nos LNM, além de ter aumentado a frequência de DCs CD11b-CD11c+ na LP e a concentração de Fas-L no intestino grosso. Considerando o uso em curto prazo com ATO isolada, foi observado aumento da frequência de DCs CD11b-CD11c+ nos LNM e de células Natural killer (NK) no baço dos camundongos doentes e diminuição dos níveis de expressão de RNAm de PPAR- no intestino grosso. O uso isolado de DX em curto prazo melhorou os aspectos histológicos, diminuiu o número de macrófagos e os níveis de IFN- no cólon, diminuiu o número de leucócitos circulantes (principalmente linfócitos), aumentou a frequência de células CD11b+ no baço e a síntese de IL-10 por esplenócitos ex vivo. Apesar da frequência de células T reguladoras (Treg) e da susceptibilidade dos esplenócitos à sinais reguladores não terem sido modificados após os diferentes tratamentos, nossos resultados sugerem que as estatinas usadas isoladamente preservaram a resposta inflamatória do organismo de modo eficiente e controlado, enquanto que o uso associado das drogas causou a imunossupressão dos animais doentes, contribuindo para as complicações clínicas decorrentes da colite experimental induzida por DSS.
Crohn\'s disease (CD) and Ulcerative colitis (UC) are the main conditions that comprise the Inflammatory Bowel Diseases (IBD). The conventional drug therapies for IBD aim to attenuate the uncontrolled inflammation in the intestinal mucosa, to treat the complications and to extend clinical remission. However, all available drugs have unpredictable or limited effects. Glucocorticoids (GCs) are commonly anti-inflammatory drugs, which are associated to refractoriness and/or dependence in over half of IBD patients. On the other hand, statins have pleiotropic properties and the concomitant use with GCs has shown good prospects in several autoimmune and inflammatory diseases, including IBD. Despite the putative clinical improvement after combined use of GCs and statins in IBD, there is a lack of data indicating their additive effects on the immune system. Therefore, the purpose of this study was to evaluate the immune modulatory effects of the concomitant use of statins and GCs in experimental colitis induced by dextran sulfate sodium (DSS). The results showed that long-term use of GCs (dexamethasone - DX), alone or associated to statins (atorvastatin - ATO), did not improve the clinical signs and increased the death rates of C57BL/6 mice exposed to DSS, while the opposite was observed after treatment with statins alone. Short-term use of ATO (3 doses), alone or associated to DX, improved the clinical signs and histological parameters in DSS-exposed mice, decreased the number of white blood cells (mainly monocytes), the number of mononuclear cells in the lamina propria (LP), the frequency of CD11b+ cells in the LP, the frequency of CD11b+CD11c+ and CD11b-CD11c+ dendritic cells (DCs) in the spleen and the frequency of IFN--producing CD4+ T cells in the mesenteric lymph nodes (MLN). However, ATO alone or associated to DX lead to increased CD8+ T lymphocytes in the spleen and MLN. Moreover, both therapies containing ATO inhibited the proliferation of in vitro-treated splenocytes, besides decreasing IL-6 and increasing IL-10 synthesis. Differentially, the association of drugs led to a more pronounced effects over the changes mentioned above than the single use of statin and additionally decreased IL-1, IL-17 and IFN- mRNA expression levels at the intestinal tissue, the number of circulating lymphocytes, the number of leukocytes in spleen and MLN and the frequency of CD4+ T lymphocytes in the MLN. In addition, statins and GCs increased the frequency of CD11b-CD11c+ DCs in LP and the Fas-L concentrations in the large intestine. Considering the short-term use of ATO there was increased frequency of CD11b-CD11c+ DCs in MLN, increased frequency of natural killer (NK) cells in the spleen and decreased mRNA expression of PPAR- in the large intestine. The short-term use of DX improved the histology parameters, decreased the number of macrophages and IFN- levels in the colon, reduced the number of circulating leukocytes (mainly lymphocytes), and increased the frequency of CD11b+ cells in spleen and IL-10 synthesis by ex vivo splenocytes. Finally, since both regulatory T cells (Treg) frequency and the splenocytes susceptibility to regulatory signals have not been modified after the different treatments, our findings suggest that single use of statins preserved an efficient and controlled inflammatory response, while the combined use of GCs and statins led to immunosuppression, which probably contributed to long-term clinical complications of DSS-induced colitis.

