Human milk contains ~ 25 µg/mL of soluble cluster of differentiation 14 (sCD14) protein, a pattern recognition receptor (PRR) that triggers the innate immune system to respond to bacterial lipopolysaccharide (LPS). To date, the role of CD14 in the digestive tract of breast fed infants has not been well characterized and is the subject of this thesis. To investigate the biodistribution of proteins such as CD14 in vivo, a novel method for 14C radiolabeling of proteins to high specific radioactivity was developed using in vacuo methylation. Bovine serum albumin (BSA) and casein were used as test proteins to determine the following: 1) The efficacy of the in vacuo radiolabeling procedure; 2) The extent of incorporation of the 14C-label into the organs of oro-gastric gavaged 10 day old Sprague Dawley rats. [14C]BSA, [14C]casein and [14C]CD14 were prepared with specific radioactivities of 10 400, 10 800 and 163 000 dpm/µg, respectively. After feeding 6.25 µg of 14C-labeled proteins, quantifiable levels of 14C were found in the stomach, jejunum, duodenum, ileum, large intestine, intestinal luminal flushes, blood, liver, spleen and kidneys of rats. The accumulation of radiolabel in the organs of [14C]CD14 fed rats was temporally and spatially distinct from [14C]BSA and [14C]casein. Most notably, the label persisted in the stomach 480 min post-gavage. To design a neonate animal model for biodistribution, the segmental and total gastrointestinal transit times (GItt) were measured in two litters of 10 and 15 day old Sprague Dawley rat pups using barium sulfate. Ten day old rat pups that remained with and without the dam had a total gastrointestinal transit time of 13.8 ± 0.9 hr and 9.3 ± 0.7 hr, respectively. This decrease (p<0.05) in total gastrointestinal transit time in the absence of the dam was age dependent, as it was not observed (p>0.05) in the 15 day old rat pup litter. The immunological impact of an exogenous sCD14 source was examined in human peripheral blood mononuclear cells (PBMC). Pre-treatment of CD14+ monocytes with sCD14 had a protective effect, one of reducing the production of proinflammatory cytokines (TNF-α, IL-6, IL-8, IL-1β) when challenged with LPS. 14C was absorbed by neonate rats upon ingestion of [14C]CD14 and exposure to relatively high concentrations of rCD14 led to a reduction in inflammation. This may be beneficial to initial gut colonization in breast-fed newborns. / Alexander Graham Bell NSERC CGS M scholarship. Japan Society for the Promotion of Sciences, Summer in Japan Fellowship. Funded by the Canadian Institutes of Health Research, Institute of Nutrition Metabolism and Diabetes Grant #82816 “Fate and function of breast milk and recombinant human CD14 at mammary and newborn gastrointestinal mucosal epithelia”.

Reproduction de : Thèse de doctorat : Médecine.Biologie cellulaire : Reims : 2007. / Bibliographie f.115-134.

Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) are the most prevalent pathogens to induce mastitis. The pathogenesis of infections induced by E. coli is sophisticatedly modulated by lipopolysaccharide (LPS), LPS binding protein, membrane CD14 (mCD14), and soluble CD14 (sCD14). In the first study, administration of recombinant bovine sCD14 (rbosCDl4) significantly reduced the fatality of LPS challenged mice and the severity of mouse mastitis in terms of clinical signs, bacterial load, and TNF-alpha production. Before investigating the potential of this strategy in dairy cows, endogenous sCD14 in milk was characterized. Based on the data of 396 quarters, the milk concentration of sCD14 was 6.67 +/- 0.44 mug/ml. The stages of lactation affected the concentration of sCD14 in milk, which was higher in transitional milk (0--4 days postpartum). Milk sCD14 also increased during an intramammary LPS challenge, which paralleled with SCC increase. The protective effect of sCD14 on bovine E. coli mastitis was then investigated. It was shown that rbosCDl4 sensitized the mammary gland to recruit leukocytes in response to LPS. To prove that the early recruitment of leukocytes plays a role in preventing intramammary E. coli infections, E. coli mastitis was induced in 9 dairy cows with or without 100 mug rbosCD14. Quarters challenged with E. coli plus rbosCD14 had a more rapid recruitment of neutrophils, a faster clearance of bacteria, reduced concentrations of TNF-alpha and IL-8 in milk, and reduced clinical symptoms than quarters injected with saline. / For S. aureus mastitis, a newly designed trivalent whole-cell vaccine being composed of the most dominant serotypes (T5, T8, and T336) was evaluated. The vaccine was immunized with or without either one of the two adjuvants, aluminum hydroxide (ALUM) and Freund's incomplete adjuvant (FICA). The vaccine, with or without the presence of adjuvants, increased antigen-specific IgG1, IgG2, but not IgM, in serum. However, all formulations only had limited effects on lymphocyte subsets, interferon (IFN)-gamma mRNA expression, and neutrophil phagocytosis in comparison with the control. / Taken together, the results indicated that increasing the concentration of sCD14 in milk might be a potential strategy to prevent or reduce severity of E. coli mastitis. On the other hand, both ALUM and FICA did not augment the immune responses when formulated with trivalent vaccine. A more immunostimulatory adjuvant will be required to improve the efficacy of the novel trivalent vaccine against S. aureus mastitis.

