Evaluation of the specific IgG- and IgA-antibody response against immundominant Aspergillus fumigatus antigens as diagnostic markers of invasive aspergillosis

An improvement in the serological diagnostic toolbox of invasive aspergillosis (IA) is necessary. So far, most laboratories do not perform antibody detection assays at all to diagnose IA, as commercial test systems are based on crude and undefined antigen mixtures of A. fumigatus. Utilizing the A. fumigatus protein mitogillin, we could demonstrate that the use of selected characterized immunodominant antigens can improve the serodiagnosis of Aspergillus-related diseases. In an animal model we were able to identify additional 36 immunodominant antigens of a cDNA library of A. fumigatus germlings. Five selected antigens were expressed recombinantly in E. coli, purified and used for Westernblot und ELISA analyses to study the kinetics of the specific antibody response in rabbits that were infected systemically with A. fumigatus. Subsequently, the specific IgG- and IgA-antibody responses against these antigens were studied in patients suffering from proven IA and compared to healthy blood donors and patients with other forms of pneumonia. Furthermore, we examined how total IgG- and IgA-levels influence the diagnostic value of antibody detection in IA patients.

IgA with "secretory piece" in bovine colostrum and saliva.

We have shown that in bovine colostrum and saliva there is a secretory IgA with a sedimentation, coefficient of 11S and a secretory piece, previously unknown and comparable with the 11S IgA described in human and rabbit external secretions. These secretory IgA contain molecules of 7S IgA with an additional protein segment called the transport or secretory piece1-5. Furthermore, the free form of the secretory piece is identified in bovine colostrum and also in mature milk which contains very little IgA.

N-Glycans on secretory component: mediators of the interaction between secretory IgA and gram-positive commensals sustaining intestinal homeostasis.

Human beings live in symbiosis with billions of microorganisms colonizing mucosal surfaces. The understanding of the mechanisms underlying this fine-tuned intestinal balance has made significant processes during the last decades. We have recently demonstrated that the interaction of SIgA with Gram-positive bacteria is essentially based on Fab-independent, glycan-mediated recognition. Results obtained using mouse hybridoma- and colostrum-derived secretory IgA (SIgA) consistently show that N-glycans present on secretory component (SC) play a crucial role in the process. Natural coating may involve specific Gram-positive cell wall components, which may explain selective recognition at the molecular level. More widely, the existence of these complexes is involved in the modulation of intestinal epithelial cell (IEC) responses in vitro and the formation of intestinal biofilms. Thus, SIgA may act as one of the pillars in homeostatic maintenance of the microbiota in the gut, adding yet another facet to its multiple roles in the mucosal environment.

In vitro combination of human and bovine free secretory component with IgA of various species.

The free form of the secretory component usually associated with secretory IgA can be isolated from human and bovine milk. These free secretory components of different origin combine in vitro with human polymeric myeloma IgA, with mouse myeloma IgA, and with the serum IgA of nine different mammalian species.

IgG subclass switch capacity is low in switched and in IgM-only, but high in IgD+IgM+, post-germinal center (CD27+) human B cells.

Recent studies have shown that in humans the germinal center reactions produce three types of V(D)J mutated B cells in similar proportions, i.e. Ig-switched, IgD-IgM+ (IgM-only) and IgD+IgM+ cells, and that together they form the CD27+ compartment of recirculating B cells. We investigated the Ig isotype switch capacity of these cells. Peripheral blood B subsets were sorted and IgG subclass secretion in presence or absence of IL-4 was compared in B cell assays which lead to Ig secretion in all (coculture with EL-4 thymoma cells) or only in CD27+ (CD40L stimulation) B cells. Already switched IgG+ B cells showed no significant sequential switch and IgM-only cells also had a low switch capacity, but IgD+CD27+ switched as much as IgD+CD27- B cells to all IgG subclasses. Thus, in switched B cells some alterations compromising further switch options occur frequently; IgM-only cells may result from aborted switch. However, IgD+CD27+ human B cells, extensively V(D)J mutated and "naive" regarding switch, build up a repertoire of B cells combining (1) novel cross-reactive specificities, (2) increased differentiation capacity (including after T-independent stimulation by Staphylococcus aureus Cowan I) and (3) the capacity to produce appropriate isotypes when they respond to novel pathogens.

