A genetic variant in osteoprotegerin is associated with progression of joint destruction in rheumatoid arthritis

Introduction

Progression of joint destruction in rheumatoid arthritis (RA) is partly heritably; 45 to 58% of the variance in joint destruction is estimated to be explained by genetic factors. The binding of RANKL (Receptor Activator for Nuclear Factor κ B Ligand) to RANK results in the activation of TRAF6 (tumor necrosis factor (TNF) receptor associated factor-6), and osteoclast formation ultimately leading to enhanced bone resorption. This bone resorption is inhibited by osteoprotegerin (OPG) which prevents RANKL-RANK interactions. The OPG/RANK/RANKL/TRAF6 pathway plays an important role in bone remodeling. Therefore, we investigated whether genetic variants in OPG, RANK, RANKL and TRAF6 are associated with the rate of joint destruction in RA.

Methods

1,418 patients with 4,885 X-rays of hands and feet derived from four independent data-sets were studied. In each data-set the relative increase of the progression rate per year in the presence of a genotype was assessed. First, explorative analyses were performed on 600 RA-patients from Leiden. 109 SNPs, tagging OPG, RANK, RANKL and TRAF6, were tested. Single nucleotide polymorphisms (SNPs) significantly associated in phase-1 were genotyped in data-sets from Groningen (Netherlands), Sheffield (United Kingdom) and Lund (Switzerland). Data were summarized in an inverse weighted variance meta-analysis. Bonferonni correction for multiple testing was applied.

Results

We found that 33 SNPs were significantly associated with the rate of joint destruction in phase-1. In phase-2, six SNPs in OPG and four SNPs in RANK were associated with progression of joint destruction with P-value <0.05. In the meta-analyses of all four data-sets, RA-patients with the minor allele of OPG-rs1485305 expressed higher rates of joint destruction compared to patients without these risk variants (P = 2.35x10⁻⁴). This variant was also significant after Bonferroni correction.

Conclusions

These results indicate that a genetic variant in OPG is associated with a more severe rate of joint destruction in RA.     

Effect of weight loss and exercise on angiogenic factors in the circulation and in adipose tissue in obese subjects

Background:

Vascular growth is a prerequisite for adipose tissue (AT) development and expansion. Some AT cytokines and hormones have effects on vascular development, like vascular endothelial growth factor (VEGF‐A), angiopoietin (ANG‐1), ANG‐2 and angiopoietin‐like protein‐4 (ANGPTL‐4).

Methods:

In this study, the independent and combined effects of diet‐induced weight loss and exercise on AT gene expression and proteins levels of those angiogenic factors were investigated. Seventy‐nine obese males and females were randomized to: 1. Exercise‐only (EXO; 12‐weeks exercise without diet‐restriction), 2. Hypocaloric diet (DIO; 8‐weeks very low energy diet (VLED) + 4‐weeks weight maintenance diet) and 3. Hypocaloric diet and exercise (DEX; 8‐weeks VLED + 4‐weeks weight maintenance diet combined with exercise throughout the 12 weeks). Blood samples and fat biopsies were taken before and after the intervention.

Results:

Weight loss was 3.5 kg in the EXO group and 12.3 kg in the DIO and DEX groups. VEGF‐A protein was non‐significantly reduced in the weight loss groups. ANG‐1 protein levels were significantly reduced 22‐25% after all three interventions (P < 0.01). The ANG‐1/ANG‐2 ratio was also decreased in all three groups (P < 0.05) by 27‐38%. ANGPTL‐4 was increased in the EXO group (15%, P < 0.05) and 9% (P < 0.05) in the DIO group. VEGF‐A, ANG‐1, and ANGPTL‐4 were all expressed in human AT, but only ANGPTL‐4 was influenced by the interventions.

Conclusions:

Our data show that serum VEGF‐A, ANG‐1, ANG‐2, and ANGPTL‐4 levels are influenced by weight changes, indicating the involvement of these factors in the obese state. Moreover, it was found that weight loss generally was associated with a reduced angiogenic activity in the circulation.     

Atomistic details of effect of disulfide bond reduction on active site of Phytase B from Aspergillus niger: A MD Study

The molecular integrity of the active site of phytases from fungi is critical for maintaining phytase function as efficient catalytic machines. In this study, the molecular dynamics (MD) of two monomers of phytase B from Aspergillus niger, the disulfide intact monomer (NAP) and a monomer with broken disulfide bonds (RAP), were simulated to explore the conformational basis of the loss of catalytic activity when disulfide bonds are broken. The simulations indicated that the overall secondary and tertiary structures of the two monomers were nearly identical but differed in some crucial secondary–structural elements in the vicinity of the disulfide bonds and catalytic site. Disulfide bonds stabilize the β-sheet that contains residue Arg66 of the active site and destabilize the α-helix that contains the catalytic residue Asp319. This stabilization and destabilization lead to changes in the shape of the active–site pocket. Functionally important hydrogen bonds and atomic fluctuations in the catalytic pocket change during the RAP simulation. None of the disulfide bonds are in or near the catalytic pocket but are most likely essential for maintaining the native conformation of the catalytic site.

