Objective Single-nucleotide polymorphisms (SNPs) have been identified in several genes encoding Toll-like receptors (TLRs) that alter immune function, inflammatory responses and disease susceptibility. The SNPs with best evidence for affecting immune function are TLR1 (1805GG), TLR2 (2258GA) and TLR5 (1174CT). Methods We studied the frequency and functional outcome of these polymorphisms in 248 patients with Lyme disease. Cytokine and chemokine levels were determined in serum of patients with erythema migrans (EM), joint fluid of patients with Lyme arthritis, and supernatants of B. burgdorferi-stimulated PBMC from Lyme arthritis patients, using multiplex assays. Results The frequency of TLR1-1805GG polymorphism was greater in patients with antibiotic-refractory arthritis compared with patients with EM or antibiotic-responsive arthritis. Early in the illness, EM patients with 1805GG, primarily those infected with B. burgdorferi RST1 strains, had higher serum levels of IFNγ, CXCL9 and CXCL10, and more severe infection than patients with 1805TG/TT. These inflammatory responses were amplified in patients with Lyme arthritis, and the highest responses were observed in antibiotic-refractory arthritis patients with 1805GG who had been infected with RST1 strains. When PBMC from Lyme arthritis patients were stimulated with a B. burgdorferi RST1 strain, the 1805GG group had significantly larger fold-increase in the levels of IFNγ, CCL2, CXCL9 and CXCL10, than the 1805TG/TT group. In contrast, the TLR2 and TLR5 polymorphisms did not vary among groups in frequency or function. Conclusion The TLR1-1805GG polymorphism in B. burgdorferi RST1-infected patients was associated with stronger TH1-like inflammatory responses, which may set the stage for antibiotic-refractory arthritis.

Objective. To investigate the possible role of chemokines and cytokines in the pathogenesis of Lyme arthritis.Methods. Using cytometric bead array and flow cytometry techniques, chemokine and cytokine levels were determined in 65 synovial fluid (SF) samples and 7 synovial tissue (ST) samples from 17 patients with antibiotic-responsive Lyme arthritis and 35 patients with antibiotic-refractory Lyme arthritis seen during the past 18 years. In the ST samples, expression of chemokine receptors was measured using immunohistochemistry.Results. Before or during antibiotic therapy, when the majority of patients had positive polymerase chain reaction (PCR) results for Borrelia burgdorferi DNA, SF from patients with antibiotic-refractory arthritis contained exceptionally high levels of Th1 chemoattractants and cytokines, particularly CXCL9 and interferon-␥ (IFN␥). Compared with the patients whose arthritis was responsive to antibiotic treatment, those with antibioticrefractory arthritis had significantly higher levels of CXCL9 and CXCL10 (both P < 0.001) and CCL3, CCL4, CXCL8, IFN␥, tumor necrosis factor ␣, interleukin-1␤ (IL-1␤), and IL-6 (all P < 0.01). During the post-antibiotic period, when the results of PCR for B burgdorferi DNA in SF and ST were uniformly negative, patients with antibiotic-refractory arthritis continued to exhibit high SF and ST levels of these chemokines and cytokines. In addition, synovial samples showed marked expression of the receptors for T cell or macrophage chemokines, CXCR3 and CCR5.Conclusion. Patients with antibiotic-refractory Lyme arthritis have high synovial fluid levels of proinflammatory chemokines and cytokines, especially CXCL9 and IFN␥, throughout the illness. Thus, even when antibiotic treatment reduces or completely clears the infection in these patients, the inflammatory response in synovium persists.

