Falko Steinbach

Dendritic cells: from ontogenetic orphans to myelomonocytic descendants

Abstract Although dendritic cells (DCs) and macrophages share a bone marrow origin, these cells were long assumed to differentiate via discrete pathways. DCs have now been clearly shown to develop from myeloid lineage precursors, and recent evidence suggests that they may even differentiate from blood monocytes. Here, J. Hinrich Peters and colleagues assess current knowledge on the myelomonocytic origin and successive differentiation of human T-cell-directed DCs.

Monocytes from systemic lupus erythematous patients are severely altered in phenotype and lineage flexibility

OBJECTIVE Cells of the myeloid lineage comprise a very heterogeneous population with many phenotypes and functional activities including macrophages and dendritic cells. To investigate the status, differentiative potential and lineage commitment of monocytic cells in systemic lupus erythematosus (SLE) patients, this study isolated and cultured peripheral blood monocytes from patients and healthy donors. METHODS Monocytes were isolated by gradient centrifugation and adherence to plastic dishes. The cells were then cultured for three days, partially supplemented with GM-CSF and interleukin 4 (IL4) to obtain dendritic cells. The differentiation status was monitored by the expression of surface markers using flow cytometry and cytokine secretion. RESULTS Monocytes from SLE patients expressed significantly lower numbers of the monocytic marker CD14 and HLA-DR while secreting significantly more tumour necrosis factor α (TNFα) than monocytes from healthy donors. The addition of GM-CSF and IL4 resulted in an inhibition of TNFα secretion, but was not sufficient to generate monocytederived dendritic cells. CONCLUSION Monocytes from SLE patients are severely altered in phenotype and function and have a limited differentiation flexibility towards the accessory type of monocytic cells.

Development of a Langerhans cell phenotype from peripheral blood monocytes

Epidermal Langerhans cells (ELC) are definitively primed to differentiate into dendritic cells (DC). It is unknown at what stage of monocyte development this priming occurs. In a culture system characterized by low paracrine stimulation, i.e. Iscoves modified Dulbecco medium (IMDM) with 2% FCS, we tested the ability of peripheral blood monocytes to turn to the route of the LC-DC lineage. In this system monocytes did not develop significant yeast cell phagocytosis, although mannose receptors were available. However, they became strong stimulators of mannan specific T cell proliferation. Phenotype development was analysed by flow cytometry using the monoclonal antibodies OKT6 (CD1a), IOT2 (HLA-DR), IOM2 (CD14) and the ligand Man-BSA-FITC. CD1a was the first marker which distinguished cultured monocytes from developing macrophages, obtained by addition of 8% human serum. Like cord blood Langerhans cells (CBLC) they internalized OKT6 in deep coated pits. They maintained a phenotype of monocyte derived Langerhans cells (MoLC) during eight days of in vitro culture, expressing CD1a, mannose receptors and HLA-DR and decreasing CD14, if left in their own conditioned medium. MoLC could be converted into macrophages by addition of human serum only within the first four days in vitro. Our data suggest that monocytes acquire an LC phenotype by autocrine stimulation.

Increased pathogenicity of European porcine reproductive and respiratory syndrome virus is associated with enhanced adaptive responses and viral clearance

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases of swine worldwide. Since its first emergence in 1987 the PRRS virus (PRRSV) has become particularly divergent with highly pathogenic strains appearing in both Europe and Asia. However, the underlying mechanisms of PRRSV pathogenesis are still unclear. This study sets out to determine the differences in pathogenesis between subtype 1 and 3 strains of European PRRSV (PRRSV-I), and compare the immune responses mounted against these strains. Piglets were infected with 3 strains of PRRSV-I: Lelystad virus, 215-06 a British field strain and SU1-bel from Belarus. Post-mortem examinations were performed at 3 and 7 days post-infection (dpi), and half of the remaining animals in each group were inoculated with an Aujeszkys disease (ADV) vaccine to investigate possible immune suppression resulting from PRRSV infection. The subtype 3 SU1-bel strain displayed greater clinical signs and lung gross pathology scores compared with the subtype 1 strains. This difference did not appear to be caused by higher virus replication, as viraemia and viral load in broncho-alveolar lavage fluid (BALF) were lower in the SU1-bel group. Infection with SU1-bel induced an enhanced adaptive immune response with greater interferon (IFN)-γ responses and an earlier PRRSV-specific antibody response. Infection with PRRSV did not affect the response to vaccination against ADV. Our results indicate that the increased clinical and pathological effect of the SU1-bel strain is more likely to be caused by an enhanced inflammatory immune response rather than higher levels of virus replication.

Challenge of pigs with classical swine fever viruses after C-strain vaccination reveals remarkably rapid protection and insights into early immunity.

Pre-emptive culling is becoming increasingly questioned as a means of controlling animal diseases, including classical swine fever (CSF). This has prompted discussions on the use of emergency vaccination to control future CSF outbreaks in domestic pigs. Despite a long history of safe use in endemic areas, there is a paucity of data on aspects important to emergency strategies, such as how rapidly CSFV vaccines would protect against transmission, and if this protection is equivalent for all viral genotypes, including highly divergent genotype 3 strains. To evaluate these questions, pigs were vaccinated with the Riemser® C-strain vaccine at 1, 3 and 5 days prior to challenge with genotype 2.1 and 3.3 challenge strains. The vaccine provided equivalent protection against clinical disease caused by for the two challenge strains and, as expected, protection was complete at 5 days post-vaccination. Substantial protection was achieved after 3 days, which was sufficient to prevent transmission of the 3.3 strain to animals in direct contact. Even by one day post-vaccination approximately half the animals were partially protected, and were able to control the infection, indicating that a reduction of the infectious potential is achieved very rapidly after vaccination. There was a close temporal correlation between T cell IFN-γ responses and protection. Interestingly, compared to responses of animals challenged 5 days after vaccination, challenge of animals 3 or 1 days post-vaccination resulted in impaired vaccine-induced T cell responses. This, together with the failure to detect a T cell IFN-γ response in unprotected and unvaccinated animals, indicates that virulent CSFV can inhibit the potent antiviral host defences primed by C-strain in the early period post vaccination.