Cystic fibrosis (CF) is the most common life-restricting, genetically inherited disease among Caucasians affecting approximately 30,000 people in the United States. Lung disease is the major cause of morbidity and mortality in CF. A number of oral, inhaled, and intravenous therapies are available to combat CF lung disease. Of these, this research project focused on inhaled dornase alfa, oral azithromycin, inhaled tobramycin, and inhaled aztreonam. Data to address three research aims were requested and obtained from the Cystic Fibrosis Foundation Patient Registry (CFFPR). The first aim examined the use of inhaled dornase alfa in younger children with CF. With no clinical efficacy data of dornase alfa in children ≤ 6 years of age, the study utilized subsequent forced expiratory volume in 1 second (FEV₁) measured between 6 - 7 years of age, to assess the effectiveness of long-term dornase alfa use ≤ 6 years of age. Propensity score methods were used to reduce the likelihood of treatment indication bias. The results suggested that receiving treatment with dornase alfa before 6 years of age did not improve FEV₁ between 6 - 7 years. Unmeasured covariates leading to treatment indication bias were likely one of the key explanations for these results. Additionally, lack of a more sensitive outcome than FEV₁ to assess lung function in young patients with early lung damage was thought to be another reason for the failure to reject the null hypothesis. The second aim assessed the long-term clinical effectiveness of chronic azithromycin use on the rate of FEV₁ decline in CF patients between 6 - 20 years of age. This study was novel in that the rate of FEV₁ decline, rather than change in FEV₁ from baseline, was the primary outcome, which was characterized using propensity score matching followed by a linear mixed model analysis. The results of the analysis suggested that the rate of FEV₁ decline was slower in patients who did not receive chronic treatment with azithromycin. Treatment indication bias was thought to play an important role in the direction of the association between treatment and outcome. Associations between FEV₁ % predicted and many of the other study variables included in the analysis were consistent with previous studies. The final aim compared the clinical effectiveness of a combination of inhaled tobramycin and aztreonam with inhaled tobramycin alone on the rate of FEV₁ decline in CF patients between 6 - 20 years of age. This aim was novel in that the effect of this combination treatment on rate of decline in FEV₁ has never been assessed. A linear mixed model analysis was used after matching patients in the two treatment groups on their propensity scores. Once again, the results were contrary to the alternative hypothesis with the combination group having a steeper rate of FEV₁ decline than the group that was treated with tobramycin alone. An important reason for this result was thought to be unresolved treatment indication bias that could not be eliminated even with the use of the propensity score methods used to test the associated hypothesis. The use of validated methods of analysis, i.e., propensity scores, to counter treatment indication bias using the largest available observational dataset for CF, was one of the key strengths of this study. Moreover, this study highlighted important weaknesses in the CFFPR with regards to lack of data on patient and physician-level variables - an area of active interest for the Cystic Fibrosis Foundation.

Thesis (Ph. D.)--Georgia State University, 2007.
Title from title page. Lindsey L. Cohen, committee chair; Lisa Armistead, Erin B. McClure, Mary K. Morris, committee members. Electronic text (113 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed Oct. 11, 2007. Includes bibliographical references (p. 73-80).

Inflammatory bowel disease (IBD; Crohn’s disease & ulcerative colitis) is a chronic illness in which medication and dietary adherence may determine disease natural history and severity of symptoms. We hypothesized that age, prospective memory (PM) and body satisfaction would predict medication and dietary adherence in adolescents with IBD and that gender and age would modify the relation between body satisfaction and adherence, with older girls being less adherent than younger children. Fifty-seven participants aged 10-21 (M = 16.5, SD = 2.3) with IBD and their caregivers were recruited. Informed consent, demographics and body satisfaction questionnaires were completed. PM was assessed using a naturalistic task. Adherence was measured by the 1-week completion of a medication and dietary log. A questionnaire was administered to evaluate coping strategies used for overcoming obstacles to dietary adherence. Two hierarchical regressions were conducted for medication and diet adherence respectively. As hypothesized, age had a significant effect (â = -.42, p < .01) on dietary adherence, accounting for approximately 17% of the variance (R2change = .17; Fchange (1,41) = 8.57, p = .006), with younger children being more adherent. Body satisfaction had a greater and more significant effect on dietary adherence than age (â = -.33, p < .01); i.e. participants more satisfied with their body reported better dietary adherence (R2change = .28; Fchange (2,35) = 6.97, p < .05). Findings remained consistent across multiple measures of body satisfaction and dietary adherence. None of the predictors had a significant effect on medication adherence. Health care providers who treat adolescents with IBD and parents should be made aware of factors affecting adherence in order to improve disease outcomes and patients’ quality of life.

