Application of phage display to high throughput antibody generation and characterization.

We have created a high quality phage display library containing over 1010 human antibodies and describe its use in the generation of antibodies on an unprecedented scale. We have selected, screened and sequenced over 38,000 recombinant antibodies to 292 antigens, yielding over 7,200 unique clones. 4,400 antibodies were characterized by specificity testing and detailed sequence analysis and the data/clones are available online. Sensitive detection was demonstrated in a bead based flow cytometry assay. Furthermore, positive staining by immunohistochemistry on tissue microarrays was found for 37% (143/381) of antibodies. Thus, we have demonstrated the potential of and illuminated the issues associated with genome-wide monoclonal antibody generation.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Diagnosis and Management of Iliac Artery Endofibrosis: Results of a Delphi Consensus Study

Objective Iliac endofibrosis is a rare condition that may result in a reduction of blood flow to the lower extremity in young, otherwise healthy individuals. The data to inform everyday clinical management are weak and therefore a Delphi consensus methodology was used to explore areas of consensus and disagreement concerning the diagnosis and management of patients with suspected iliac endofibrosis. Methods A three-round Delphi questionnaire approach was used among vascular surgeons, sports physicians, sports scientists, radiologists, and clinical vascular scientists with experience of treating this condition to explore diagnosis and clinical management issues for patients with suspected iliac artery endofibrosis. Analysis is based on 18 responses to round 2 and 14 responses to round 3, with agreement reported when 70% of respondents were in agreement. Results Initially there was agreement on the typical symptoms at presentation and the need for an exercise test in the diagnosis. Round 3 clarified that duplex ultrasound was a useful tool in the diagnosis of endofibrosis. There was consensus on the most appropriate type of surgery (endarterectomy and vein patch) and that endovascular interventions were inadvisable. The final round helped to inform aspects of the natural history and post-operative surveillance. Progression of the disease was likely with continued exercise but cessation may prevent progression. Surveillance after surgery is generally recommended yearly with at least a clinical assessment. Conclusions There is broad agreement about the presenting symptoms and the investigations required to confirm (or exclude) the diagnosis of iliac endofibrosis. There was consensus on the surgical approach to repair. Disagreement existed about the specific diagnostic criteria that should be applied during non-invasive testing and about post-operative care and resumption of exercise

Breeding oat for resistance to the crown rust pathogen Puccinia coronata f. sp. avenae: achievements and prospects

Crown rust, caused by Puccinia coronata f. sp. avenae (Pca), is a significant impediment to global oat production. Some 98 alleles at 92 loci conferring resistance to Pca in Avena have been designated; however, allelic relationships and chromosomal locations of many of these are unknown. Long-term monitoring of Pca in Australia, North America and elsewhere has shown that it is highly variable even in the absence of sexual recombination, likely due to large pathogen populations that cycle between wild oat communities and oat crops. Efforts to develop cultivars with genetic resistance to Pca began in the 1950s. Based almost solely on all all-stage resistance, this has had temporary benefits but very limited success. The inability to eradicate wild oats, and their common occurrence in many oat growing regions, means that future strategies to control Pca must be based on the assumption of a large and variable prevailing pathogen population with high evolutionary potential, even if cultivars with durable resistance are deployed and grown widely. The presence of minor gene, additive APR to Pca in hexaploid oat germplasm opens the possibility of pyramiding several such genes to give high levels of resistance. The recent availability of reference genomes for diploid and hexaploid oat will undoubtedly accelerate efforts to discover, characterise and develop high throughput diagnostic markers to introgress and pyramid resistance to Pca in high yielding adapted oat germplasm.Financial support from Judith and David Coffey and family, the Grains Research and Development Corporation (GRDC: DAS00133, UOS1707-003RTX, UOS2104-001RTX) and the University of Sydney is gratefully acknowledged. Some of the unpublished research reported on was undertaken as part of a long running program on national cereal rust surveillance, conducted at the University of Sydney since 1921. EP is funded by Spanish Ministry of Science and Innovation [PID2019-104518RB-100], (AEI/FEDER, UE) and regional government through the AGR-253 group, the European Regional and Social Development Funds.Peer reviewe

Evidence for the presence of multilineage chimerism and progenitors of donor dendritic cells in the peripheral blood of bone marrow-augmented organ transplant recipients