Prevalence of Hepatitis E Virus Infection in Pigs at the Time of Slaughter, United Kingdom, 2013.

Pigs raised in the United Kingdom are unlikely to be the source of UK human infections.

Complete Genome Sequences of Elephant Endotheliotropic Herpesviruses 1A and 1B Determined Directly from Fatal Cases

ABSTRACT A highly lethal hemorrhagic disease associated with infection by elephant endotheliotropic herpesvirus (EEHV) poses a severe threat to Asian elephant husbandry. We have used high-throughput methods to sequence the genomes of the two genotypes that are involved in most fatalities, namely, EEHV1A and EEHV1B (species Elephantid herpesvirus 1, genus Proboscivirus, subfamily Betaherpesvirinae, family Herpesviridae). The sequences were determined from postmortem tissue samples, despite the data containing tiny proportions of viral reads among reads from a host for which the genome sequence was not available. The EEHV1A genome is 180,421 bp in size and consists of a unique sequence (174,601 bp) flanked by a terminal direct repeat (2,910 bp). The genome contains 116 predicted protein-coding genes, of which six are fragmented, and seven paralogous gene families are present. The EEHV1B genome is very similar to that of EEHV1A in structure, size, and gene layout. Half of the EEHV1A genes lack orthologs in other members of subfamily Betaherpesvirinae, such as human cytomegalovirus (genus Cytomegalovirus) and human herpesvirus 6A (genus Roseolovirus). Notable among these are 23 genes encoding type 3 membrane proteins containing seven transmembrane domains (the 7TM family) and seven genes encoding related type 2 membrane proteins (the EE50 family). The EE50 family appears to be under intense evolutionary selection, as it is highly diverged between the two genotypes, exhibits evidence of sequence duplications or deletions, and contains several fragmented genes. The availability of the genome sequences will facilitate future research on the epidemiology, pathogenesis, diagnosis, and treatment of EEHV-associated disease.

IL-4 activates equine neutrophils and induces a mixed inflammatory cytokine expression profile with enhanced neutrophil chemotactic mediator release ex vivo

Neutrophils are potent contributors to the lung pathophysiological changes occurring in allergic airway inflammation, which typically involve T helper type 2 (Th2) cytokine overexpression. We have previously reported that equine pulmonary endothelial cells are activated by the Th2 cytokine IL-4 and express chemotactic factors for neutrophils after stimulation. We have further explored the possible mechanisms linking Th2-driven inflammation and neutrophilia by studying the effects of recombinant equine IL-4, a prototypical Th2 cytokine, on peripheral blood neutrophils (PBN) isolated from normal animals and from horses with asthmatic airway inflammation (equine heaves). We found that IL-4 induced morphological changes in PBN, dose- and time-dependent expression of IL-8 mRNA, as well as the release of chemotactic factors for neutrophils in culture supernatants. Also, IL-4 induced a mixed inflammatory response in PBN from control and asthmatic-animals with increased expression of proinflammatory IL-8 and TNF-α but a marked inhibition of IL-1β. IL-4 type I receptor (IL-4Rα) and CD23 (FcεRII) expression were also upregulated by IL-4. Importantly, disease as well as chronic antigenic exposure modified gene expression by PBN. Finally, we found that activation of equine neutrophils with IL-4 involved STAT6 phosphorylation and p38 MAPK and phosphatidylinositol 3-kinase (PI3K); the pharmacological inhibitors, SB-203580 and LY-294002, respectively, significantly reversed IL-4-induced gene modulation in PBN. Overall, results from this study add to the link between Th2-driven inflammation and neutrophilia in the equine model and further extend the characterization of IL-4 effects on neutrophils.

Monocyte-derived dendritic cells from horses differ from dendritic cells of humans and mice.

Dendritic cells (DC) are the initiators of immune responses and are present in most tissues in vivo. To generate myeloid DC from monocytes (MoDC) in vitro the necessary cytokines are granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and interleukin‐4 (IL‐4). Using degenerated primers delineated from other species and rapid amplification of cDNA ends reverse transcription–polymerase chain reaction (RACE RT‐PCR), the cDNA of equine (eq.) GM‐CSF was cloned and found to have a point deletion at the 3′‐end of eq.GM‐CSF, resulting in a 24‐nucleotide extended open reading frame not described in any species thus far. For differentiating eq.MoDC, monocytes were stimulated with eq.GM‐CSF and eq.IL‐4. The eq.MoDC was analysed by both light and electron microscopy and by flow cytometry and mixed lymphocyte reaction. The eq.MoDC obtained had the typical morphology and function of DC, including the ability to stimulate allogeneic T cells in a mixed lymphocyte reaction. In contrast to the human system, however, monocytes had to be differentiated for 6–7 days before immature DC were obtained. Our data also indicate that lipopolysaccharide or poly(I:C) alone are not sufficient to confer the full phenotypic transition into mature DC. Thus our study contributes to understanding the heterogeneity of immunity and adds important information on the equine immune system, which is clearly distinct from those of mice or man.

Incursion of RHDV2-like variant in Great Britain.

RABBIT haemorrhagic disease virus (RHDV) causes an acute, fulminating and generally fatal disease in the European rabbit ( Oryctolagus cuniculus ). It was first discovered in China in 1984 and then confirmed in the UK in 1992, at …