Cryptic Speciation in Western North America and Eastern Eurasia of the Pathogens Responsible for Laminated Root Rot.

Coniferiporia sulphurascens is a facultative fungal pathogen that causes laminated root rot (LRR) in commercially important coniferous species worldwide. This fungus spreads primarily by way of vegetative mycelium transferring at points of contact between infected and healthy roots. Successful intervention to control LRR requires a better understanding of the population structure and genetic variability of C. sulphurascens. In this study, we investigated the population genetic structure and origin of C. sulphurascens populations in western North America and eastern Eurasia collected from multiple coniferous hosts. By analyzing the small and large mitochondrial ribosomal RNA subunit genes combined with six nuclear loci (internal transcribed spacer region, actin, RNA polymerase II largest subunit, RNA polymerase II second-largest subunit, laccase-like multicopper oxidase, and translation elongation factor 1-α), we observed that none of the alleles among the loci were shared between North American (NA) and Eurasian C. sulphurascens populations. In total, 55 multilocus genotypes (MLGs) were retrieved in C. sulphurascens isolates occurring in these two continental regions. Of these, 41 MLGs were observed among 58 isolates collected from widespread locations in British Columbia (Canada) and the northwestern United States, while 14 MLGs were observed among 16 isolates sampled in Siberia and Japan. Our data showed that the levels of genetic differentiation between the NA and Eurasian populations are much greater than the populations from within each continental region; the two continental populations formed clearly divergent phylogenetic clades or lineages since they were separated approximately 7.5 million years ago. Moreover, the Eurasian population could be the source of the NA population. Our study indicates the existence of cryptic diversity in this pathogen species, and strongly suggests that the NA and Eurasian populations represent two lineages, which have progressively diverged from each other in allopatry.

Elucidating the chromosome 9 association with AS; CARD9 is a candidate gene.

Ankylosing spondylitis (AS) is polygenic with contributions from the immunologically relevant genes HLA-B*27, ERAP1 and IL23R. A recent genome-wide association screen (GWAS) identified associations (P approximately 0.005) with the non-synonymous single-nucleotide polymorphisms (nsSNPs), rs4077515 and rs3812571, in caspase recruitment domain-containing protein 9 (CARD9) and small nuclear RNA-activating complex polypeptide 4 (SNAPC4) on chromosome 9q that had previously been linked to AS. We replicated these associations in a study of 730 AS patients compared with 2879 historic disease controls (rs4077515 P=0.0004, odds ratio (OR)=1.2, 95% confidence interval (CI)=1.1-1.4; rs3812571 P=0.0003, OR=1.2, 95% CI=1.1-1.4). Meta-analysis revealed strong associations of both SNPs with AS, rs4077515 P=0.000005, OR=1.2, 95% CI=1.1-1.3 and rs3812571 P=0.000006, OR=1.2, 95% CI=1.1-1.3. We then typed 1604 AS cases and 1020 controls for 13 tagging SNPs; 6 showed at least nominal association, 5 of which were in CARD9. We imputed genotypes for 13 additional SNPs but none was more strongly associated with AS than the tagging SNPs. Finally, interrogation of an mRNA expression database revealed that the SNPs most strongly associated with AS (or in strong linkage disequilibrium) were those most associated with CARD9 expression. CARD9 is a plausible candidate for AS given its central role in the innate immune response.

Identification, characterization, and expression analyses of class II and IV chitinase genes from Douglas-fir seedlings infected by Phellinus sulphurascens.

Laminated root rot (LRR) disease, caused by the fungus Phellinus sulphurascens, is a major threat to coastal Douglas-fir (DF) (Pseudotsuga menziesii) forests in western North America. Understanding host-pathogen interactions of this pathosystem is essential to manage this important conifer root disease. Our research objectives were to identify DF pathogenesis-related (PR) genes and analyze their expression patterns over the course of infection. We constructed a cDNA library of Phellinus sulphurascens-infected DF seedling roots and sequenced a total of 3,600 random cDNA clones from this library. One of the largest groups of identified genes (203 cDNA clones) matched with chitinase genes reported in other plant species. We identified at least three class II and six class IV chitinase genes from DF seedlings. Quantitative reverse-transcriptase polymerase chain reaction analyses showed significant differential expression patterns locally in root tissues and systemically in needle tissues after fungal invasion. Nonetheless, there was a common trend in gene expression patterns for most of the chitinase genes: an upregulation within 12 h of pathogen inoculation followed by down-regulation within 2 to 3 days postinoculation (dpi), and then further upregulation within 5 to 7 dpi. Western immunoblot data showed differential accumulation of class IV chitinases in Phellinus sulphurascens-infected DF seedlings. Further detailed functional analyses will help us to understand the specific role of DF chitinases in defense against Phellinus sulphurascens infection.