Objective. In a murine model of antibioticrefractory Lyme arthritis, the numbers of Treg cells are dramatically reduced. The aim of this study was to examine Treg cell numbers and function in patients with antibiotic-refractory Lyme arthritis.Methods. CD4؉ T cell subsets were enumerated in the peripheral blood (PB) and synovial fluid (SF) of 12 patients with antibiotic-refractory arthritis and 6 patients with antibiotic-responsive arthritis. Treg cell function was examined using Borrelia-specific and nonspecific Treg cell proliferation assays.Results. In both patient groups, interferon-␥-positive Th1 cells in SF were abundant and enriched (ϳ50% of CD4؉ T cells). In patients with antibioticrefractory arthritis, the median percentages of FoxP3-positive Treg cells were significantly higher in SF than in PB (12% versus 6%; P ‫؍‬ 0.03) or in SF from patients with antibiotic-responsive arthritis (12% versus 5%; P ‫؍‬ 0.04). Moreover, in the antibiotic-refractory group, a higher percentage of Treg cells in SF correlated with a shorter duration until resolution of arthritis (r ‫؍‬ ؊0.74, P ‫؍‬ 0.006). In contrast, patients with fewer Treg cells had suboptimal responses to disease-modifying antirheumatic drugs and a longer duration of arthritis after antibiotic treatment, and they often required synovectomies for arthritis resolution. In each group, Treg cells in SF dampened Borrelia burgdorferi-specific proliferative responses, and in 2 patients with antibioticrefractory arthritis, Treg cells were functional in nonspecific suppression assays.Conclusion. Treg cells were functional in patients with antibiotic-refractory arthritis, and in some patients, higher numbers of these cells in SF appeared to participate in arthritis resolution. However, as in the murine model, patients with antibiotic-refractory arthritis and lower numbers of Treg cells seemed unable to achieve resolution of synovial inflammation.

Completion of the sequencing of the Brassica rapa genome enabled us to undertake a genome-wide identification and functional study of the gene families related to the morphological diversity and agronomic traits of Brassica crops. In this study, we identified the auxin response factor (ARF) gene family, which is one of the key regulators of auxin-mediated plant growth and development in the B. rapa genome. A total of 31 ARF genes were identified in the genome. Phylogenetic and evolutionary analyses suggest that ARF genes fell into four major classes and were amplified in the B. rapa genome as a result of a recent whole genome triplication after speciation from Arabidopsis thaliana. Despite its recent hexaploid ancestry, B. rapa includes a relatively small number of ARF genes compared with the 23 members in A. thaliana, presumably due to a paralog reduction related to repetitive sequence insertion into promoter and non-coding transcribed region of the genes. Comparative genomic and mRNA sequencing analyses demonstrated that 27 of the 31 BrARF genes were transcriptionally active, and their expression was affected by either auxin treatment or floral development stage, although 4 genes were inactive, suggesting that the generation and pseudogenization of ARF members are likely to be an ongoing process. This study will provide a fundamental basis for the modification and evolution of the gene family after a polyploidy event, as well as a functional study of ARF genes in a polyploidy crop species.Electronic supplementary materialThe online version of this article (doi:10.1007/s00438-012-0718-4) contains supplementary material, which is available to authorized users.

The three skin disorders of Lyme borreliosis in Europe include erythema migrans, an acute, self-limited lesion; borrelial lymphocytoma, a subacute lesion; and acrodermatitis chronica atrophicans, a chronic lesion. Using quantitative reverse transcription-PCR, we determined mRNA expression of selected chemokines, cytokines, and leukocyte markers in skin samples from 100 patients with erythema migrans, borrelial lymphocytoma, or acrodermatitis chronica atrophicans and from 25 control subjects. Chemokine patterns in lesional skin in each of the three skin disorders included low but significant mRNA levels of the neutrophil chemoattractant CXCL1 and the dendritic cell chemoattractant CCL20 and intermediate levels of the macrophage chemoattractant CCL2. Erythema migrans and particularly acrodermatitis lesions had high mRNA expression of the T-cell-active chemokines CXCL9 and CXCL10 and low levels of the B-cell-active chemokine CXCL13, whereas lymphocytoma lesions had high levels of CXCL13 and lower levels of CXCL9 and CXCL10. This pattern of chemokine expression was consistent with leukocyte marker mRNA in lesional skin. Moreover, using immunohistologic methods, CD3؉ T cells and CXCL9 were visualized in erythema migrans and acrodermatitis lesions, and CD20؉ B cells and CXCL13 were seen in lymphocytoma lesions. Thus, erythema migrans and acrodermatitis chronica atrophicans have high levels of the T-cell-active chemokines CXCL9 and CXCL10, whereas borrelial lymphocytoma has high levels of the B-cell-active chemokine CXCL13.