We have postulated that the donor leukocyte microchimerism plays a seminal role in the acceptance of allografts by inducing and perpetuating variable degree of donor-specific nonreactivity in long-surviving organ recipients. Limited information is available, however, concerning the phenotype and function of these chimeric cells in humans. The unequivocal presence of donor dendritic cells (DCs), a prominent lineage in the microchimerism observed in rodents and clinical organ recipients, was difficult to demonstrate in bone marrow (BM)-augmented organ transplant recipients. This enigma was resolved by the recent description of a method for propagating circulating human DCs from their progenitors by culture in a medium enriched with granulocyte-macrophage colony-stimulating factor and interleukin 4, a condition known to inhibit outgrowth of monocytes, thus providing a selective growth advantage to committed progenitors of the myeloid lineage. Cells from BM-augmented organ recipients and normal control subjects harvested from 12- to 14-day cultures exhibited dendritic morphology and potent allostimulatory capacity. Using appropriate primers, the presence of donor DNA was verified by polymerase chain reaction within the lineage(null)/class II(bright) sorted DC. Phenotypic analysis of cultured DCs from BM-augmented patients, unlike that of controls, exhibited a marked down- regulation of B7-1 (CD80) while retaining normal levels of expression of B7- 2 (CD86) cell surface molecules. The presence of donor DNA was also confirmed by polymerase chain reaction in individually sorted lineage+ (T, B, and NK) cells and macrophages, suggesting that the chimerism in BM-augmented patients is multilineage. The presence of progenitors of donor DCs in the peripheral blood of BM-augmented patients further substantiates the already convincing evidence of stem cell engraftment

Perfluorocarbon Particle Size Influences Magnetic Resonance Signal and Immunological Properties of Dendritic Cells

The development of cellular tracking by fluorine (19F) magnetic resonance imaging (MRI) has introduced a number of advantages for following immune cell therapies in vivo. These include improved signal selectivity and a possibility to correlate cells labeled with fluorine-rich particles with conventional anatomic proton (1H) imaging. While the optimization of the cellular labeling method is clearly important, the impact of labeling on cellular dynamics should be kept in mind. We show by 19F MR spectroscopy (MRS) that the efficiency in labeling cells of the murine immune system (dendritic cells) by perfluoro-15-crown-5-ether (PFCE) particles increases with increasing particle size (560>365>245>130 nm). Dendritic cells (DC) are professional antigen presenting cells and with respect to impact of PFCE particles on DC function, we observed that markers of maturation for these cells (CD80, CD86) were also significantly elevated following labeling with larger PFCE particles (560 nm). When labeled with these larger particles that also gave an optimal signal in MRS, DC presented whole antigen more robustly to CD8+ T cells than control cells. Our data suggest that increasing particle size is one important feature for optimizing cell labeling by PFCE particles, but may also present possible pitfalls such as alteration of the immunological status of these cells. Therefore depending on the clinical scenario in which the 19F-labeled cellular vaccines will be applied (cancer, autoimmune disease, transplantation), it will be interesting to monitor the fate of these cells in vivo in the relevant preclinical mouse models

A Novel DC Therapy with Manipulation of MKK6 Gene on Nickel Allergy in Mice

BACKGROUND: Although the activation of dermal dendritic cells (DCs) or Langerhans cells (LCs) via p38 mitogen-activated protein kinase (MAPK) plays a crucial role in the pathogenesis of metal allergy, the in vivo molecular mechanisms have not been identified and a possible therapeutic strategy using the control of dermal DCs or LCs has not been established. In this study, we focused on dermal DCs to define the in vivo mechanisms of metal allergy pathogenesis in a mouse nickel (Ni) allergy model. The effects of DC therapy on Ni allergic responses were also investigated. METHODS AND FINDING: The activation of dermal DCs via p38 MAPK triggered a T cell-mediated allergic immune response in this model. In the MAPK signaling cascade in DCs, Ni potently phosphorylated MAP kinase kinase 6 (MKK6) following increased DC activation. Ni-stimulated DCs could prime T cell activation to induce Ni allergy. Interestingly, when MKK6 gene-transfected DCs were transferred into the model mice, a more pronounced allergic reaction was observed. In addition, injection of short interfering (si) RNA targeting the MKK6 gene protected against a hypersensitivity reaction after Ni immunization. The cooperative action between T cell activation and MKK6-mediated DC activation by Ni played an important role in the development of Ni allergy. CONCLUSIONS: DC activation by Ni played an important role in the development of Ni allergy. Manipulating the MKK6 gene in DCs may be a good therapeutic strategy for dermal Ni allergy

Early Myeloid Dendritic Cell Dysregulation is Predictive of Disease Progression in Simian Immunodeficiency Virus Infection

