Franco H. Falcone

Molecular Characterization of an Interleukin-4-inducing Factor from Schistosoma mansoni Eggs

The eggs of the parasitic trematodeSchistosoma mansoni are powerful inducers of a T helper type 2 (Th2) immune response and immunoglobulin E (IgE) production.S. mansoni egg extract (SmEA) stimulates human basophils to rapidly release large amounts of interleukin (IL)-4, the key promoter of a Th2 response. Here we show purification and sequence of the IL-4-inducing principle of S. mansoni eggs (IPSE). Stimulation studies with human basophils using SmEA fractions and natural and recombinant IPSE as well as neutralization and immunodepletion studies using antibodies to recombinant IPSE demonstrate that IPSE is the bioactive principle in SmEA leading to activation of basophils and to expression of IL-4 and IL-13. Regarding the mechanism of action, blot analysis showed that IPSE is an IgE-binding factor, suggesting that it becomes effective via cross-linking receptor-bound IgE on basophils. Immunohistology revealed that IPSE is enriched in and secreted from the subshell area of the schistosome egg. We conclude from these data that IPSE may be an important parasite-derived component for skewing the immune response toward Th2.

Experimental hookworm infection: a randomized placebo-controlled trial in asthma

Background Epidemiological studies suggest that hookworm infection protects against asthma, and therefore that hookworm infection may have a direct or an indirect therapeutic potential in this disease. We now report the first clinical trial of experimental hookworm infection in people with allergic asthma.

A Brugia malayi Homolog of Macrophage Migration Inhibitory Factor Reveals an Important Link Between Macrophages and Eosinophil Recruitment During Nematode Infection

Infections with the helminth parasite Brugia malayi share many key features with Th2-mediated allergic diseases, including recruitment of eosinophils. We have investigated the dynamics of inflammatory cell recruitment under type 2 cytokine conditions in mice infected with B. malayi. Among the cells recruited to the site of infection is a novel population of “alternatively activated” macrophages that ablate cell proliferation and enhance Th2 differentiation. By profiling gene expression in this macrophage population, we found a dramatic up-regulation of a recently described eosinophil chemotactic factor, eosinophil chemotactic factor-L/Ym1, representing over 9% of clones randomly selected from a cDNA library. Because B. malayi is known to secrete homologs (Bm macrophage migration inhibitory factor (MIF)-1 and -2) of the human cytokine MIF, we chose to investigate the role this cytokine mimic may play in the development of the novel macrophage phenotype observed during infection. Strikingly, administration of soluble recombinant Bm-MIF-1 was able to reproduce the effects of live parasites, leading both to the up-regulation of Ym1 by macrophages and a marked recruitment of eosinophils in vivo. Because activity of Bm-MIF-1 is dependent upon an amino-terminal proline, this residue was mutated to glycine; the resultant recombinant (Bm-MIF-1G) was unable to induce Ym1 transcription in macrophages or to mediate the recruitment of eosinophils. These data suggest that macrophages may provide a crucial link between helminth parasites, their active cytokine mimics, and the recruitment of eosinophils in infection.

Alginate encapsulation technology supports embryonic stem cells differentiation into insulin-producing cells.

This work investigates an application of the alginate encapsulation technology to the differentiation of embryonic stem (ES) cells into insulin-producing cells. It shows that the ES cells can efficiently be encapsulated within the alginate beads, retaining a high level of cell viability. The alginate encapsulation achieves approximately 10-fold increase in the cell density in the culture, in comparison to the two-dimensional conditions, opening a potential benefit of the technology in large-scale cell culture applications. Manipulations of encapsulation conditions, particularly of the initial alginate concentration, allow the control over both the diffusion of molecules into the alginate matrix (e.g. differentiation factors) as well as control over the matrix porosity/flexibility to permit the proliferation and growth of encapsulated ES aggregates within the bead. Post-differentiation analysis confirms the presence of insulin-positive cells, as judged from immunostaining, insulin ELISA and RT-PCR analysis. The functionality of the encapsulated and differentiated cells was confirmed by their insulin production capability, whereby on glucose challenge the insulin production by the cells differentiated within alginate beads was found to be statistically significantly higher than for the cells from conventional two-dimensional differentiation system.

