Anti-microbial proteins (AMP) are the key effector arm of the innate immune system. The prevalence of AMP in single-celled eukaryotes to humans shows its importance during the course of evolution. The first report for the role of the anti-microbial peptide in clearing infection was given by Alexander Fleming in 1990’s through the discovery of Penicillin and Lysozyme. The search for antimicrobial agents in human granulocytes was begun by Ehrlich in 1870’s but the first successful isolation of an antimicrobial agent from rabbit neutrophils was done by Zeya and Spitznagel in 1969. Later work by Peter Elshbach and his group on AMPs in rabbit neutrophils brought to light an AMP that can increase the permeability of the bacterial membrane. This AMP named as Bactericidal/permeability-increasing protein (BPI) was further isolated from human neutrophils. Since then many studies have been carried out to understand the mode of action of BPI, which culminated in understanding the new functional activity of this protein viz opsonisation, LPS neutralization and anti-angiogenic function. Knowing to the role of BPI as an anti-inflammatory agent, multiple studies have tried to use BPI for treating endotoxic shock. Dysregulation of BPI expression is associated with various inflammatory diseases like Crohn’s Disease (CD), Ulcerative colitis (UC) and Infectious enteritis’s. Mutations in BPI are also linked to susceptibility to various infections. Even though there are several studies focusing on the functional aspects of BPI, the regulation of BPI expression is poorly understood. Knowing the clinical importance of dysregulation of BPI, it is vital to understand the regulation of BPI expression during the course of bacterial infection. The Thesis is divided into four chapters. As the main aim of this study is to understand the regulation of BPI expression, in Chapter 1 we introduce the known facts about the protein. A brief overview of the mode of action and regulation of BPI is discussed in this chapter. The subsequent sections describe the diseases associated with Dysregulation of BPI and the use of BPI as a therapeutic agent in various diseases. Towards the end, the objective of the present study is discussed. BPI is primarily known to be expressed in human neutrophils and epithelial cells. Previous studies have shown that among innate immune cells, murine BPI is expressed only in dendritic cells and neutrophils, but not in macrophages. Based on these results, it was presumed that BPI is not expressed in human macrophages. In Chapter 2, we report the presence of BPI in human macrophages. Our studies revealed increased expression of BPI in human macrophages stimulated with various PAMPs (Pathogen-associated molecular patterns) viz., LPS, flagellin as well as during bacterial infection. Further, during the course of an infection, BPI interacted with Gram-negative bacteria, resulting in enhanced phagocytosis and subsequent control of the bacterial replication. However, it was observed that bacteria which can maintain an active replicating niche (Salmonella Typhimurium) avoid the interaction with BPI during later stages of infection. On the other hand Salmonella mutants, which cannot maintain a replicating niche, as well as Shigella flexneri, which quit the endosomal vesicle, showed interaction with BPI. BPI was induced in both M1 and M2 differentiated macrophages suggesting its role in limiting Gram-negative bacteria and parasitic infection. These results propose an active role of BPI in Gram-negative bacterial clearance by human macrophages. This chapter concludes with a discussion on the importance of BPI expression in human but not murine macrophages. The importance of maintaining an active replicating niche by STM to evade interaction with BPI is also discussed. As the first line of defense against invading pathogens, intestinal epithelium produces various antimicrobial proteins (AMP) that help with clearance of pathogen. The precise mechanism of AMP regulation in intestinal epithelium is not clear. Intestinal epithelium being a primary entry point for various pathogens, we tried to understand the regulation of BPI expression in the intestine during the course of bacterial infection. In Chapter 3, we report a direct correlation between intestinal damage and BPI expression. In Caco-2 cells, we see a significant increase in BPI levels upon membrane damage mediated by S.aureus infection and pore-forming toxins (Streptolysin and Listeriolysin). Cells detect changes in potassium levels as a Danger-associated molecular pattern (DAMP) associated with cell damage and induce BPI expression in a p38 dependent manner. These results are further supported by in vivo findings that BPI expression in the murine intestinal epithelium is induced upon infection with bacteria which cause intestinal damage (Salmonella Typhimurium & Shigella flexneri) whereas mutants which don’t cause intestinal damage (STM fliC & STM invC), didn’t induce BPI expression. These findings