BackgroundThe species Brassica rapa includes important vegetable and oil crops. It also serves as an excellent model system to study polyploidy-related genome evolution because of its paleohexaploid ancestry and its close evolutionary relationships with Arabidopsis thaliana and other Brassica species with larger genomes. Therefore, its genome sequence will be used to accelerate both basic research on genome evolution and applied research across the cultivated Brassica species.ResultsWe have determined and analyzed the sequence of B. rapa chromosome A3. We obtained 31.9 Mb of sequences, organized into nine contigs, which incorporated 348 overlapping BAC clones. Annotation revealed 7,058 protein-coding genes, with an average gene density of 4.6 kb per gene. Analysis of chromosome collinearity with the A. thaliana genome identified conserved synteny blocks encompassing the whole of the B. rapa chromosome A3 and sections of four A. thaliana chromosomes. The frequency of tandem duplication of genes differed between the conserved genome segments in B. rapa and A. thaliana, indicating differential rates of occurrence/retention of such duplicate copies of genes. Analysis of 'ancestral karyotype' genome building blocks enabled the development of a hypothetical model for the derivation of the B. rapa chromosome A3.ConclusionsWe report the near-complete chromosome sequence from a dicotyledonous crop species. This provides an example of the complexity of genome evolution following polyploidy. The high degree of contiguity afforded by the clone-by-clone approach provides a benchmark for the performance of whole genome shotgun approaches presently being applied in B. rapa and other species with complex genomes.

BackgroundMicroRNAs (miRNAs) are one of the functional non-coding small RNAs involved in the epigenetic control of the plant genome. Although plants contain both evolutionary conserved miRNAs and species-specific miRNAs within their genomes, computational methods often only identify evolutionary conserved miRNAs. The recent sequencing of the Brassica rapa genome enables us to identify miRNAs and their putative target genes. In this study, we sought to provide a more comprehensive prediction of B. rapa miRNAs based on high throughput small RNA deep sequencing.ResultsWe sequenced small RNAs from five types of tissue: seedlings, roots, petioles, leaves, and flowers. By analyzing 2.75 million unique reads that mapped to the B. rapa genome, we identified 216 novel and 196 conserved miRNAs that were predicted to target approximately 20% of the genome’s protein coding genes. Quantitative analysis of miRNAs from the five types of tissue revealed that novel miRNAs were expressed in diverse tissues but their expression levels were lower than those of the conserved miRNAs. Comparative analysis of the miRNAs between the B. rapa and Arabidopsis thaliana genomes demonstrated that redundant copies of conserved miRNAs in the B. rapa genome may have been deleted after whole genome triplication. Novel miRNA members seemed to have spontaneously arisen from the B. rapa and A. thaliana genomes, suggesting the species-specific expansion of miRNAs. We have made this data publicly available in a miRNA database of B. rapa called BraMRs. The database allows the user to retrieve miRNA sequences, their expression profiles, and a description of their target genes from the five tissue types investigated here.ConclusionsThis is the first report to identify novel miRNAs from Brassica crops using genome-wide high throughput techniques. The combination of computational methods and small RNA deep sequencing provides robust predictions of miRNAs in the genome. The finding of numerous novel miRNAs, many with few target genes and low expression levels, suggests the rapid evolution of miRNA genes. The development of a miRNA database, BraMRs, enables us to integrate miRNA identification, target prediction, and functional annotation of target genes. BraMRs will represent a valuable public resource with which to study the epigenetic control of B. rapa and other closely related Brassica species. The database is available at the following link: http://bramrs.rna.kr [1].

Low sL-selectin may be an early indicator of enhanced risk for BPD. Low levels of sL-selectin and increasing levels of sE-selectin may be risk factors for BPD. The effects of dexamethasone treatment include significant modulation of adhesion molecules.