<p>Granulocyte adhesion and release of their granule proteins are key steps during selective accumulation of a certain cell to an inflammatory site. Eosinophils are specifically recruited to sites of allergic inflammation and parasitic infection, whereas neutrophil influx predominates in bacterial infection and rheumatoid arthritis. </p><p>A simple, reliable and convenient method was developed for the measurement of granulocyte adhesion and release of granule proteins by using the normal population of granulocytes. The design allows simultaneous quantitative assessment of eosinophil and neutrophil adhesion to proteins and degranulation. </p><p>Using this method, manganese ions (Mn²⁺) induced a higher level of eosinophil adhesion to fibronectin, fibrinogen and albumin as compared with neutrophils. PMA induced comparable levels of eosinophil and neutrophil adhesion. F-MLP stimulated a rapid, short-term adhesion of neutrophils to fibrinogen. </p><p>In the same conditions PMA alone stimulated a dose-dependent release of ECP from cells that adhered to both fibronectin and fibrinogen. Meanwhile, Mn²⁺ amplified the release of ECP induced by PMA. Furthermore, release of ECP was shown to be associated with cell death.</p><p>PMA, in combination with Mn²⁺, induced a marked release of ~ 80%of the intracellular content of lactoferrin and HNL in neutrophils. PMA or f-MLP alone induced 30-40% release of lactoferrin and HNL. A maximal release of MPO of 15-20% was obtained from neutrophils stimulated by PMA and Mn²⁺. Release of lactoferrin and HNL showed a significant negative relationship to the viability of cells.</p><p>Stimulated by PMA, eosinophils from pollen-atopic patients during early pollen season displayed a markedly enhanced adhesion and release of ECP of eosinophils compared with eosinophils from the references. Priming with IL-5 caused a significantly higher adhesion and release of ECP by eosinophils in response to PMA. GM-CSF priming enhanced eosinophil adhesion in response to PAF and PMA plus Mn²⁺, but did not enhance the release of ECP.</p><p>In conclusion, the assay allows a simple quantification of eosinophil and neutrophil adhesion, as well as degranulation by using the normal population of granulocytes. Cellular adhesion plays an important role in the regulation of both eosinophil and neutrophil degranulation, but adhesion and degranulation can be induced separately.</p>

Neonates are generally thought to be more likely to suffer from gastrointestinal disease, owing in part to diminished immune cell function. To gain insight into the development of mucosal immune responses during early life, we developed a model of orogastric infection with the Gram-negative bacterium Yersinia enterocolitica using murine neonates. Remarkably, neonatal mice of either the BALB/c or C57BL/6 mouse strains showed markedly enhanced survival after infection compared to adult mice. Both innate and adaptive immune components appear to contribute to this phenomenon. First, the increased resistance of neonates coincided with containment of the bacteria in the intestinal tissue with low dissemination into the spleen and liver. In contrast, the bacteria readily disseminated to the peripheral tissues in adult mice. Flow cytometric and histological studies revealed increased levels of neutrophils and macrophages in the neonatal mesenteric lymph nodes (MLN) compared to adult mice. Similar results were obtained using two different high virulence Y. enterocolitica strains. The rapid mobilization of innate cells sequestered the bacteria to the intestinal tissue, since in vivo neutrophil depletion led to efficient dissemination of Y. enterocolitica to the spleen and liver of neonates. Together, these results support the hypothesis that the neonatal intestinal immune system is competent to mount a strong antibacterial response by rapidly mobilizing innate phagocytes and thereby confining the bacterial infection to the gut, resulting in a high level of resistance. Second, we have also demonstrated that the adaptive immune system was mobilized during primary and secondary infection with this pathogen and that some of these factors may contribute to the enhanced resistance of neonatal mice to infection. Primary infection in neonates led to increased levels of antigen presenting cells, B and T cells with an activated phenotype in the MLN. MLN CD4+ Th cells from infected neonates were found to produce greater levels of IFN-gamma and IL-17A, compared to CD4+ Th cells from adult mice. These Th responses are likely to be functionally significant because neonatal mice deficient in CD4+ T cells were found to be more susceptible than adult mice to primary infection. CD4+ T cells adoptively transferred into CD4 deficient mice rescued the majority of mice from lethal infection and led to the production of IFN-gamma and IL-17A by MLN cells. In addition, primary T cell-dependent IgG1 and IgG2a serum antibodies specific for the Yersinia immunogen LcrV were increased compared to adult mice, and the absence of B cells partially increased the susceptibility of neonatal mice to primary infection. During secondary infection, however, neonatal and adult mice mounted quantitatively and qualitatively similar Yersinia-specific memory antibody responses, demonstrating that infection with Y. enterocolitica promotes mature B cell responses in neonatal mice. Finally, primed neonatal and adult mice were protected from colonization of the Peyer's patches, weight loss and mortality after a lethal infection in adulthood, demonstrating the development of long-lived protective memory responses at the intestinal interface. Together, these results indicate that both B and T cell responses, in particular Th1 and Th17 associated immunity, are important for the development of long lasting immunity to this pathogen in early life. Third, infection of neonatal mice with a Y. enterocolitica strain deleted of the anti-inflammatory protein YopP led to massive infiltration and/or accumulation of innate phagocytes in the intestine and MLN. This effect was not detectable in infected adult mice. Thus, we have identified a novel negative regulator of intestinal inflammation which might be valuable in preventing or ameliorating inflammatory conditions. This model system has revealed the unprecedented potential of neonatal mice to develop protective inflammatory innate and adaptive immunity at mucosal surfaces. The combined results presented here demonstrate that neonatal mice may be well equipped to mount robust innate and adaptive intestinal inflammatory responses that are highly protective toward Y. enterocolitica. These findings have implications for understanding how pediatric intestinal adaptive immune responses develop in response to naturally occurring gastroenteric pathogens and offer a new biological platform for development of vaccines aimed at improving mucosal and systemic immunity in early life.