Abbreviations

PhyB - 2.5 pH acid phophatese from Aspergillus niger, NAP - disulphide intact monomer of Phytase B, RAP - disulphide reduced monomer of Phytase B, Rg - radius of gyration, RMSD - root mean square deviation, MD - molecular dynamics.     

Microfluorometry of pectic materials in the dehiscence zone of almond (Prunus dulcis [Mill.] DA Webb) fruits

We examined the middle lamella and the primary and secondary walls in almond pericarp dehiscence zone cells using a fluorescent cytochemical method which permitted specific, quantitative detection of pectic cell wall materials. Glycol methacrylate-embedded sections were stained with coriphosphine and pectin-specific fluorescent emissions at 630 nm were quantified using green excitation (546 nm). Examination of sectioned material extracted with purified pecto-lytic enzyme preparations was used to demonstrate the relative specificity of the staining reaction for pectic substances.     

The human collagen beta(1-O)galactosyltransferase,GLT25D1, is a soluble endoplasmic reticulum localized protein

Background  

Glycosyl transferases transfer glycosyl groups onto their substrate. Localization partially defines their function. Glycosyl transferase 25 domain 1 (GLT25D1) was recently shown to have galactosyltransferase activity towards collagens and another well known substrate, mannose binding lectin (MBL). To gain more insight in the role of galactosylation of lysines in the Gly-X-Lys repeats of collagenous proteins, we investigated the subcellular localization of GLT25D1.     

Vascular Occlusions in Grapevines with Pierce’s Disease Make Disease Symptom Development Worse