Comparison of cardiovascular risk in ankylosing spondylitis and rheumatoid arthritis.

Cardiovascular co-morbidity is now a recognised complication of chronic inflammation and an elevated acute phase response predisposes to hypertension, stroke and myocardial infarction. Dyslipidaemia is a feature of inflammatory joint diseases and is closely related to elevated CRP and Il-6 levels. Rheumatoid arthritis (RA) has an increased standardised mortality ratio largely attributable to cardiovascular risk. An increased although lesser, cardiovascular morbidity has also been observed in ankylosing spondylitis (AS) which has a similar abnormal lipid profile to that seen in RA. There is some evidence that therapeutic agents such as anti-tumour necrosis factor-alpha (TNF-alpha) drugs that down-regulate the acute phase response, also have an effect in reducing cardiovascular complications in RA and AS.

The development of Assessment of SpondyloArthritis international Society classification criteria for axial spondyloarthritis (part I): classification of paper patients by expert opinion including uncertainty appraisal.

OBJECTIVE: Non-radiographic axial spondyloarthritis (SpA) is characterised by a lack of definitive radiographic sacroiliitis and is considered an early stage of ankylosing spondylitis. The objective of this study was to develop candidate classification criteria for axial SpA that include patients with but also without radiographic sacroiliitis. METHODS: Seventy-one patients with possible axial SpA, most of whom were lacking definite radiographic sacroiliitis, were reviewed as "paper patients" by 20 experts from the Assessment of SpondyloArthritis international Society (ASAS). Unequivocally classifiable patients were identified based on the aggregate expert opinion in conjunction with the expert-reported level of certainty of their judgement. Draft criteria for axial SpA were formulated and tested using classifiable patients. RESULTS: Active sacroiliitis on magnetic resonance imaging (MRI) (odds ratio 45, 95% CI 5.3 to 383; p<0.001) was strongly associated with the classification of axial SpA. The knowledge of MRI findings led to a change in the classification of 21.1% of patients. According to the first set of candidate criteria (sensitivity 97.1%; specificity 94.7%) a patient with chronic back pain is classified as axial SpA in the presence of sacroiliitis by MRI or x rays in conjunction with one SpA feature or, if sacroilitiis is absent, in the presence of at least three SpA features. In a second set of candidate criteria, inflammatory back pain is obligatory in the clinical arm (sensitivity 86.1%; specificity 94.7%). CONCLUSION: The ASAS group has developed candidate criteria for the classification of axial SpA that include patients without radiographic sacroiliitis. The candidate criteria need to be validated in an independent international study.

Ultrastructural studies of Phellinus sulphurascens infection of Douglas-fir roots and immunolocalization of host pathogenesis-related proteins.

Interactions between roots of Douglas-fir (DF; Pseudotsuga menziesii) seedlings and the laminated root rot fungus Phellinus sulphurascens were investigated using scanning and transmission electron microscopy and immunogold labelling techniques. Scanning electron micrographs revealed that P. sulphurascens hyphae colonize root surfaces and initiate the penetration of root epidermal tissues by developing appressoria within 2 d postinoculation (dpi). During early colonization, intra- and intercellular fungal hyphae were detected. They efficiently disintegrate cellular components of the host including cell walls and membranes. P. sulphurascens hyphae penetrate host cell walls by forming narrow hyphal tips and a variety of haustoria-like structures which may play important roles in pathogenic interactions. Ovomucoid-WGA (wheat germ agglutinin) conjugated gold particles (10 nm) confirmed the occurrence and location of P. sulphurascens hyphae, while four specific host pathogenesis-related (PR) protein antibodies conjugated with protein A-gold complex (20 nm) showed the localization and abundance of these PR proteins in infected root tissues. A thaumatin-like protein and an endochitinase-like protein were both strongly evident and localized in host cell membranes. A DF-PR10 protein was localized in the cell walls and cytoplasm of host cells while an antimicrobial peptide occurred in host cell walls. A close association of some PR proteins with P. sulphurascens hyphae suggests their potential antifungal activities in DF roots.

Angiotensin II type 1 receptor as a novel therapeutic target in rheumatoid arthritis: in vivo analyses in rodent models of arthritis and ex vivo analyses in human inflammatory synovitis.