Background: Sexually transmitted diseases (STD's) have a major impact on sexual and reproductive health worldwide. Each year, the World Health Organization (WHO) estimates 448 million new cases of curable STD's are diagnosed. The emergence of drug resistance in STD related microorganisms and potential side effects demand the discovery of newer drugs. The exploration of newer anti-microbial substances from natural sources may serve as promising alternatives. In this study, twelve medicinal plant species used traditionally in the treatment of STD's are investigated in this regard. Methods: Ethanol plant extracts and three flavonoids were evaluated for their antimicrobial properties against one fungi and three bacteria, through the micro-dilution assay. To determine the anti-inflammatory activities of the extracts and compounds, the inhibitory effect was measured on the pro-inflammatory enzyme lipoxygenase, 15-LOX. Extracts were further evaluated for their inhibitory effect on the supercoiling activity of bacterial DNA gyrase by using the DNA gyrase kit. The extracts and compounds were lastly investigated for their anti-HIV activities against recombinant HIV-1 enzyme using non-radioactive HIV-RT colorimetric assay. Results: Acacia karroo and Rhoicissus tridentata extracts showed good antimicrobial activity with MIC values ranging between 0.4 and 3.1 mg/ml. Extracts of Jasminum fluminense, Solanum tomentosum and flavonoid 2 and 3 had good anti-inflammatory activity with IC50 less than the positive control quercetin (IC50 = 48.86 ug/ml). Extracts of Diospyros mespiliformis, Peltophorum africanum, Rhoicissus tridentata and flavonoids 1 and 2 showed the best inhibitory activity against the bacterial DNA gyrase. A. karroo and flavonoid 3 exhibited moderate HIV RT inhibition activity of 66.8 and 63.7 % respectively. R. tridentata and Terminalia sericea had the best RT inhibition activity (75.7 and 100 %) compared to the positive control doxorubicin (96.5%) at 100 ug/ml concentration. Conclusion: The observed activities may lead to new multi-target drugs against sexually transmitted diseases.
Dissertation (MSc)--University of Pretoria, 2017.
Plant Science
MSc
Unrestricted

Objective: Vedolizumab (VDZ) is a monoclonal antibody directed against α4β7 integrin heterodimer, approved for patients with inflammatory bowel disease (IBD). This study aimed at identifying circulating and mucosal immunologic predictors of response in patients with active ulcerative colitis (UC) and Crohn’s disease (CD). Design: This is an explorative, prospective, phase IV interventional trial (Eudract n. 2015- 003270-32). Consecutive CD and UC patients received open-label VDZ at weeks 0, 2, 6 and 14. A week 10 infusion was performed in CD patients. Patients with clinical response at week 14 were maintained with VDZ every 8 weeks up to 54 weeks. At week 0 and 14 peripheral blood was obtained and endoscopy with biopsies was performed. The expression of surface markers, chemokine receptors and α4β7 heterodimer on peripheral blood and lamina propria lymphocytes was assessed by flow cytometry. Results: 38 IBD patients (20 UC, 18 CD) were included in the study. At week 14, clinical response and remission rates were 87% and 66%, respectively. Endoscopic response rate was 47%. Among week 14 responders, clinical remission rate at week 54 was 69%. No clinical variables were found to predict either clinical or endoscopic outcomes. On the contrary, higher baseline levels of circulating memory CXCR3+CCR6- CD4+ T cells (Th1 cells) were strongly associated with week 14 clinical response (P=0.0001). Reduced baseline levels of lamina propria memory CXCR3-CCR6+ CD4+ T cells (Th17 cells) and CXCR3+CCR6+ CD4+ T cells (Th1/17 cells) were predictive of endoscopic response (P=0.012 and P=0.005 respectively). Circulating levels of Th1 memory T cells predicted clinical remission in IBD patients at week 54. Conclusions: The results of this exploratory study uncovered a panel of circulating and mucosal immunological predictors of response to vedolizumab treatment. These data provide further insights on the mechanism of action of vedolizumab in IBD patients.