have a huge impact on our current understanding of AMP response during inflammatory bowel diseases (IBD). Our results suggest that dysregulation of BPI expression might be an effect rather than a cause of IBD. This chapter concludes with a discussion on the importance of potassium efflux associated with membrane damage as an important signal that helps in discriminating the invading pathogen from the pool of gut microflora. Bactericidal/permeability-increasing protein had been shown to possess anti-inflammatory and endotoxin neutralizing activity by interacting with LPS of Gram-negative bacteria. Even though rBPI (recombinant BPI) has cleared phase III clinical trials for treating endotoxemia, the high cost of purified BPI provided by pharmaceutical companies makes it inaccessible or unavailable for the common man. In Chapter 4, we examined the feasibility of using murine BPI (mBPI) expressed on halophilic Archaeal gas vesicle nanoparticles (GVNPs) for the treatment of endotoxemia in high-risk patients, using a murine model of D-galactosamine-induced endotoxic shock. Halobacterium sp. NRC-1 was used to express the N-terminal 199 amino acid residues of mBPI fused to the GVNP GvpC protein, and bound to the surface of the haloarchaeal GVNPs. Our results indicate that delivery of mBPIN-GVNPs increase the survival rate of mice challenged with lethal concentrations of lipopolysaccharide (LPS) and D-galactosamine. Additionally, the mBPIN-GVNP-treated mice displayed reduced symptoms of inflammation including inflammatory anemia, recruitment of neutrophils, liver apoptosis and pro-inflammatory serum cytokine levels. This chapter concludes with a discussion of the advantages of using mBPIN-GVNPs over purified protein in treating endotoxic shock.

Les objectifs de cette thèse étaient de produire des connaissances nouvelles sur les méthodologies d’évaluation de l’optimalité des soins avec l’exemple des délais diagnostiques et des infections bactériennes sévères de l’enfant (IBS). Nous avons mis en évidence, dans deux revues systématiques de la littérature, que les points méthodologiques potentiellement associés à des risques de biais et d’obstacles à la transportabilité des résultats étaient rarement rapportés dans les études primaires sur les délais diagnostiques ou rarement évalués par les auteurs de méta-analyses. Nous avons donc construit et validé internationalement une reporting guideline pour aider les scientifiques à prendre en compte ces points méthodologiques critiques. Nous avons montré par une enquête confidentielle avec comité d’experts en population que : (i) les prises en charge étaient suboptimales pour 76% des enfants décédés d’IBS, (ii) un retard au recours médical, une sous-évaluation de la gravité ou un retard à l’antibiothérapie étaient retrouvés dans la prise en charge de respectivement 20%, 20% et 24% des enfants atteints d’IBS, (iii) les soins suboptimaux étaient indépendamment et fortement associés au risque de décès et (iv) les soins suboptimaux étaient plus fréquents chez les enfants de moins d’un an ou lorsque qu’ils n’étaient pas administrés par un médecin spécifiquement formé. La minimisation des biais dans la sélection des participants et la mesure de l’optimalité et la prise en compte de facteurs de confusion comme la sévérité intrinsèque de la maladie sont des éléments clefs de l’évaluation de l’optimalité des soins afin de produire des messages cliniques correctifs valides. / The aim of this thesis was to product new knowledge about the methodology on how to assess the optimality of care with the examples of time to diagnosis and serious bacterial infection (SBI). In two systematic reviews, we found that the key methodological points potentially related to risks of bias or threats to transportability were rarely reported in the primary studies and rarely evaluated by authors of systematic reviews. Then, we developed and internationally validated a reporting guideline to help scientists to better take into consideration these critical methodological points. In a population-based confidential inquiry, we found that: (i) care was suboptimal in 76% of the initial management of children who died from SBI, (ii) delayed first medical contact, undervaluation of severity or delayed antibiotic administration were detected in the management of 20%, 20% and 24% of children admitted to intensive care for a SBI, respectively, (iii) the total number of suboptimal cares delivered during the management was independently associated with death, and (iv) suboptimal cares were more frequent in children younger than one year old and if the care was delivered by a non specialist physician. Minimizing the risks of bias both in the selection process of the study population and in the assessment of the optimality of care, and taking into account confounding factors such as the intrinsic severity of the disease are keys elements to ensure a reliable evaluation of optimality of care in order to produce effective corrective clinical messages.