Secretory IgA, a major immunoglobulin in most bovine external secretions.

Bovine secretory IgA (SIgA), recently identified in colostrum, was shown to be homologous to human SIgA by immunologic cross-reaction. A quantitative study indicated that bovine SIgA, a minor component of colostrum, is a major immunoglobulin in most other external secretions including saliva, spermatic fluid, lacrimal, nasal and gastrointestinal secretions. SIgA was isolated from saliva. The free form of secretory component was found to be abundant in milk. A normal lactating cow produces about 1.2 g of this protein per day. Two forms of IgA were identified in serum: a normal serum IgA with no secretory antigenic determinant, and a small amount of SIgA. In vitro synthesis of SIgA by the salivary gland was studied by tissue cultures with incorporation of labeled amino acids.

Cocoa flavonoid-enriched diet modulates systemic and intestinal immunoglobulin synthesis in adult Lewis rats

Previous studies have reported that a diet containing 10% cocoa, a rich source of flavonoids, has immunomodulatory effects on rats and, among others effects, is able to attenuate the immunoglobulin (Ig) synthesis in both systemic and intestinal compartments. The purpose of the present study was focused on investigating whether these effects were attributed exclusively to the flavonoid content or to other compounds present in cocoa. To this end, eight-week-old Lewis rats were fed, for two weeks, either a standard diet or three isoenergetic diets containing increasing proportions of cocoa flavonoids from different sources: one with 0.2% polyphenols from conventional defatted cocoa, and two others with 0.4% and 0.8% polyphenols, respectively, from non-fermented cocoa. Diet intake and body weight were monitored and fecal samples were obtained throughout the study to determine fecal pH, IgA, bacteria proportions, and IgA-coated bacteria. Moreover, IgG and IgM concentrations in serum samples collected during the study were quantified. At the end of the dietary intervention no clear changes of serum IgG or IgM concentrations were quantified, showing few effects of cocoa polyphenol diets at the systemic level. However, in the intestine, all cocoa polyphenol-enriched diets attenuated the age-related increase of both fecal IgA and IgA-coated bacteria, as well as the proportion of bacteria in feces. As these effects were not dependent on the dose of polyphenol present in the diets, other compounds and/or the precise polyphenol composition present in cocoa raw material used for the diets could be key factors in this effect.

Transient suppression of Shigella flexneri type 3 secretion by a protective O-antigen-specific monoclonal IgA.

Mucosal immunity to the enteric pathogen Shigella flexneri is mediated by secretory IgA (S-IgA) antibodies directed against the O-antigen (O-Ag) side chain of lipopolysaccharide. While secretory antibodies against the O-Ag are known to prevent bacterial invasion of the intestinal epithelium, the mechanisms by which this occurs are not fully understood. In this study, we report that the binding of a murine monoclonal IgA (IgAC5) to the O-Ag of S. flexneri serotype 5a suppresses activity of the type 3 secretion (T3S) system, which is necessary for S. flexneri to gain entry into intestinal epithelial cells. IgAC5's effects on the T3S were rapid (5 to 15 min) and were coincident with a partial reduction in the bacterial membrane potential and a decrease in intracellular ATP levels. Activity of the T3S system returned to normal levels 45 to 90 min following antibody treatment, demonstrating that IgAC5's effects were transient. Nonetheless, these data suggest a model in which the association of IgA with the O-Ag of S. flexneri partially de-energizes the T3S system and temporarily renders the bacterium incapable of invading intestinal epithelial cells. IMPORTANCE: Secretory IgA (S-IgA) serves as the first line of defense against enteric infections. However, despite its well-recognized role in mucosal immunity, relatively little is known at the molecular level about how this class of antibody functions to prevent pathogenic bacteria from penetrating the epithelial barrier. It is generally assumed that S-IgA functions primarily by "immune exclusion," a phenomenon in which the antibody binds to microbial surface antigens and thereby promotes bacterial agglutination, entrapment in mucus, and physical clearance from the gastrointestinal tract via peristalsis. The results of the present study suggest that in addition to serving as a physical barrier, S-IgA may have a direct impact on the ability of microbial pathogens to secrete virulence factors required for invasion of intestinal epithelial cells.