Alzheimer's disease (AD) is a neurological disorder characterized by plaque deposition and an elevated immune response. Epidemiological studies have shown that use of non-steroidal anti-inflammatory drugs (NSAIDs) by the elderly is associated with a decreased relative risk and a delayed onset of AD. Moreover, the apolipoprotein E (apoE) gene has been proven to be a risk factor for AD with apoE &egr;4 AD patients having been found to show lower levels of brain apoE. In the present study, treatment of primary rat mixed glial cell cultures with indomethacin, aspirin, and interleukin-1beta resulted in significant increases in extracellular apoE protein. Furthermore, treatment of primary rat astrocyte cell cultures with aspirin, interleukin-6, and a cyclooxygenase-2 selective aspirin derivative was found to result in increased levels of apoE. Consequently, NSAID-induced increases in apoE protein may enhance apoE-mediated immunosuppression and compensatory synaptic plasticity, potentially resulting in decreased risk and delay of disease onset.

Introduction: Tissue-engineered oral mucosa (TEOM) is increasingly being used to assess drug delivery and toxicity, as well as for modeling oral diseases. Current TEOM models are constructed using primary oral fibroblasts and keratinocytes that display donor-to-donor variability and whose widespread use is restricted by availability and ethic limitations. To address these issues, an attractive approach is the development of TEOM using immortalised cells. Aim: This study aimed to construct and characterise TEOM based on TERT2-immortalised oral keratinocytes (FNB6) cells and use these TEOM to assess the toxicity and delivery of corticosteroids using a novel electrospun-based oral patch. Methods: TEOM were constructed by culturing immortalised FNB6 oral keratinocytes on top of a normal oral fibroblast (NOF)-populated collagen type 1 hydrogel in tissue culture transwell inserts at an air-to-liquid interface (ALI) for up to 14 days. The TEOM were characterised using histological, immunohistological, ultrastructural (TEM), tissue viability (AlamarBlue), trans-electrical resistance (TEER), and permeability (FITC-dextran) analysis. Cytotoxicity assessment of seven corticosteroids was performed using MTT assay on monolayer cultures (FNB6 and NOF cells) and TEOM. Novel mucoadhesive bilayer patches containing clobetasol 17-propionate (CP) were subjected to morphological, physicochemical, drug release, swelling and cytotoxicity analysis. In vitro permeation studies of the corticosteroids against TEOM was measured using HPLC. The immunosuppressive effect of delivered CP against activated Jurkat T-cells was assessed by measuring changes in interleukin-2 (IL-2) release. Results: Histologically, TEOM mimicked native oral mucosa displaying a stratified epithelium, fibroblast-containing connective tissue and basement membrane. IHC revealed the expression markers for differentiation (cytokeratin 4,13,14), proliferation (Ki-67), cell adhesion (E-cadherin, claudin-4). Furthermore, TEM confirmed the presence of desmosomes and hemidesmosomes in the epithelium. Maximal TEOM viability was found up to day 25 and maximal TEER value was exhibited at day 20 (155.8 Ω.cm2). Permeability analysis showed that only small molecules (3 kDa) could pass through the epithelium. Differential drug sensitivity of corticosteroids against monolayer cultures was ranked as follow; CP > BU > BD > BV > TA > HV > HB by IC50 value, and this was similar for TEOM although IC50 values were higher for 3D models. Novel mucoadhesive bilayer patches containing CP exhibited good physicochemical characteristics and drug release profiles. Toxicity testing to the OECD standard revealed that patch delivered CP was considered a non-irritant. Oral mucosal delivered CP using liquid or patch formulation into the TEOM tissue or receptive medium was both dose and time-dependent. In addition, both liquid and patch delivered CP significantly reduced the secretion of IL-2 by activated Jurkat T cells in a TEOM model replicating an oral inflammatory disease. Conclusion: FNB6 TEOM models are able to mimic the native oral mucosa and have the potential to be used for drug delivery and toxicity evaluation. Oral patch-delivered CP was able to cross the TEOM and inhibit the IL-2 secretion of activated T cells, suggesting that this mode of drug delivery could be used to treat oral inflammatory diseases.

