No abstract

Background: Phagosomal pH is thought to play an important role in the antimicrobial activity of polymorphonuclear leukocytes (PMNs). In this study, we set up a method for a rapid and accurate measurement of phagosomal pH in PMNs with the use of Candida albicans doubly labeled with a pH-insensitive and a pH-sensitive probe and flow cytometry. Methods: Heat-killed, serum-opsonized C. albicans were doubly labeled with fluorescein, a pH-sensitive probe, and rhodamine, a pH-insensitive probe, and incubated with human PMNs. Flow cytometric readings of PMN-associated Candida were then taken, and the intraphagosomal pH was calculated on the basis of the ratio of fluorescein: rhodamine fluorescence by using a calibration curve obtained after equilibration of phagosomal pH with different external pH values after addition of digitonin. Results: A rapid rise in phagosomal pH, which reached pH 7.8, was observed 2 min after initiation of phagocytosis and progressively declined to pH 6.9 after 15 min. Such a rise was not observed in PMNs with defective microbi-

The mannose receptor (MR) plays an important role in the recognition of some pathogens in nonopsonic phagocytosis and in antigen presentation to T cells. We found that Borrelia burgdorferi, the agent of Lyme borreliosis, adheres to monocyte-derived macrophages and to rat MR-transfected cells but not to untransfected cells. Antibodies to MR and sugars such as mannose, mannan, fucose, and some lectins significantly lowered the adhesion, confirming participation of the MR in the binding.The etiological agent of Lyme disease, Borrelia burgdorferi, is able to disseminate widely and avoid clearance by innate immunity and by the immune system, thereby establishing chronic infection. Spirochetes undertake complex interactions with a variety of mammalian cells, which contribute to the establishment of infection in the host. Such interactions consist of adhesion to cells and tissues: B. burgdorferi binds to platelets via the integrins ␣ II ␤ 3 and to endothelial cells through the integrins ␣ V ␤ 3 and ␣ 5 ␤ 1 (7,8) and to a variety of cells which express glycosaminoglycans such as heparin, heparan sulfate, and dermatan sulfate (19, 2). Lyme borreliosis Borrelia also recognizes other substrates like fibronectin (17, 23) and decorins (12), which explains the attachment of B. burgdorferi to the extracellular matrix of the skin and other tissues. Binding to such receptors as integrins, which are true cell-associated receptors, not only provides adhesion to cells, allowing B. burgdorferi to attach itself firmly to the tissues, but also can induce effector mechanisms, normally triggered by the integrin under appropriate stimuli. This is the case with integrin ␣ M ␤ 2 , a dynamic molecule also known as CR3 or CD11b/CD18 receptor; this molecule was shown to bind borrelias (4) and trigger phagocytosis by neutrophils in the absence of specific opsonization; such binding activates the molecule to up-regulation and increases adhesion to fibronectin (6). The binding seems to involve not only the I domain of the molecule, specific for the iC3b and the RGD motif, but also the lectin-like domain, which is known to recognize lipopolysaccharide, mannose, and other sugar residues (5).Another cell receptor which is involved in microbe recognition and phagocytosis in the absence of specific opsonization is the mannose receptor (MR), which acts as a true lectin in the lectin phagocytosis of microorganisms (22). It is a type I transmembrane glycoprotein of 165 kDa containing as many as eight adjoining carbohydrate recognition domains, a fibronectin type II domain, and a cysteine-rich domain (2); it is expressed on tissue macrophages, dendritic cells (mostly on Langerhans cells), endothelium, and rat microglia. Besides acting as a scavenger of mannose-containing glycoconjugates on the surface of a wide spectrum of microorganisms, such as Escherichia coli, Klebsiella pneumoniae, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Candida albicans, Leishmania, and Pneumocystis carinii (21), it mediates their ingestion by macrophages. Moreover, it ...

The authors' results permit only to suppose an etiological role of FAG and ROI deficiencies of polymorphonuclear neutrophils in the genesis of pediatric RRI, irrespective of the age of the patients, and further studies are necessary for confirmation.

Coeliac disease (CD) is a T-cell mediated immunological disease of the small intestine which is triggered in susceptible individuals by ingestion of gluten. The pathogenic mechanism of coeliac disease, and the role that a-gliadin specific T cells play in mucosal lesions and their involvement in peripheral blood is not yet explained at all. Previous studies have reported proliferative response to a-gliadin measured with the classic assay of 3HTdR incorporation. We analysed the activation antigen CD69 on T cells from CD patients and normal individuals following stimulation with a-gliadin and different antigens (tetanus toxoid, peptides unrelated to gliadin and PHA). CD69 coexpression with T cell CD3+ and proliferation marker Ki67 was evaluated with time. CD69 coexpression with T cell CD3+, CD4+ and CD8+ was also evaluated. It was found that peripheral blood mononuclear cells (PBMC) of coeliac patients increased their percentage of CD69 positive T cells when stimulated with a-gliadin, in comparison with cells from controls. Significant T cell activation was found only in subjects not treated with the gluten free diet; a positive response was found also in two coeliac patients with selective IgA deficiency, anti-endomisium negative, without circulat- ing IgA anti a-gliadin or anti-tissue transglutaminase antibodies. The CD69 expression after stimulation was compared with the standard method of 3HTdR incorporation. Our data show that CD69 expression is useful to asses a specific T cell response to a-gliadin in coeliac disease. in a very short time. Moreover, the method allows to investigate T cell response at the lymphocyte subsets level, which represents a useful tool in the diagnosis of coeliac disease