Secretory IgA: an actor of the interplay between the commensal flora and the intestinal immune system?

In the gastro-intestinal tract,Peyers patches have been describedas a major inductive site for mucosalsecretory IgA (SIgA) responses directedagainst pathogens. The classicalview is that SIgAserves as the firstline of defense against microorganismsby agglutining potential invadersand faciliting their clearance byperistaltic and mucociliary movements,a mechanism called immuneexclusion. Our laboratory has shownthat SIgA is not only able to be"retrotransported" into Peyers patchesvia the associated M cells, but also todeliver sizeable cargos in the form ofSIgA-based immune complexes, resultingin the onset of non-inflammatorytype of responses. Such a novelfunction raises the question of thepossible role of mucosal SIgA in theinterplay with commensal bacteriaand the contribution of the antibody inbacterial homeostasis. To address thisquestion, Lactobacillus rhamnosus(LPR) was administered into a mouseligated loop comprising a Peyerspatch, in association or not with SIgA.The fate of fluorescently labelled bacteriawas followed by laser scanningconfocal microscopy at different incubationtimes. After 2 hours of incubationin the loop, LPR bacteria arefound more abundantly in thesubepithelial dome (SED) regionwhen they are coated with SIgA thanLPR administered alone despite theyare absent from neighboring villi.Herein, it is shown that this mechanismof entry involves M cells inPeyers pathes. After their sampling byM cells, bacteria are engulfed by thedendritic cells of the subjacent SEDregion. Interestingly, LPR bacteriaare found coated by the endogenousnatural SIgA present in mice intestinalsecretions, confirming the requirementof SIgA for this type of entry.The subsequent effect on the maturationof dendritic cells after interactionwith LPR was investigated in vitroin presence or not of SIgA by measuringthe expression of CD40, CD80and CD86 surface markers with flowcytometry analyses. Results show thatDCs respond in the same way in presenceof SIgA than with LPR bacteriaalone, indicating that SIgA does notmodulate the interaction betweenDCs and bacteria in this context. Thiswork gives new evidences about theinvolvement of SIgA in the mechanismby which the intestinal immunesystem permanently checks the contentof the intestine.

The Rose Bengal test in human brucellosis: a neglected test for the diagnosis of a neglected disease

Brucellosis is a highly contagious zoonosis affecting livestock and human beings. The human disease lacks pathognomonic symptoms and laboratory tests are essential for its diagnosis. However, most tests are difficult to implement in the areas and countries were brucellosis is endemic. Here, we compared the simple and cheap Rose Bengal Test (RBT) with serum agglutination, Coombs, competitive ELISA, Brucellacapt, lateral flow immunochromatography for IgM and IgG detection and immunoprecipitation with Brucella proteins. We tested 208 sera from patients with brucellosis proved by bacteriological isolation, 20 contacts with no brucellosis, and 1559 sera of persons with no recent contact or brucellosis symptoms. RBT was highly sensitive in acute and long evolution brucellosis cases and this related to its ability to detect IgM, IgG and IgA, to the absence of prozones, and to the agglutinating activity of blocking IgA at the pH of the test. RBT was also highly specific in the sera of persons with no contact with Brucella. No test in this study outperformed RBT, and none was fully satisfactory in distinguishing contacts from infected patients. When modified to test serum dilutions, a diagnostic titer >4 in RBT resulted in 87.4% sensitivity (infected patients) and 100% specificity (contacts). We discuss the limitations of serological tests in the diagnosis of human brucellosis, particularly in the more chronic forms, and conclude that simplicity and affordability of RBT make it close to the ideal test for small and understaffed hospitals and laboratories.