Vascular occlusions are common structural modifications made by many plant species in response to pathogen infection. However, the functional role(s) of occlusions in host plant disease resistance/susceptibility remains controversial. This study focuses on vascular occlusions that form in stem secondary xylem of grapevines (Vitis vinifera) infected with Pierce’s disease (PD) and the impact of occlusions on the hosts’ water transport and the systemic spread of the causal bacterium Xylella fastidiosa in infected vines. Tyloses are the predominant type of occlusion that forms in grapevine genotypes with differing PD resistances. Tyloses form throughout PD-susceptible grapevines with over 60% of the vessels in transverse sections of all examined internodes becoming fully blocked. By contrast, tylose development was mainly limited to a few internodes close to the point of inoculation in PD-resistant grapevines, impacting only 20% or less of the vessels. The extensive vessel blockage in PD-susceptible grapevines was correlated to a greater than 90% decrease in stem hydraulic conductivity, compared with an approximately 30% reduction in the stems of PD-resistant vines. Despite the systemic spread of X. fastidiosa in PD-susceptible grapevines, the pathogen colonized only 15% or less of the vessels in any internode and occurred in relatively small numbers, amounts much too small to directly block the vessels. Therefore, we concluded that the extensive formation of vascular occlusions in PD-susceptible grapevines does not prevent the pathogen’s systemic spread in them, but may significantly suppress the vines’ water conduction, contributing to PD symptom development and the vines’ eventual death.Pierce’s disease (PD) of grapevines (Vitis vinifera), currently jeopardizing the wine and table grape industries in the southern United States and California, as well as in many other countries, is a vascular disease caused by the xylem-limited bacterium Xylella fastidiosa (Hopkins, 1989; Varela et al., 2001). The pathogen is transmitted mostly via xylem sap-feeding sharpshooters (e.g. Homalodisca vitripennis; Redak et al., 2004) and inhabits, proliferates, and spreads within the vessel system of a host grapevine (Fry and Milholland, 1990a; Hill and Purcell, 1995). PD symptom development in grapevines depends on the interactions between the pathogen and the host vine’s xylem tissue, through which the pathogen may achieve its systemic spread (Purcell and Hopkins, 1996; Krivanek and Walker, 2005; Pérez-Donoso et al., 2010; Sun et al., 2011). Since the path for this spread is the host’s xylem system, xylem tissue and its vessels have become the major focus for studying potential X. fastidiosa-host vine interactions at the cellular or tissue levels (Fry and Milholland, 1990b; Stevenson et al., 2004a; Sun et al., 2006, 2007; Thorne et al., 2006).One major issue related to this host-pathogen interaction is the relationship of a vine’s xylem anatomy to the X. fastidiosa population’s spread. Sun et al. (2006) did a detailed anatomical analysis of the stem secondary xylem, especially the vessel system. Stevenson et al. (2004b) described xylem connection patterns between a stem and the attached leaves. Other studies reported the presence of open continuous vessels connecting stems and leaves, which represent conduits that might facilitate the pathogen’s stem-to-leaf movement (Thorne et al., 2006; Chatelet et al., 2006, 2011). Chatelet et al. (2011) also suggested that vessel size and ray density were the two xylem features that were most relevant to the restriction of X. fastidiosa’s movement. These studies indicate the importance of understanding the grapevine’s xylem anatomy in order to characterize the grapevine host’s susceptibility or resistance to PD.Another focus of PD-related xylem studies is the tylose, a developmental modification that has important impacts on a vessel’s role in water transport and, potentially, its availability as a path for X. fastidiosa’s systemic spread through a vine. Tyloses are outgrowths into a vessel lumen from living parenchyma cells that are adjacent to the vessel and can transfer solutes into the transpiration stream via vessel-parenchyma (V-P) pit pairs (Esau, 1977). Tylose development involves the expansion of the portions of the parenchyma cell’s wall that are shared with the neighboring vessels, specifically the so-called pit membranes (PMs). Intensive tylose development may eventually block the affected vessel (Sun et al., 2006). Since tyloses occur in the vessel system of PD-infected grapevines (Esau, 1948; Mollenhauer and Hopkins, 1976; Stevenson et al., 2004a; Krivanek et al., 2005) that is also the avenue of X. fastidiosa’s spread and water transport, a great deal of effort has been made to understand tyloses and their possible relations to grapevine PD as well as to diseases caused by other vascular system-localized pathogens. One major aspect is to clarify the process of tylose development itself, in which an open vessel may be gradually sealed (Sun et al., 2006, 2008). Our investigations of the initiation of tylose formation in grapevines have identified ethylene as an important factor (Pérez-Donoso et al., 2007; Sun et al., 2007). In terms of the relationship of tyloses to grapevine PD, studies have so far led to several controversial viewpoints that are discussed below (Mollenhauer and Hopkins, 1976; Fry and Milholland, 1990b; Stevenson et al., 2004a; Krivanek et al., 2005). However, more convincing evidence is still needed to support any of them.Another issue potentially relevant to PD symptom development is the possibility that X. fastidiosa cells and/or their secretions contribute to the blockage of water transport in host vines. The bacteria secrete an exopolysaccharide (Roper et al., 2007a) that contributes to the formation of cellular aggregates. Accumulations of X. fastidiosa cells embedded in an exopolysaccharide matrix (occasionally identified as biofilms, gums, or gels) have been reported in PD-infected grapevines (Mollenhauer and Hopkins, 1974; Fry and Milholland, 1990a; Newman et al., 2003; Stevenson et al., 2004b). However, a more detailed investigation is still needed to clarify if and to what extent these aggregates affect water transport in infected grapevines.The xylem tissue in which X. fastidiosa spreads can be classified as primary xylem or secondary xylem, being derived from procambium or vascular cambium, respectively. Primary xylem is located in and responsible for material transport and structural support in young organs (i.e. leaves, young stems, and roots), while secondary xylem is the conductive and supportive tissue in more mature stems and roots (Esau, 1977). It should be noted that most of the earlier experimental results have been based on examinations of leaves (petioles or veins) or young stems of grapevines, which contain mostly primary xylem with little or no secondary xylem. However, X. fastidiosa’s systemic spread generally occurs after introduction during the insect vector’s feeding from an internode of one shoot. The pathogen then moves upward along that shoot and also downward toward the shoot base. The downward movement allows the bacteria to enter the vine’s other shoots via the shared trunk and then move upward (Stevenson et al., 2004a; Sun et al., 2011). These upward and downward bacterial movements occur through stems that contain significant amounts of secondary xylem but relatively dysfunctional primary xylem. Secondary and primary xylem show some major differences in the structure and arrangement of their cell components (Esau, 1977). In terms of the vessel system that is the path of X. fastidiosa’s spread, the secondary xylem has a large number of much bigger vessels with scalariform (ladder-like) PMs (and pit pairs) as the sole intervessel (I-V) PM type, compared with the primary xylem, which contains only a limited number of smaller vessels with multiple types of I-V PMs (Esau, 1948; Sun et al., 2006). Vessels in secondary xylem are also different from those in primary xylem in forming vessel groups and in the number of parenchyma cells associated with a vessel (as seen in transverse sections of xylem tissue). These features of secondary xylem can affect the initial entry and subsequent I-V movement of the pathogen and the formation of vascular occlusions, respectively, in stems containing significant amounts of secondary xylem. Recently, the X. fastidiosa population size only in stems with secondary xylem was found to correlate with the grapevine’s resistance to PD (Baccari and Lindow, 2011), indicating an important role of stem secondary xylem in determining a host vine’s disease resistance. Despite these facts, little is known about the pathogen-grapevine interactions in the stem secondary xylem and their possible impacts on disease development.This study addresses X. fastidiosa-grapevine interactions in stem secondary xylem and examines the resulting impacts on overall vine physiology, with a primary focus on vine water transport. We have made use of grapevine genotypes displaying different PD resistances and explored whether differences in the pathogen’s induction of vascular occlusions occur among the genotypes and, if so, how the differences impact X. fastidiosa’s systemic spread. Our overall, longer-term aim is to elucidate the functional role of vascular occlusions in PD development, an understanding that we view to be essential for identifying effective approaches for controlling this devastating disease.     