Myeloid dendritic cells (mDC) are lost from blood in individuals with human immunodeficiency virus (HIV) infection but the mechanism for this loss and its relationship to disease progression are not known. We studied the mDC response in blood and lymph nodes of simian immunodeficiency virus (SIV)-infected rhesus macaques with different disease outcomes. Early changes in blood mDC number were inversely correlated with virus load and reflective of eventual disease outcome, as animals with stable infection that remained disease-free for more than one year had average increases in blood mDC of 200% over preinfection levels at virus set-point, whereas animals that progressed rapidly to AIDS had significant loss of mDC at this time. Short term antiretroviral therapy (ART) transiently reversed mDC loss in progressor animals, whereas discontinuation of ART resulted in a 3.5-fold increase in mDC over preinfection levels only in stable animals, approaching 10-fold in some cases. Progressive SIV infection was associated with increased CCR7 expression on blood mDC and an 8-fold increase in expression of CCL19 mRNA in lymph nodes, consistent with increased mDC recruitment. Paradoxically, lymph node mDC did not accumulate in progressive infection but rather died from caspase-8-dependent apoptosis that was reduced by ART, indicating that increased recruitment is offset by increased death. Lymph node mDC from both stable and progressor animals remained responsive to exogenous stimulation with a TLR7/8 agonist. These data suggest that mDC are mobilized in SIV infection but that an increase in the CCR7-CCL19 chemokine axis associated with high virus burden in progressive infection promotes exodus of activated mDC from blood into lymph nodes where they die from apoptosis. We suggest that inflamed lymph nodes serve as a sink for mDC through recruitment, activation and death that contributes to AIDS pathogenesis

The role of dendritic cells in the immunopathogenesis of psoriasis

Psoriasis vulgaris is a chronic inflammatory skin disease that is marked by a complex interplay of dendritic cells (DCs), T-cells, cytokines, and downstream transcription factors as part of a self-sustaining type 1 cytokine network. As integral players of the immune system, DCs represent antigen-presenting cells that are crucial for efficient activation of T-cells and B-cells. DCs have also been linked to distinct chronic inflammatory conditions, including psoriasis. In the setting of psoriasis therapy, DC/T cell interactions serve as a potential target for biologic response modifiers. Here we describe the major DC subsets as well as the immunologic involvement of DCs within the context of psoriatic lesions

Novel immunomodulators from hard ticks selectively reprogramme human dendritic cell responses

Hard ticks subvert the immune responses of their vertebrate hosts in order to feed for much longer periods than other blood-feeding ectoparasites; this may be one reason why they transmit perhaps the greatest diversity of pathogens of any arthropod vector. Tick-induced immunomodulation is mediated by salivary components, some of which neutralise elements of innate immunity or inhibit the development of adaptive immunity. As dendritic cells (DC) trigger and help to regulate adaptive immunity, they are an ideal target for immunomodulation. However, previously described immunoactive components of tick saliva are either highly promiscuous in their cellular and molecular targets or have limited effects on DC. Here we address the question of whether the largest and globally most important group of ticks (the ixodid metastriates) produce salivary molecules that specifically modulate DC activity. We used chromatography to isolate a salivary gland protein (Japanin) from Rhipicephalus appendiculatus ticks. Japanin was cloned, and recombinant protein was produced in a baculoviral expression system. We found that Japanin specifically reprogrammes DC responses to a wide variety of stimuli in vitro, radically altering their expression of co-stimulatory and co-inhibitory transmembrane molecules (measured by flow cytometry) and their secretion of pro-inflammatory, anti-inflammatory and T cell polarising cytokines (assessed by Luminex multiplex assays); it also inhibits the differentiation of DC from monocytes. Sequence alignments and enzymatic deglycosylation revealed Japanin to be a 17.7 kDa, N-glycosylated lipocalin. Using molecular cloning and database searches, we have identified a group of homologous proteins in R. appendiculatus and related species, three of which we have expressed and shown to possess DC-modulatory activity. All data were obtained using DC generated from at least four human blood donors, with rigorous statistical analysis. Our results suggest a previously unknown mechanism for parasite-induced subversion of adaptive immunity, one which may also facilitate pathogen transmission

Migratory Dermal Dendritic Cells Act as Rapid Sensors of Protozoan Parasites

Dendritic cells (DC), including those of the skin, act as sentinels for intruding microorganisms. In the epidermis, DC (termed Langerhans cells, LC) are sessile and screen their microenvironment through occasional movements of their dendrites. The spatio-temporal orchestration of antigen encounter by dermal DC (DDC) is not known. Since these cells are thought to be instrumental in the initiation of immune responses during infection, we investigated their behavior directly within their natural microenvironment using intravital two-photon microscopy. Surprisingly, we found that, under homeostatic conditions, DDC were highly motile, continuously crawling through the interstitial space in a Gαi protein-coupled receptor–dependent manner. However, within minutes after intradermal delivery of the protozoan parasite Leishmania major, DDC became immobile and incorporated multiple parasites into cytosolic vacuoles. Parasite uptake occurred through the extension of long, highly dynamic pseudopods capable of tracking and engulfing parasites. This was then followed by rapid dendrite retraction towards the cell body. DDC were proficient at discriminating between parasites and inert particles, and parasite uptake was independent of the presence of neutrophils. Together, our study has visualized the dynamics and microenvironmental context of parasite encounter by an innate immune cell subset during the initiation of the immune response. Our results uncover a unique migratory tissue surveillance program of DDC that ensures the rapid detection of pathogens