The influence of the Internet on immunology education.

The potential of the Internet as a medium through which to teach basic and applied immunology lies in the ability to illustrate complex concepts in new ways for audiences that are diverse and often geographically dispersed. This article explores two collaborative Internet-based learning projects (also known as e-learning projects) that are under development: Immunology Online, which will present an Internet-based curriculum in basic and clinical immunology to Swiss undergraduate and graduate students across five campuses; and the OCTAVE project, which will offer online training to an international cadre of new investigators, the members of which are carrying out clinical trials of vaccines against HIV infection.

Customized online and onsite training for rabies-control officers.

PROBLEM: It is difficult to deliver adequate training for people working in rabies control in low and middle-income countries. Popular e-learning systems for low-income settings are not well suited to developing and testing practical skills, including laboratory methods. APPROACH: We customized training in rabies control methods for African professionals and students from different disciplines. Trainees participated in preparatory online sessions, evaluations and exercises before and after a 12-day workshop. Trainees and mentors continued to interact through an online forum up to one year after the workshop. LOCAL SETTING: In Africa, 15,000 deaths from rabies occur each year due to a lack of awareness, inaccessibility of post-exposure prophylaxis, inadequate or absent canine rabies-control programmes and lack of governmental financial support. RELEVANT CHANGES: Thirty two trainees - working in health departments, hospitals, veterinary stations and research institutes - were selected to participate; 28 completed the course and passed the final evaluation. Pilot rabies investigation programmes were developed, and two manuscripts submitted for publication. An online forum facilitated further progress for a year after the workshop. LESSONS LEARNT: A combination of customized online and onsite training is suitable for teaching disease-control personnel in low-income countries. Participation in this course enabled trainees to advocate for the development of national disease-control strategies. Mentoring is needed to develop a strong network of experts in similar settings.

Aerosol immunization with NYVAC and MVA vectored vaccines is safe, simple, and immunogenic.

Each year, approximately five million people die worldwide from putatively vaccine-preventable mucosally transmitted diseases. With respect to mass vaccination campaigns, one strategy to cope with this formidable challenge is aerosol vaccine delivery, which offers potential safety, logistical, and cost-saving advantages over traditional vaccination routes. Additionally, aerosol vaccination may elicit pivotal mucosal immune responses that could contain or eliminate mucosally transmitted pathogens in a preventative or therapeutic vaccine context. In this current preclinical non-human primate investigation, we demonstrate the feasibility of aerosol vaccination with the recombinant poxvirus-based vaccine vectors NYVAC and MVA. Real-time in vivo scintigraphy experiments with radiolabeled, aerosol-administered NYVAC-C (Clade C, HIV-1 vaccine) and MVA-HPV vaccines revealed consistent mucosal delivery to the respiratory tract. Furthermore, aerosol delivery of the vaccines was safe, inducing no vaccine-associated pathology, in particular in the brain and lungs, and was immunogenic. Administration of a DNA-C/NYVAC-C prime/boost regime resulted in both systemic and anal-genital HIV-specific immune responses that were still detectable 5 months after immunization. Thus, aerosol vaccination with NYVAC and MVA vectored vaccines constitutes a tool for large-scale vaccine efforts against mucosally transmitted pathogens.

A new standard nomenclature for proteins related to Apx and Shroom.

Shroom is a recently-described regulator of cell shape changes in the developing nervous system. This protein is a member of a small family of related proteins that are defined by sequence similarity and in most cases by some link to the actin cytoskeleton. At present these proteins are named Shroom, APX, APXL, and KIAA1202. In light of the growing interest in this family of proteins, we propose here a new standard nomenclature.

Mucosal interplay among commensal and pathogenic bacteria: lessons from flagellin and Toll-like receptor 5.

Toll-like receptors (TLR) detect pathogen-associated molecular patterns (PAMP) and play a crucial role in triggering immunity. Due to their large surfaces in direct contact with the environment, mucosal tissues are the major sites of PAMP-TLR signalling. How innate and adaptive immunity are triggered through flagellin-TLR5 interaction is the main focus of the review. In view of recent reports on genetic polymorphism, we will summarize the impact of TLR5 on the susceptibility to mucosal infections and on various immuno-pathologies. Finally, the contribution of TLRs in the induction and maintenance of mucosal homeostasis and commensal discrimination is discussed.