Human milk contains ~ 25 µg/mL of soluble cluster of differentiation 14 (sCD14) protein, a pattern recognition receptor (PRR) that triggers the innate immune system to respond to bacterial lipopolysaccharide (LPS). To date, the role of CD14 in the digestive tract of breast fed infants has not been well characterized and is the subject of this thesis. To investigate the biodistribution of proteins such as CD14 in vivo, a novel method for 14C radiolabeling of proteins to high specific radioactivity was developed using in vacuo methylation. Bovine serum albumin (BSA) and casein were used as test proteins to determine the following: 1) The efficacy of the in vacuo radiolabeling procedure; 2) The extent of incorporation of the 14C-label into the organs of oro-gastric gavaged 10 day old Sprague Dawley rats. [14C]BSA, [14C]casein and [14C]CD14 were prepared with specific radioactivities of 10 400, 10 800 and 163 000 dpm/µg, respectively. After feeding 6.25 µg of 14C-labeled proteins, quantifiable levels of 14C were found in the stomach, jejunum, duodenum, ileum, large intestine, intestinal luminal flushes, blood, liver, spleen and kidneys of rats. The accumulation of radiolabel in the organs of [14C]CD14 fed rats was temporally and spatially distinct from [14C]BSA and [14C]casein. Most notably, the label persisted in the stomach 480 min post-gavage. To design a neonate animal model for biodistribution, the segmental and total gastrointestinal transit times (GItt) were measured in two litters of 10 and 15 day old Sprague Dawley rat pups using barium sulfate. Ten day old rat pups that remained with and without the dam had a total gastrointestinal transit time of 13.8 ± 0.9 hr and 9.3 ± 0.7 hr, respectively. This decrease (p<0.05) in total gastrointestinal transit time in the absence of the dam was age dependent, as it was not observed (p>0.05) in the 15 day old rat pup litter. The immunological impact of an exogenous sCD14 source was examined in human peripheral blood mononuclear cells (PBMC). Pre-treatment of CD14+ monocytes with sCD14 had a protective effect, one of reducing the production of proinflammatory cytokines (TNF-α, IL-6, IL-8, IL-1β) when challenged with LPS. 14C was absorbed by neonate rats upon ingestion of [14C]CD14 and exposure to relatively high concentrations of rCD14 led to a reduction in inflammation. This may be beneficial to initial gut colonization in breast-fed newborns. / Alexander Graham Bell NSERC CGS M scholarship. Japan Society for the Promotion of Sciences, Summer in Japan Fellowship. Funded by the Canadian Institutes of Health Research, Institute of Nutrition Metabolism and Diabetes Grant #82816 “Fate and function of breast milk and recombinant human CD14 at mammary and newborn gastrointestinal mucosal epithelia”.