Granulocyte adhesion and release of their granule proteins are key steps during selective accumulation of a certain cell to an inflammatory site. Eosinophils are specifically recruited to sites of allergic inflammation and parasitic infection, whereas neutrophil influx predominates in bacterial infection and rheumatoid arthritis. A simple, reliable and convenient method was developed for the measurement of granulocyte adhesion and release of granule proteins by using the normal population of granulocytes. The design allows simultaneous quantitative assessment of eosinophil and neutrophil adhesion to proteins and degranulation. Using this method, manganese ions (Mn2+) induced a higher level of eosinophil adhesion to fibronectin, fibrinogen and albumin as compared with neutrophils. PMA induced comparable levels of eosinophil and neutrophil adhesion. F-MLP stimulated a rapid, short-term adhesion of neutrophils to fibrinogen. In the same conditions PMA alone stimulated a dose-dependent release of ECP from cells that adhered to both fibronectin and fibrinogen. Meanwhile, Mn2+ amplified the release of ECP induced by PMA. Furthermore, release of ECP was shown to be associated with cell death. PMA, in combination with Mn2+, induced a marked release of ~ 80%of the intracellular content of lactoferrin and HNL in neutrophils. PMA or f-MLP alone induced 30-40% release of lactoferrin and HNL. A maximal release of MPO of 15-20% was obtained from neutrophils stimulated by PMA and Mn2+. Release of lactoferrin and HNL showed a significant negative relationship to the viability of cells. Stimulated by PMA, eosinophils from pollen-atopic patients during early pollen season displayed a markedly enhanced adhesion and release of ECP of eosinophils compared with eosinophils from the references. Priming with IL-5 caused a significantly higher adhesion and release of ECP by eosinophils in response to PMA. GM-CSF priming enhanced eosinophil adhesion in response to PAF and PMA plus Mn2+, but did not enhance the release of ECP. In conclusion, the assay allows a simple quantification of eosinophil and neutrophil adhesion, as well as degranulation by using the normal population of granulocytes. Cellular adhesion plays an important role in the regulation of both eosinophil and neutrophil degranulation, but adhesion and degranulation can be induced separately.

Neutrophil granulocytes play an important role in the host defence against invading microorganisms and constitute the frontline of defence within the innate immune system and are among the first cells to arrive at the site of inflammation. Effective phagocytosis and killing of invading pathogens by neutrophils is of significant importance for successful resistance to infectious diseases. An important complication in diabetes mellitus is an increased sensitivity to infections and increased tissue damage, leading to many secondary diseases. This may in part be explained by an impaired function of neutrophil granulocytes. Since the exact mechanisms underlying defective neutrophil function in diabetes mellitus are not fully understood, the aim of the present study was to investigate the effects of elevated glucose and insulin concentrations on phagocytosis of opsonized yeast and on production of reactive oxygen metabolites (ROS) in normal human neutrophils. Elevated D-glucose concentrations (15-25 mM) inhibited the phagocytosis of C3bi- or IgG-opsonized yeast particles, which was neither an osmotic effect nor an effect due to reduced binding of opsonized yeast particles to the neutrophils. Inhibition of protein kinase C (PKC) by GF109203X or Go6976 could completely reverse the inhibitory effect of 25 mM D-glucose on phagocytosis. Diacylglycerol (DAG) dose-dependently inhibited phagocytosis and suboptimal inhibitory concentrations of DAG and glucose showed an additive inhibitory effect. Elevated concentrations of insulin (80-160 μU/ml) also inhibited neutrophil phagocytosis, an effect shown in part to be due to a delayed phagocytosis process. Insulin was found to increase the accumulation of cortical F-actin, without affecting the total cellular F-actin content. The PKCalpha/beta inhibitor, Go6976, abolished the insulin-mediated increase in cortical F-actin content and both Go6976 and the PKCalpha/beta/delta/epsilon-specific inhibitor GF109203X reversed the inhibitory effects of insulin on phagocytosis. The inhibition of phagocytosis by either glucose or insulin resulted in an expected reduction of intracellular respiratory burst. However, the extracellular release of ROS during phagocytosis was increased by insulin, but inhibited by glucose. The ability of insulin to enhance ROS production was found to be F-actin dependent. Data suggests that glucose inhibited intracellular respiratory burst activation by interfering with intracellular signaling downstream of PKC activation, whereas extracellular release of ROS was inhibited by glucose upstream of PKC signaling. Taken together these results suggest that both hyperglycemia and hyperinsulinemia inhibit complement receptor and Fc receptor-mediated phagocytosis in human neutrophils. Insulin, but not glucose, also induced an enhanced extracellular release of ROS during phagocytosis. The combination of reduced phagocytosis and alterations in ROS production may possibly explain both the increased sensitivity to infections and tissue damage seen in type 2 diabetes.