Dendritic cells: the host Achille's heel for mucosal pathogens?

Mucosal surfaces represent the main sites of interaction with environmental microorganisms and antigens. Sentinel cells, including epithelial cells and dendritic cells (DCs), continuously sense the environment and coordinate defenses for the protection of mucosal tissues. DCs play a central role in the control of adaptive immune responses owing to their capacity to internalize foreign materials, to migrate into lymph nodes and to present antigens to naive lymphocytes. Some pathogenic microorganisms trigger epithelial responses that result in the recruitment of DCs. These pathogens hijack the recruited DCs to enable them to infect the host, escape the host's defense mechanisms and establish niches at remote sites.

How the gut links innate and adaptive immunity.

Mucosal surfaces represent the main sites in which environmental microorganisms and antigens interact with the host. Sentinel cells, including epithelial cells, lumenal macrophages, and intraepithelial dendritic cells, continuously sense the environment and coordinate defenses for the protection of mucosal tissues. The mucosal epithelial cells are crucial actors in coordinating defenses. They sense the outside world and respond to environmental signals by releasing chemokines and cytokines that recruit inflammatory and immune cells to control potential infectious agents and to attract cells able to trigger immune responses. Among immune cells, dendritic cells (DC) play a key role in controlling adaptive immune responses, due to their capacity to internalize foreign materials and to present antigens to naive T and B lymphocytes, locally or in draining organized lymphoid tissues. Immune cells recruited in epithelial tissues can, in turn, act upon the epithelial cells and change their phenotype in a process referred to as epithelial metaplasia.

Mucosal vaccines: where do we stand?

Mucosal vaccinology is a relatively young but rapidly expanding discipline. At present the vast majority of vaccines are administered by injection, including vaccines that protect against mucosally acquired pathogens such as influenza virus and human papilloma virus. However, mucosal immune responses are most efficiently induced by the administration of vaccines onto mucosal surfaces. The small number of currently licensed mucosal vaccines have reduced the burden of disease and mortality caused by enteric pathogens including rotavirus, V. cholerae and S. typhi, or those that spread to affect distal organs such as poliovirus. Expanding knowledge about the special features of the mucosal immune system promises to accelerate development of mucosal vaccines that could contribute significantly to protection against pathogens that colonize or invade via mucosal surfaces including HIV, Shigella, ETEC, Campylobacter jejuni, Helicobacter pylori and many others.

A European approach to clinical investigator training.

A better education and training of clinical investigators and their teams is one of the factors that could foster the development of clinical research in Europe, a key objective of the Innovative Medicines Initiative (IMI). PharmaTrain (an IMI programme on training in medicines development), and European Clinical Research Infrastructures Network (ECRIN) have joined forces to address this issue. An advisory group composed of representatives of universities, pharmaceutical companies and other organisations met four times between June 2011 and July 2012. This resulted in a position paper proposing a strategy to improve and harmonize clinical investigator training in Europe, and including a detailed syllabus and list of learning outcomes. Major recommendations are the establishment of minimal and mutually recognized certification requirement for investigators throughout the EU and the creation of a European platform to provide a suitable course and examination infrastructure.

An HIV-1 clade C DNA prime, NYVAC boost vaccine regimen induces reliable, polyfunctional, and long-lasting T cell responses.

The EuroVacc 02 phase I trial has evaluated the safety and immunogenicity of a prime-boost regimen comprising recombinant DNA and the poxvirus vector NYVAC, both expressing a common immunogen consisting of Env, Gag, Pol, and Nef polypeptide domain from human immunodeficiency virus (HIV)-1 clade C isolate, CN54. 40 volunteers were randomized to receive DNA C or nothing on day 0 and at week 4, followed by NYVAC C at weeks 20 and 24. The primary immunogenicity endpoints were measured at weeks 26 and 28 by the quantification of T cell responses using the interferon gamma enzyme-linked immunospot assay. Our results indicate that the DNA C plus NYVAC C vaccine regimen was highly immunogenic, as indicated by the detection of T cell responses in 90% of vaccinees and was superior to responses induced by NYVAC C alone (33% of responders). The vaccine-induced T cell responses were (a) vigorous in the case of the env response (mean 480 spot-forming units/10(6) mononuclear cells at weeks 26/28), (b) polyfunctional for both CD4 and CD8 T cell responses, (c) broad (the average number of epitopes was 4.2 per responder), and (d) durable (T cell responses were present in 70% of vaccinees at week 72). The vaccine-induced T cell responses were strongest and most frequently directed against Env (91% of vaccines), but smaller responses against Gag-Pol-Nef were also observed in 48% of vaccinees. These results support the development of the poxvirus platform in the HIV vaccine field and the further clinical development of the DNA C plus NYVAC C vaccine regimen.