2010 - 2011
The aim of the present PhD project was to design inhalable powder-based formulations for pharmaceutical products that may improve the treatment of pulmonary diseases, mainly cystic fibrosis, and may be easier for patients to use. Particularly, the present project aims to supply CF patients with flavonoids (Naringin) and aminoglycosides (Gentamicin sulfate) in a respirable form as a valid alternative over more conventional (oral or parenteral) anti-inflammatory and antibiotic therapy. As a matter of fact, in CF epithelial cells, antioxidant defense systems appear to be defective in their ability to control the amount of ROS produced and over abundance of ROS may cause tissue injury-events and modify intracellular signalling pathways leading to enhanced inflammatory processes, typical of CF airways. Overall, evidence suggests improved CFTR function in vitro when flavonoids, such as genistein, are used. For chronic Pseudomonas aeruginosa (Pa) infections in CF, gentamicin given by pulmonary route may plays important role. In fact, it was observed daily inhalation of some aminoglycosides from nebulized solution delays the acquisition of chronic Pa infections and decreases CF progression. The project address a number of the key features that are outstanding in inhaled delivery, mainly - characteristic of the active drug; - properties of the drug formulation, particularly powder flow, particle size, shape, surface properties and drug/carrier interaction; - consistent dose delivery and high proportion of dose getting to the lung; - performance of the inhaler device, including aerosol generation and delivery. A balance among these characteristics is necessary in the design of a drug formulation intended for pulmonary administration. Utilizing proven (Spray-drying) or innovative (Supercritical Assisted Atomization) technology, stable and micronized powders usefull for dry powder inhaler (DPI) production have been developed. Moreover, the research has been based on in vitro product test methods to evaluate the health effects of produced powders and their aerodynamic behaviour through the pulmonary system. Optimized stability and bioavailability of the selected drugs, the achieving of therapeutically effective concentrations for the pulmonary care of cystic fibrosis have been other goals of the research. Technologies and products that the research is aimed to develop would be of interest to a number of pharmaceutical companies either in the respiratory area or trying to get a toehold in this market. Specific objectives of this research have been: design and development of Dry Powder Inhalers (DPIs) containing flavonoids (Naringin) or aminoglycoside antibiotics (Gentamicin sulfate) micronized powder by spray drying production or by Supercritical Assisted Atomization (SAA); optimization of the aerodynamic characteristics of the powders, through the use of excipients (amino acids) not toxic for lung but able to improve the powder flow properties and dispersion which, in turn, may increase lung deposition of the drugs; in vitro evaluation of the biological activity of the engineered particles on a model of bronchial epithelial cell lines from patients with cystic fibrosis (CuFi1, F508del/F508del CFTR), in comparison to the activity of the same products on normal bronchial epithelial cell lines (NuLi1). (edited by author)
X n.s.

The primary aim of this research is to test the therapeutic potential of certain new generation corticosteroid drugs in order to develop safe and effective treatment for eye diseases that result in oedema, or swelling. The rising incidence of diabetes and the ageing population of developed countries mean that the prevalence of uveitis, diabetic retinopathy and age related macular degeneration will rise. Often, oedema is one of the reasons for vision loss. Corticosteroids are often used to reduce inflammation. Inflammation is one of several sources of oedema. Glucocorticoids, a class of corticosteroids that have anti-inflammatory properties, are thus used to treat ocular oedema. There is an unmet need to support clinical experience of the efficacy of steroids for ocular inflammation and oedema with more substantial scientific evidence. None of the drugs under investigation, with the exceptions of dexamethasone and triamcinolone, have been used for any ocular therapeutic purpose before. This thesis investigates “repurposing” fludrocortisone to the ophthalmic area. 11-Desoxycorticosterone (11D) and Deoxycorticosterone (DCS), other potentially valuable mineralocorticoids, remain completely untested. Lastly, Kenacort ®, or triamcinolone acetonide (TCA), is only used off-label by ophthalmologists. Methods: In the first study, corticosteroids, and especially mineralocorticoids, were investigated for their treatment efficacy in experimental uveitis, or intraocular inflammation (using a model known as endotoxin induced uveitis). In the second study, endothelial cells from choroidal blood vessels in the back of the eye were used in vitro to study whether corticosteroids reduce paracellular (between cells) permeability. Lastly, since endophthalmitis due to frequent injections is a side effect of corticosteroid use, the pharmacokinetics of different size formulations of corticosteroids were studied in an effort to find a formula that would have a prolonged dwell time within the eye.