OBJECTIVE: Angiotensin II (Ang II) is known to have proinflammatory actions, and Ang II type 1 (AT(1)) receptors are up-regulated in the rheumatoid synovium, suggesting that this receptor could be a therapeutic target. The purpose of this study was to investigate the antiinflammatory potential of the selective AT(1) receptor antagonist losartan, which is currently used for the treatment of cardiovascular disease. METHODS: Dose-ranging studies of losartan (1-50 mg/kg) were initially conducted in a rat model of acute (carrageenan/kaolin) arthritis, with subsequent evaluation in a rat model of adjuvant-induced arthritis (Freund's complete adjuvant). Losartan (10(-10) to 10(-6)M) was further tested ex vivo in human inflammatory synovitis, using collagenase-digested synovium. RESULTS: Western blot and immunohistochemical analyses both revealed a substantial increase in AT(1) receptor protein content in synovium from acutely and chronically inflamed rat knee joints. Similarly, synovial Ang I/II protein content was elevated during inflammation. Losartan inhibited acute joint inflammation in a dose-dependent manner, with 15 mg/kg being the optimal dose (and used in subsequent studies). Both prophylactic and therapeutic administration of 15 mg/kg of losartan substantially reduced knee joint swelling in rats with adjuvant monarthritis (> or =50%; P < 0.0001). Losartan also suppressed tumor necrosis factor alpha generation from inflamed human synovium in a dose-dependent manner (P < 0.05). CONCLUSION: Targeting the angiotensin pathway, particularly AT(1) receptors, could have significant therapeutic potential. Randomized placebo-controlled trials are now warranted to establish the extent to which angiotensin receptor blockers may provide antiinflammatory benefits.

Host-Pathogen Interactions in Douglas-Fir Seedlings Infected by Phellinus sulphurascens.

ABSTRACT Several aspects of the host-pathogen interaction between Douglas-fir (Pseudotsuga menziesii) and the fungal pathogen Phellinus sulphurascens were investigated in an in vitro inoculation system using young seedlings and fungal mycelia. Light microscopy confirmed that P. sulphurascens mycelia can successfully penetrate host epidermal cells within 3 days postinoculation (dpi). Extensive fungal colonization and cortical cell decay occurred within 14 dpi. Western immunoblot studies showed significant upregulation (five to sixfold) of four specific pathogenesis-related (PR) proteins in infected roots. These proteins were a Douglas-fir thaumatin-like protein (PmTLP), an endochitinase protein (ECP), a Douglas-fir PR10 (DF-PR10) protein (PsemI), and a 10.6-kDa antimicrobial peptide (PmAMP1). The highest accumulation of PmTLP and PmAMP1 occurred at 12 dpi, whereas accumulations of the ECP and DF-PR10 proteins peaked at 7 dpi. For both inoculated and control Douglas-fir seedlings, only one of the four PR proteins, PmAMP1, was clearly detectable in needles. Immunolocalization experiments using fluorescein isothiocyanate-conjugated secondary antibodies confirmed accumulation of all four PR proteins mainly in and around cell walls of root cortical tissues. Overall, the highest immunofluorescence was observed in infected roots at 12 dpi, whereas labeling in control roots was negligible at all sample times. The ECP produced the highest fluorescence; the DF-PR10 the lowest. Upregulation and localization of these PR proteins in cortical tissues of inoculated roots suggest that they play a defensive role in response to infection by P. sulphurascens. This in vitro inoculation system will facilitate further proteomic and genomic studies of this important pathosystem.

TLR4 mutations (Asp299Gly and Thr399Ile) are not associated with ankylosing spondylitis.

BACKGROUND: Immunoregulatory genes and Gram negative gut bacteria are thought to be important in disease expression in ankylosing spondylitis (AS). OBJECTIVE: To compare the frequency of two common and functional TLR4 mutations (Asp299Gly, and Thr399Ile) between patients with AS and HLA-B27 healthy controls. METHODS: The TLR4 genotypes of patients and healthy HLA-B27 controls were determined using allele-specific PCR and restriction fragment length polymorphism analysis. Asp299Gly genotype was determined in 193 patients and 125 HLA-B27 positive controls and Thr399Ile genotype in 184 patients and 113 HLA-B27 controls. Allele frequencies were compared using a chi(2) test of association. RESULTS: 29/193 (15%) patients with AS had a polymorphism in the Asp299 site compared with 18/125 (14.4%) healthy HLA-B27 controls. Of the patients genotyped for the Thr399Ile allele, 29/184 (15.8%) carried the polymorphism compared with 19/113 (16.8%) HLA-B27 controls. No significant difference between the frequencies of the Asp299Gly genotype or the Thr399Ile genotype between patients with AS and healthy HLA-B27 controls was found. No significant difference in allele frequency was found at either site. CONCLUSION: Two common TLR4 polymorphisms, which cause a functional deficiency in host immune response to Gram negative bacteria, are not overrepresented in patients with AS.

ASAS/EULAR recommendations for the management of ankylosing spondylitis.