Airway cellularity, lipid laden macrophages and microbiology of gastric juice and airways in children with reflux oesophagitis

Background and methods

Human metapneumovirus (hMPV) is a recently discovered respiratory virus associated with bronchiolitis, pneumonia, croup and exacerbations of asthma. Since respiratory viruses are frequently detected in patients with acute exacerbations of COPD (AE-COPD) it was our aim to investigate the frequency of hMPV detection in a prospective cohort of hospitalized patients with AE-COPD compared to patients with stable COPD and to smokers without by means of quantitative real-time RT-PCR.

Results

We analysed nasal lavage and induced sputum of 130 patients with AE-COPD, 65 patients with stable COPD and 34 smokers without COPD. HMPV was detected in 3/130 (2.3%) AE-COPD patients with a mean of 6.5 × 10⁵ viral copies/ml in nasal lavage and 1.88 × 10⁵ viral copies/ml in induced sputum. It was not found in patients with stable COPD or smokers without COPD.

Conclusion

HMPV is only found in a very small number of patients with AE-COPD. However it should be considered as a further possible viral trigger of AE-COPD because asymptomatic carriage is unlikely.     

A new brace treatment similar for adolescent scoliosis and kyphosis based on restoration of thoracolumbar lordosis. Radiological and subjective clinical results after at least one year of treatment

Study design

A prospective treatment study with a new brace was conducted Objective. To evaluate radiological and subjective clinical results after one year conservative brace treatment with pressure onto lordosis at the thoracolumbar joint in children with scoliosis and kyphosis.

Summary of background data

Conservative brace treatment of adolescent scoliosis is not proven to be effective in terms of lasting correction. Conservative treatment in kyphotic deformities may lead to satisfactory correction. None of the brace or casting techniques is based on sagittal forces only applied at the thoracolumbar spine (TLI= thoracolumbar lordotic intervention). Previously we showed in patients with scoliosis after forced lordosis at the thoracolumbar spine a radiological instantaneous reduction in both coronal curves of double major scoliosis.

Methods

A consecutive series of 91 children with adolescent scoliosis and kyphosis were treated with a modified symmetric 30 degrees Boston brace to ensure only forced lordosis at the thoracolumbar spine. Scoliosis was defined with a Cobb angle of at least one of the curves [greater than or equal to] 25 degrees and kyphosis with or without a curve <25 degrees in the coronal plane. Standing radiographs were made i) at start, ii) in brace at beginning and iii) after one year treatment without brace.

Results

Before treatment start ??in brace?? radiographs showed a strong reduction of the Cobb angles in different curves in kyphosis and scoliosis groups (sagittal n = 5 all p < 0.001, pelvic obliquity p < 0.001). After one year of brace treatment in scoliosis and kyphosis group the measurements on radiographs made without brace revealed an improvement in 3 Cobb angles each.

Conclusion

Conservative treatment using thoracolumbar lordotic intervention in scoliotic and kyphotic deformities in adolescence demonstrates a marked improvement after one year also in clinical and postural criteria. An effect not obtained with current brace techniques.     

Long-term effects of graduated compression stockings on cardiorespiratory performance

The use of graduated compression stockings (GCS) in sport has been increasing in the last years due to their potential positive effects for athletes. However, there is little evidence to support whether these types of garments actually improve cardiorespiratory performance. The aim of this study was to examine the cardiorespiratory responses of GCS during running after three weeks of regular use. Twenty recreational runners performed three tests on different days: test 1) – a 5-min maximal effort run in order to determine the participants’ maximal aerobic speed; and tests 2) and 3) – a fatigue running test of 30 minutes at 80% of their maximal aerobic speed with either GCS or PLACEBO stockings at random. Cardiorespiratory parameters (minute ventilation, heart rate, relative oxygen consumption, relative carbon dioxide production, ventilatory equivalents for oxygen and carbon dioxide, and oxygen pulse) were measured. Before each test in the laboratory, the participants trained with the randomly assigned stockings (GCS or PLACEBO) for three weeks. No significant differences between GCS and PLACEBO were found in any of the cardiorespiratory parameters. In conclusion, the present study provides evidence that running with GCS for three weeks does not influence cardiorespiratory parameters in recreational runners.