Reproduction de : Thèse doctorat : Médecine. Biochimie et biologie moléculaire : Reims : 2005. / Titre provenant de l'écran-titre. Bibliogr. p 205-227.

Infective endocarditis (IE) is a rare but complex disease that is fatal if untreated. With a modern combination of antimicrobial therapy and heart valve surgery, mortality is still 10-20 %. The structure of the endocarditis vegetation impedes the penetration of phagocytic cells such as monocytes and granulocytes. This leads to high bacterial counts inside the vegetation and the need for long treatment courses with a combination of intravenously administered bactericidal antibiotics. The aim of this thesis was to study the changes in epidemiology, management, and mortality at our hospital between 1980 and 2001, and to identify prognostic factors associated with mortality. To assess the issue of referral bias, differences between referred episodes and episodes from our local community were studied. Additional aims were to study the occurrence of the pro-chemotactic cytokines IL-8 and TNF-α in heart valves and vegetations during the active phase of IE, and to study the effect of the glycopeptide antibiotic vancomycin in dense staphylococcal cultures in vitro. As it is a rare and complex disease, management of IE is usually complicated for non-specialists. For this reason a computerised decision support system for IE was developed and evaluated. Between 1980 and 2001, the occurrence of Staphylococcus aureus IE and the use of early heart valve surgery increased significantly, regardless of whether the episodes were referred or of local origin. Glycopeptide antibiotics, mainly vancomycin, were used more frequently, especially among referred patients. Referred patients were younger, predominantly male, had more complications, and received surgical treatment more often than patients from our local community. The reason for the lower frequency of female patients in the referral cohort cannot be explained by more comorbidity or fewer complications. The differences between referred and local episodes seen in our study highlight the need for assessment and adjustment for referral bias in IE studies (Paper I). In six patients who needed early heart valve surgery, the largest numbers of IL-8-containing cells, and the greatest amount of inflammation, were seen in patients with short preoperative antimicrobial treatment courses. No such relationships were seen with respect to TNF-α-containing cells. The IL-8-containing cells and the inflammatory cells were predominantly scattered in the heart valve stroma or in the margin of the vegetation (Paper II). The primary effect of IL-8 is to stimulate chemotaxis of polymorphonuclear neutrophil granulocytes. This indicates that there is no deficiency of IL-8 in the area close to the vegetation as a cause of the localised agranulocytosis often present inside the vegetation. Our study revealed a need for computerised decision support systems (DSSs) in the field of IE, but to be used in clinical practice these DSSs need be part of knowledge bases covering larger domains (Paper IV). Some of our initial ideas described in Paper III, especially the use of Internet technology and the combination of rule-based advice and explanatory hypertext, will probably be included in these knowledge bases. In vitro, there is a rapid reduction of free vancomycin in broth containing dense staphylococcal cultures. Consequently, there is a simultaneous increase in broth MICs, particularly in high inocula, which is not caused by a development of resistance (Paper V). These findings need further evaluation in vivo, but indicate that the dosing regimen of vancomycin is of particular importance in staphylococcal infections with dense inocula, e.g. infective endocarditis. Diabetes mellitus and moderate to severe heart failure were independent risk factors for 6-month mortality in left-sided, Duke definite IE episodes, regardless of referral or local origin of the episodes. Early heart valve surgery had a positive impact on the 6-month mortality in the referral cohort of episodes, which may be due to referral bias (Paper VI).

Diss. (sammanfattning) Linköping : Linköpings universitet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning). Uppsala : Sveriges lantbruksuniv., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.