OBJECTIVE: To develop evidence based recommendations for the management of ankylosing spondylitis (AS) as a combined effort of the 'ASsessment in AS' international working group and the European League Against Rheumatism. METHODS: Each of the 22 participants was asked to contribute up to 15 propositions describing key clinical aspects of AS management. A Delphi process was used to select 10 final propositions. A systematic literature search was then performed to obtain scientific evidence for each proposition. Outcome data for efficacy, adverse effects, and cost effectiveness were abstracted. The effect size, relative risk, number needed to treat, and incremental cost effectiveness ratio were calculated. On the basis of the search results, 10 major recommendations for the management of AS were constructed. The strength of recommendation was assessed based on the strength of the literature evidence, risk-benefit trade-off, and clinical expertise. RESULTS: The final recommendations considered the use of non-steroidal anti-inflammatory drugs (NSAIDs) (conventional NSAIDs, coxibs, and co-prescription of gastroprotective agents), disease modifying antirheumatic drugs, treatments with biological agents, simple analgesics, local and systemic steroids, non-pharmacological treatment (including education, exercise, and physiotherapy), and surgical interventions. Three general recommendations were also included. Research evidence (categories I-IV) supported 11 interventions in the treatment of AS. Strength of recommendation varied, depending on the category of evidence and expert opinion. CONCLUSION: Ten key recommendations for the treatment of AS were developed and assessed using a combination of research based evidence and expert consensus. Regular updating will be carried out to keep abreast of new developments in the management of AS.

Musculoskeletal ultrasound--a state of the art review in rheumatology. Part 1: Current controversies and issues in the development of musculoskeletal ultrasound in rheumatology.

As we begin the 21st century, musculoskeletal ultrasound (MSUS) is routinely used by an increasing number of rheumatologists throughout Europe and there is a growing interest in the application of MSUS in rheumatological practice in the UK. MSUS allows high-resolution, real-time imaging of articular and periarticular structures and has the advantages of being non-radioactive, inexpensive, portable, highly acceptable to patients and repeatable. There are a number of critical issues that need to be addressed in order to develop the role of MSUS within rheumatology. These include issues of equipment costs, training and certification and the relationship of rheumatologists and radiologists in advancing the field of MSUS. Rheumatologists must demonstrate the relevance of MSUS in their clinical practice through high-quality research. Emerging technologies such as power Doppler and 3D imaging will further improve imaging capabilities and the range of clinical applications of MSUS systems. This paper reviews how MSUS in rheumatology has evolved and the controversies and issues that rheumatologists must now address in developing MSUS as an indispensable, everyday clinical tool.

Musculoskeletal ultrasound--a state of the art review in rheumatology. Part 2: Clinical indications for musculoskeletal ultrasound in rheumatology.

Rheumatologists remain divided on whether they should introduce musculoskeletal ultrasound (MSUS) into their clinical practice. A central issue in the application of MSUS in clinical rheumatology is the need for proof of clinical relevance and improved patient care. There is now accumulating evidence that MSUS improves clinical diagnosis and intervention skills. High-resolution ultrasound is superior to clinical examination in the diagnosis and localization of joint and bursal effusion and synovitis. MSUS is the imaging modality of choice for the diagnosis of tendon pathology. MSUS is seven times more sensitive than plain radiography in the detection of rheumatoid erosions, allowing earlier diagnosis of progressive rheumatoid arthritis. Ligament, muscle, peripheral nerve and cartilage pathology can also be readily demonstrated by MSUS. There is exciting evidence that MSUS may potentially be used by rheumatologists to non-invasively diagnose and monitor not just joint and muscle disease but also nerve compression syndromes, scleroderma, vasculitis and Sjögren's syndrome. Joint aspiration and injection accuracy can be improved by MSUS, with initial evidence confirming improved efficacy. As the number of rheumatologists performing MSUS increases and the technical capabilities of MSUS improve, there is likely to be a growing number of proven clinical indications for the application of MSUS in rheumatology practice. This paper reviews the evidence for the application of MSUS in rheumatology.

The detection of antibodies against Schistosoma mansoni soluble egg antigens (SEA) and CEF6 in ELISA, before and after chemotherapy.

Circulating IgG antibody reactivity and excreted egg counts were investigated in 489 Kenyans given chemotherapy for schistosomiasis mansoni. Antibody reactivity was measured in ELISA, using either unfractionated aqueous soluble constituents of Schistosoma mansoni eggs (SEA) or CEF6 (a soluble fraction of S. mansoni eggs containing two cationic antigens) as the antigen source. Antibody reactivity for each antigen source was strongly associated with egg counts, both pre- and post-treatment. Approximately 6 months after chemotherapy, egg counts were zero in 84% of the subjects. The mean optical densities (OD) measured in the post-treatment ELISA were 60% (CEF6) or 45% (SEA) lower than the pre-treatment values, the reduction in the OD with CEF6 as antigen source being significantly greater than that observed with SEA (P <0.001). The usefulness of an assay for antibody reactivity in monitoring the effects of the treatment of schistosomiasis is discussed.