Endotoxin recognition by the innate immune receptor CD14 is a critical part of the innate immune system’s early detection and activation of the inflammatory response during microbial invasion. The differential recognition and high affinity binding of endotoxins from gram-negative and gram-positive bacteria is performed by the innate immune receptor CD14. Upon endotoxin binding, CD14 transfers the specific endotoxins to a Toll-like receptor signaling complex, which is responsible for initiating the intracellular signaling cascade. In the presence of overwhelming infection, the effects of CD14 lead to the over-activation of the inflammatory response, which results in the life threatening condition known as sepsis. Preparation of a 15N isotopically labeled truncated version of soluble CD14, using Pichia pastoris, allowed direct structural observation of the binding interaction between CD14 and two endotoxin ligands, lipopolysaccharide (LPS) and lipoteichoic acid (LTA), from gram-negative and gram-positive bacteria, respectively using solution NMR spectroscopy. These studies revealed that CD14 uses both a common set of residues, and endotoxin specific subsets of residues, to bind LPS and LTA. To further investigate the structural features of each endotoxin recognized by CD14, 13C 15N isotopically labeled Kdo2–Lipid A, a fully active chemically defined gram-negative endotoxin, and LTA lipid anchor, the minimal unit of LTA, were produced. This allowed detailed NMR spectral mapping of these agonist ligands bound to sCD14 which identified, for the first time, structural regions and features in each that are strongly affected during complex formation with sCD14. Additionally, the presence of differential dynamic behavior was seen in both CD14 and the ligands upon complexation. This behavior suggests a likely role for dynamics in the mechanism of pattern recognition by CD14, which uses the dynamic ability of specific residue combinations to differentially affect endotoxin binding. Using NMR, the dynamic behavior of CD14 was further investigated using temperature and pH-dependence studies of isotopically labeled CD14. These studies clearly demonstrated the presence of multiple conformations for several residues, and may provide a possible explanation for the broad specificity of ligand binding by CD14. In addition, the spin-labeling of isotopically labeled lipid A enabled the collection of intermolecular distances on CD14 bound lipid A.

[Truncated abstract] Chronic liver injury results from many etiologies ranging from viral infection to inborn errors of metabolism. A common result of liver injury is activation of hepatic stellate cells and portal fibroblasts to myofibroblasts. In chronic injury, production of extracellular matrix by activated myofibroblasts results in liver fibrosis and ultimately cirrhosis. Kupffer cells and monocytes may play an important role in the pathogenesis of certain liver diseases. Endotoxin-responsive macrophages and recruited monocytes (CD14-positive cells) are potential sources of profibrogenic factors but their potential role in the pathogenesis of liver disease has not previously been examined. The first aim of this thesis described in chapter 3 was to evaluate the hypothesis that CD14-positive macrophages/monocytes are present in the livers of patients with hereditary haemochromatosis (HH), primary biliary cirrhosis (PBC), chronic hepatitis C (HCV) and nonalcoholic steatohepatitis (NASH) and contribute to the pathogenesis of fibrosis as evidenced by co-localization of these cells with activated myofibroblasts. Liver specimens from control subjects and those with HH, PBC, HCV and NASH were immunostained for CD14, CD68 and α-smooth muscle actin and the number of cells expressing these antigens was determined. The total number of hepatic CD68-positive cells was similar in diseased and control livers. The number of CD14-positive cells correlated with advanced fibrosis in HH, PBC, HCV but not in NASH. The number of CD14-positive cells was increased with advanced inflammatory activity in HCV. CD14-positive cells were often associated with α-smooth muscle actin-positive myofibroblasts in fibrous septa. In conclusion, many forms of human chronic liver disease demonstrate increased numbers of CD14-positive macrophages/monocytes which are associated with fibrous septa and myofibroblasts. To determine whether CD14-positive cells contribute to fibrogenesis, experimental models of liver injury were used in chapters 5 and 6. The aim of chapter 5 was to determine whether CD14-positive macrophages/monocytes are detected in a bile duct ligation model of liver injury. To accomplish this aim, a novel antibody to rat CD14 was developed as described in chapter 4. A time-course study was undertaken in rats following bile duct ligation for up to 14 days. An increase in the number of hepatic CD14-positive cells was detected early following bile duct ligation, and was associated with increased gene expression of α-smooth muscle actin and procollagen I. Thus, myofibroblastic transformation in this model was associated with increased numbers of CD14-positive cells suggesting a possible relationship between the two phenomena. In order to specifically evaluate the role of CD14 in myofibroblastic transformation, a final study in CD14 knockout (KO) mice was undertaken in chapter 6

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Thèse (Ph. D.)--Université Laval, 2004. / Bibliogr.: f. 158-199. Publié aussi en version électronique.

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