Role of fiberscopic transbronchial needle aspiration in the staging of N2 disease due to non–small cell lung cancer

BACKGROUND Transbronchoscopic needle aspiration (TBNA) can offer a unique opportunity to identify surgically unresectable lung cancer and to avoid surgical mediastinal exploration in many patients with mediastinal lymph node extension of the tumor. The aim of this study was to assess the yield of TBNA performed with either histology or cytology needles in mediastinal staging of N2 disease due to non-small cell lung cancer (NSCLC). METHODS Retrospective chart review was carried out on 194 TBNA procedures performed between January 1997 and September 2000 at a single institution. Inclusion criteria were pathologic evidence of NSCLC; contrast enhancement computed tomography scan of the chest suggesting N2 disease; and negative bronchoscopic examination for possible neoplastic lesions at the site of RESULTS Overall sensitivity and diagnostic accuracy were 71% and 73%, respectively, with no significant differences between 19-gauge and 22-gauge cytology needles. Procedures performed for right paratracheal and subcarinal lymph node stations had a significantly higher yield than those for the left paratracheal station. CONCLUSIONS TBNA mediastinal staging, performed during the initial diagnostic evaluation of NSCLC, can spare costs and risks of more invasive procedures in patients with inoperable tumors, in patients who are not candidates for operation because of coexistent significant comorbidities, and in patients with N2 disease.

Dietary lectins can induce in vitro release of IL-4 and IL-13 from human basophils

Dietary lectins, present in beans and other edible plant products, pose a potential threat to consumers due to their capacity to induce histamine release from basophils. In this study, we analyzed the capacity of 16 common, in particular dietary, lectins to induce human basophils to secrete IL‐4 and IL‐13, the key promoters of Th2 responses and IgE synthesis. Several of the lectins, especially concanavalin A, lentil lectin, phytohemagglutinin, Pisum sativum agglutinin and Sambucus nigra agglutinin, triggered basophils to release IL‐4 at concentrations of up to 1 ng / 106 basophils. Lectins with high IL‐4‐inducing capacity also stimulated the release of IL‐13 and histamine. Lectin‐induced IL‐4 and IL‐13 release reached a maximum after 4 – 6 h and more than 18 h, respectively. Affinoblotting revealed that lectins with the capacity to induce mediator release bind to IgE, suggesting IgE binding as initial step of signal generation. In conclusion, several dietary lectins can trigger human basophils to release IL‐4 and IL‐13. Since lectins can enter the circulation after oral uptake, they might play a role in inducing the so‐called early IL‐4 required to switch the immune response towards a Th2 response and type I allergy.

The 21st century renaissance of the basophil? Current insights into its role in allergic responses and innate immunity

Abstract:  Basophils and mast cells express all the three subchains of the high‐affinity immunoglobulin E (IgE) receptor FcɛRI and contain preformed histamine in the cytoplasmic granules. However, it is increasingly clear that these cells play distinct roles in allergic inflammatory disease. Despite their presence throughout much of the animal kingdom, the physiological function of basophils remains obscure. As rodent mast cells are more numerous than basophils, and generate an assortment of inflammatory cytokines, basophils have often been regarded as minor players in allergic inflammation. In humans, however, basophils are the prime early producers of interleukin (IL)‐4 and IL‐13, T helper (Th)2‐type cytokines crucial for initiating and maintaining allergic responses. Basophils also express CD40 ligand which, in combination with IL‐4 and IL‐13, facilitates IgE class switching in B cells. They are the main cellular source for early IL‐4 production, which is vital for the development of Th2 responses. The localization of basophils in various tissues affected by allergic inflammation has now been clearly demonstrated by using specific staining techniques and the new research is shedding light on their selective recruitment to the tissues. Finally, recent studies have shown that basophil activation is not restricted to antigen‐specific IgE crosslinking, but can be caused in non‐sensitized individuals by a growing list of parasitic antigens, lectins and viral superantigens, binding to non‐specific IgE antibodies. This, together with novel IgE‐independent routes of activation, imparts important new insights into the potential role of basophils in both adaptive and innate immunity.