EV01: a phase I trial in healthy HIV negative volunteers to evaluate a clade C HIV vaccine, NYVAC-C undertaken by the EuroVacc Consortium.

NYVAC-C (vP2010), a recombinant vector expressing HIV subtype C gag, pol, env and nef antigens, was tested in a phase I study in healthy, HIV negative volunteers in London and Lausanne. Twenty-four participants were randomised to receive NYVAC-C (20) or matching placebo (4) at weeks 0 and 4, and assessed for safety and immunogenicity over 48 weeks. There were no serious adverse events, and no clinical or laboratory abnormalities or other events that led to withdrawal, interruption or dose reduction of the NYVAC-C/placebo. Half of the 10 assessed responded in the ELISpot assay under stringent criteria, which informed the sample size for a DNA-NYVAC-C comparison to NYVAC-C alone.

Molecular and cellular basis of microflora-host interactions.

Mucosal surfaces represent the main sites in which environmental microorganisms and antigens interact with the host. In particular the intestinal mucosal surfaces are in continuous contact with a heterogeneous population of microorganisms of the endogenous flora and are exposed to food and microbes. As a result, the immune system of the host has to discriminate between pathogenic and commensal microorganisms. This article reviews the types of sentinel cells that continuously sense the environment and coordinate immune defenses as well as the mechanisms of the innate and adaptive immune systems that are activated by bacterial and viral molecular patterns leading to inflammatory, allergic, or regulatory immune response with special emphasis on probiotic bacteria.

Adhesin-dependent binding and uptake of Salmonella enterica serovar Typhimurium by dendritic cells.

Salmonella enterica serovar Typhimurium can be internalized by immature dendritic cells (DCs). The interacting host and bacterial molecules initiating this process remain uncharacterized. The objective of this study was to investigate whether specific fimbriae are involved in the early step of binding and uptake of Salmonella by DCs. Type 1 fimbriated S. enterica serovar Typhimurium or recombinant Escherichia coli expressing the type 1 fimbriae showed a significantly greater ability to attach to murine bone-marrow-derived DCs than non-fimbriated bacteria. The FimH adhesin was required for efficient interactions with DCs, since fimbriated fimH mutants were impaired in both binding and internalization. Finally, the internalization involved a FimH-dependent process but did not require sipB, a gene essential for Salmonella-mediated invasion of mammalian epithelial cells. Collectively, these data suggest that the bacterial interaction of DCs through the type 1 fimbrial adhesin FimH is sufficient to target S. enterica serovar Typhimurium for cellular uptake.

Novel markers of the human follicle-associated epithelium identified by genomic profiling and microdissection.

BACKGROUND & AIMS: Regulation of gene expression in the follicle-associated epithelium (FAE) over Peyer's patches is largely unknown. CCL20, a chemokine that recruits immature dendritic cells, is one of the few FAE-specific markers described so far. Lymphotoxin beta (LTalpha1beta2) expressed on the membrane of immune cells triggers CCL20 expression in enterocytes. In this study, we measured expression profiles of LTalpha1beta2-treated intestinal epithelial cells and selected CCL20 -coregulated genes to identify new FAE markers. METHODS: Genomic profiles of T84 and Caco-2 cell lines treated with either LTalpha1beta2, flagellin, or tumor necrosis factor alpha were measured using the Affymetrix GeneChip U133A. Clustering analysis was used to select CCL20 -coregulated genes, and laser dissection microscopy and real-time polymerase chain reaction on human biopsy specimens was used to assess the expression of the selected markers. RESULTS: Applying a 2-way analysis of variance, we identified regulated genes upon the different treatments. A subset of genes involved in inflammation and related to the nuclear factor kappaB pathway was coregulated with CCL20 . Among these genes, the antiapoptotic factor TNFAIP3 was highly expressed in the FAE. CCL23 , which was not coregulated in vitro with CCL20 , was also specifically expressed in the FAE. CONCLUSIONS: We have identified 2 novel human FAE specifically expressed genes. Most of the CCL20 -coregulated genes did not show FAE-specific expression, suggesting that other signaling pathways are critical to modulate FAE-specific gene expression.