Mechanisms involved in down-regulation of intestinal IgA in rats by high cocoa intake

Previous studies have shown that rat intestinal immunoglobulin A (IgA) concentration and lymphocyte composition of the intestinal immune system were influenced by a highly enriched cocoa diet. The aim of this study was to dissect the mechanisms by which a long-term high cocoa intake was capable of modifying gut secretory IgA in Wistar rats. After 7 weeks of nutritional intervention, Peyer's patches, mesenteric lymph nodes and the small intestine were excised for gene expression assessment of IgA, transforming growth factor ß, C-C chemokine receptor-9 (CCR9), interleukin (IL)-6, CD40, retinoic acid receptors (RAR¿ and RARß), C-C chemokine ligand (CCL)-25 and CCL28 chemokines, polymeric immunoglobulin receptor and toll-like receptors (TLR) expression by real-time polymerase chain reaction. As in previous studies, secretory IgA concentration decreased in intestinal wash and fecal samples after cocoa intake. Results from the gene expression showed that cocoa intake reduced IgA and IL¿6 in Peyer's patches and mesenteric lymph nodes, whereas in small intestine, cocoa decreased IgA, CCR9, CCL28, RAR¿ and RARß. Moreover, cocoa-fed animals presented an altered TLR expression pattern in the three compartments studied. In conclusion, a high-cocoa diet down-regulated cytokines such as IL-6, which is required for the activation of B cells to become IgA-secreting cells, chemokines and chemokine receptors, such as CCL28 and CCR9 together with RAR¿ and RARß, which are involved in the gut homing of IgA-secreting cells. Moreover, cocoa modified the cross-talk between microbiota and intestinal cells as was detected by an altered TLR pattern. These overall effects in the intestine may explain the intestinal IgA down-regulatory effect after the consumption of a long-term cocoa-enriched diet.

The regulation of the mucosal immune response by secretory IgA

L'immunoglobuline A sécrétoire (SlgA) est l'anticorps qui est dominant dans la réponse humorale des muqueuses. IgA est produit localement sous la forme de dimère ou polymère. Ces derniers sont ensuite relâchés dans les sécrétions muqueuses sous forme d'un complexe avec la pièce sécrétoire (SC). SlgA est capable d'identifier des antigènes microbiens dans l'environnement des muqueuses et de prévenir leur diffusion en bloquant l'adhésion ou la surface des cellules épithéliales. Au-delà de sa fonction classique d'exclusion immune, SlgA peut aussi adhérer aux cellules M présente au niveau des follicules associés à l'épithélium dans des structures organisées appelées plaques de Peyer (PPs) dans le système gastrointestinal. L'interaction sélective avec les SlgA amène cette dernière à être transportée à travers les cellules M Ce procès facilite l'association de l'anticorps avec les cellules dendritiques (DCs) CDllc+CDllb+, localisées dans la région sous-épithéliale des PPs. L'entrée de SlgA ou de microorganismes couverts par la SlgA via ce chemin est cruciale pour la modulation de la réponse immunitaire locale. Dans la première partie du travail, nous avons établi les conditions pour l'analyse de l'interaction entre SlgA et une lignes de DCs dérivée in vitro. Nous avons aussi montré que l'internalisation de la SlgA par les DCs est affecté après traitement avec des inhibiteurs de l'endocytose ou par l'utilisation de compétiteurs moléculaires. En utilisant le microscope confocal, on a observé qu'après internalisation la SlgA suit la voie endocytique, vers le compartiment lysozomal. Dans la deuxième partie du travail, une partie des résultats a été confirmée pour les DCs CDllc+CDllb+ des muqueuses. En outre, l'importance des sucres présents sur la SlgA a été mis en évidence par la réduction de l'interaction avec les DCs des muqueuses après déglycosylation. On a montré pour la première fois à notre connaissance que Dectin-1 et SIGNR3 sont des récepteurs potentiels pour la SlgA sur les DCs des muqueuses de la souris. De plus, les DCs de la souris prélevées des PPs, des ganglions lymphatiques mésentériques (MLNs) et de la rate ont été infectés avec Shigella flexneri (Sf) seule ou en complexe avec SlgA. Les DCs infectées ont montré une augmentation de l'expression des marqueurs co-stimulateurs, une forte production des cytokines pro¬inflammatoires et une induction de la prolifération des cellules T. Après l'association des Sf avec SlgA, les profils pro-inflammatoires des DCs ont diminués. En particulier, SlgA a un effet sur les cytokines sécrétées et sur la prolifération des cellules T. Ces données démontrent le rôle de la SlgA dans la réponse immunitaire par les DCs des muqueuses.