Vaccination against helminth parasites - The ultimate challenge for vaccinologists?

Summary: Helminths are multicellular pathogens which infect vast numbers of human and animal hosts, causing widspread chronic disease and morbidity, Vaccination against these parasites requires more than identification of effective target antigens, because without understanding the immunology of the host–parasite relationship, ineffective immune mechanisms may he invoked, and there is a danger of amplifying immuno‐pathogenic responses. The fundamental features of the immune response to helminths are therefore summarised in the context of vaccines to helminths parasites. The contention between type‐1 and type‐2 responses is a central issue in helminth infections, which bias the immune system strongly to the type‐2 pathway. Evidence from both human and experimental animal infections indicates that both lineages contribute to immunity in differing circumstances, and that a balanced, response leads to the most favourable outcome. A diversity of immune mechanisms can be brought to bear on various helminth species, ranging from antibody‐independent macrophages, antibody‐dependent granulocyte killing, and non‐lymphoid actions, particularly in the gut. This diversity is highlighted by analysis of rodent infections, particularly in comparisons of cytokine‐depleted and gene‐targeted animals. This knowledge of protective mechanisms needs to he combined with a careful choice of parasite antigens for vaccines. Many existing candidates have been selected with host antibodies, rather than T‐cell responses, and include a preponderance of highly conserved proteins with similarities to mammalian or invertebrate antigens. Advantage has yet to he taken of parasite genome projects, or of directed searches for novel, parasite‐specific antigens and targets expressed only by infective stages and not mature forms which may generate immunopathology. With advances under way in parasite genomics and new vaccine delivery systems offering more rapid assessment and development, there are now excellent opportunities for new antihelminth vaccines.

Safety of hookworm infection in individuals with measurable airway responsiveness: a randomized placebo-controlled feasibility study

Background Epidemiological evidence suggests that hookworm infection protects against asthma. However, for ethical and safety reasons, before testing this hypothesis in a clinical trial in asthma it is necessary to establish whether experimental hookworm infection might exacerbate airway responsiveness during larval lung migration.

Helminth Allergens, Parasite-Specific IgE, and Its Protective Role in Human Immunity.

The Th2 immune response, culminating in eosinophilia and IgE production, is not only characteristic of allergy but also of infection by parasitic worms (helminths). Anti-parasite IgE has been associated with immunity against a range of helminth infections and many believe that IgE and its receptors evolved to help counter metazoan parasites. Allergens (IgE-antigens) are present in only a small minority of protein families and known IgE targets in helminths belong to these same families (e.g., EF-hand proteins, tropomyosin, and PR-1 proteins). During some helminth infection, especially with the well adapted hookworm, the Th2 response is moderated by parasite-expressed molecules. This has been associated with reduced allergy in helminth endemic areas and worm infection or products have been proposed as treatments for allergic conditions. However, some infections (especially Ascaris) are associated with increased allergy and this has been linked to cross-reactivity between worm proteins (e.g., tropomyosins) and highly similar molecules in dust-mites and insects. The overlap between allergy and helminth infection is best illustrated in Anisakis simplex, a nematode that when consumed in under-cooked fish can be both an infective helminth and a food allergen. Nearly 20 molecular allergens have been isolated from this species, including tropomyosin (Ani s 3) and the EF-hand protein, Ani s troponin. In this review, we highlight aspects of the biology and biochemistry of helminths that may have influenced the evolution of the IgE response. We compare dominant IgE-antigens in worms with clinically important environmental allergens and suggest that arrays of such molecules will provide important information on anti-worm immunity as well as allergy.