Passive immunity in Helicobacter-challenged neonatal mice conferred by immunized dams lasts until weaning.

The objective of this study was to examine the effect of breast-feeding by immunized dams on Helicobacter colonization in newborns. Urease-based immunization regimens failed to protect nursing pups against H. felis, whereas H. felis lysate-cholera toxin resulted in protection. This observation correlated with a high recognition of cell surface-expressed bacterial antigens by milk antibodies. Protection lasted until weaning, indicating that infection is maintained at undetectable levels by passive immunity but then resumes when breast-feeding stops.

Lymphotoxin beta receptor signaling induces the chemokine CCL20 in intestinal epithelium.

BACKGROUND & AIMS: The follicle-associated epithelium (FAE) that overlies Peyer's patches (PPs) exhibits distinct features compared with the adjacent villus epithelium. Besides the presence of antigen-sampling membranous M cells and the down-regulation of digestive functions, it constitutively expresses the chemokine CCL20. The mechanisms that induce FAE differentiation and CCL20 expression are poorly understood. The aim of this work was to test whether lymphotoxin beta receptor signaling (LTbetaR), which plays a central role in PPs' organogenesis, mediates CCL20 gene expression in intestinal epithelial cells. METHODS: CCL20, lymphotoxin beta (LTbeta) and LTbetaR expression were monitored during embryonic development by in situ hybridization of mouse intestine. The human intestinal epithelial cell line T84 was used to study CCL20 expression following LTalpha(1)/beta(2) stimulation. In vivo CCL20 expression following agonistic anti-LTbetaR antibody treatment was studied by laser microdissection and quantitative RT-PCR. RESULTS: CCL20 was expressed in the FAE before birth at the time when the first hematopoietic CD4(+)CD3(-) appeared in the PP anlage. LTbetaR was expressed in the epithelium during PP organogenesis, making it a putative target for LTalpha(1)beta(2)signals. In vitro, CCL20 was induced in T84 cells upon LTbetaR signaling, either using an agonistic ligand or anti-LTbeta receptor agonistic antibody. LTalpha(1)beta(2)-induced CCL20 expression was found to be NF-kappaB dependent. LTbetaR signaling up-regulated CCL20 expression in the small intestinal epithelium in vivo. CONCLUSIONS: Our results show that LTbetaR signaling induces CCL20 expression in intestinal epithelial cells, suggesting that this pathway triggers constitutive production of CCL20 in the FAE.

Transepithelial transport of HIV-1 by M cells is receptor-mediated.

Human colon carcinoma Caco-2 cell monolayers undergo conversion into cells that share morphological and functional features of M cells when allowed to interact with B lymphocytes. A lymphotropic (X4) HIV-1 strain crosses M cell monolayers and infects underlying CD4(+) target cells. Transport requires both lactosyl cerebroside and CXCR4 receptors, which are expressed on the apical surface of Caco-2 and M cells. Antibodies specific for each receptor block transport. In contrast, a monotropic (R5) HIV-1 strain is unable to cross M cell monolayers and infect underlying monocytes, despite efficient transport of latex beads. Caco-2 and M cells do not express CCR5, but transfection of these cells with CCR5 cDNA restores transport of R5 virus, which demonstrates that HIV-1 transport across M cells is receptor-mediated. The follicle-associated epithelium covering human gut lymphoid follicles expresses CCR5, but not CXCR4, and lactosyl cerebroside, suggesting that HIV-1 infection may occur through M cells and enterocytes at these sites.