The wMel Wolbachia strain blocks dengue and invades caged Aedes aegypti populations.

Dengue fever is the most important mosquito-borne viral disease of humans with more than 50 million cases estimated annually in more than 100 countries. Disturbingly, the geographic range of dengue is currently expanding and the severity of outbreaks is increasing. Control options for dengue are very limited and currently focus on reducing population abundance of the major mosquito vector, Aedes aegypti. These strategies are failing to reduce dengue incidence in tropical communities and there is an urgent need for effective alternatives. It has been proposed that endosymbiotic bacterial Wolbachia infections of insects might be used in novel strategies for dengue control. For example, the wMelPop-CLA Wolbachia strain reduces the lifespan of adult A. aegypti mosquitoes in stably transinfected lines. This life-shortening phenotype was predicted to reduce the potential for dengue transmission. The recent discovery that several Wolbachia infections, including wMelPop-CLA, can also directly influence the susceptibility of insects to infection with a range of insect and human pathogens has markedly changed the potential for Wolbachia infections to control human diseases. Here we describe the successful transinfection of A. aegypti with the avirulent wMel strain of Wolbachia, which induces the reproductive phenotype cytoplasmic incompatibility with minimal apparent fitness costs and high maternal transmission, providing optimal phenotypic effects for invasion. Under semi-field conditions, the wMel strain increased from an initial starting frequency of 0.65 to near fixation within a few generations, invading A. aegypti populations at an accelerated rate relative to trials with the wMelPop-CLA strain. We also show that wMel and wMelPop-CLA strains block transmission of dengue serotype 2 (DENV-2) in A. aegypti, forming the basis of a practical approach to dengue suppression.

Successful establishment of Wolbachia in Aedes populations to suppress dengue transmission.

Genetic manipulations of insect populations for pest control have been advocated for some time, but there are few cases where manipulated individuals have been released in the field and no cases where they have successfully invaded target populations. Population transformation using the intracellular bacterium Wolbachia is particularly attractive because this maternally-inherited agent provides a powerful mechanism to invade natural populations through cytoplasmic incompatibility. When Wolbachia are introduced into mosquitoes, they interfere with pathogen transmission and influence key life history traits such as lifespan. Here we describe how the wMel Wolbachia infection, introduced into the dengue vector Aedes aegypti from Drosophila melanogaster, successfully invaded two natural A. aegypti populations in Australia, reaching near-fixation in a few months following releases of wMel-infected A. aegypti adults. Models with plausible parameter values indicate that Wolbachia-infected mosquitoes suffered relatively small fitness costs, leading to an unstable equilibrium frequency <30% that must be exceeded for invasion. These findings demonstrate that Wolbachia-based strategies can be deployed as a practical approach to dengue suppression with potential for area-wide implementation.

An open-label dosing study to evaluate the safety and effects of a dietary plant-derived polysaccharide supplement on the N-glycosylation status of serum glycoproteins in healthy subjects.

BACKGROUND: The functional role of dietary carbohydrates in nutrition is one of the most complex and at times controversial areas in nutritional science. In-vitro and in-vivo studies suggest that certain dietary saccharide biopolymers can have bifidogenic and or immunomodulatory effects, and that some could represent preferential substrates or precursors that can impact cellular glycosylation. OBJECTIVE: Examine the impact of oral ingestion of a standardized dietary plant-derived polydisperse polysaccharide supplement (Advanced Ambrotose powder (AA)) on the N-glycosylation status of serum glycoproteins in a cohort of healthy individuals. DESIGN: An open-label study was carried out. This study was in two phases: pilot study (n=6 individuals) to assess safety and dose, and a larger study (n=12) to evaluate specific glycosylation changes. Serum N-glycosylation profiles, using mass spectrometry, were monitored at weekly intervals, for 7 weeks, to evaluate baseline levels and normal fluctuations. The individuals were then monitored for a further 7 weeks, during which time increasing doses of AA were ingested (1.3-5.2 g/day). RESULTS: No adverse events were encountered. AA supplementation resulted in distinct changes in the relative intensities of seven biantennary N-glycans (P<0.001), and a significant overall shift towards increased sialylation. Regression analysis revealed a dose-dependent decrease in mono- and di-galactosylated structures (coefficient -0.130 decrease/week: P=0.02 and -0.690: P=0.005), and a concomitant increase in disialylated glycans ( × 1.083: P<0.05). CONCLUSIONS: Supplementation with the dietary plant-derived polysaccharides in AA resulted in significant changes in serum protein N-glycosylation in healthy individuals. How this occurs and whether it has biological significance remains to be evaluated.

A role for human endogenous retrovirus-K (HML-2) in rheumatoid arthritis: investigating mechanisms of pathogenesis.

Human endogenous retroviruses (HERVs) are remnants of ancient retroviral infections within the human genome. These molecular fossils draw parallels with present-day exogenous retroviruses and have been linked previously with immunopathology within rheumatoid arthritis (RA). Mechanisms of pathogenesis for HERV-K in RA such as molecular mimicry were investigated. To clarify a role for HERVs in RA, potential autoantigens implicated in autoimmunity were scanned for sequence identity with retroviral epitopes. Short retroviral peptides modelling shared epitopes were synthesized, to survey anti-serum of RA patients and disease controls. A novel real-time polymerase chain reaction (PCR) assay was also developed to quantify accurately levels of HERV-K (HML-2) gag expression, relative to normalized housekeeping gene expression. Both serological and molecular assays showed significant increases in HERV-K (HML-2) gag activity in RA patients, compared to disease controls. The real-time PCR assay identified significant up-regulation in HERV-K mRNA levels in RA patients compared to inflammatory and healthy controls. Exogenous viral protein expression and proinflammatory cytokines were also shown to exert modulatory effects over HERV-K (HML-2) transcription. From our data, it can be concluded that RA patients exhibited significantly elevated levels of HERV-K (HML-2) gag activity compared to controls. Additional factors influencing HERV activity within the synovium were also identified. The significant variation in RA patients, both serologically and transcriptionally, may be an indication that RA is an umbrella term for a number of separate disease entities, of which particular HERV polymorphisms may play a role in development.

Sweet and sour: the impact of sugars on disease.

The clinical relevance of glycobiology has become the focus of considerable research, as the role of glycosylation in the development, regulation and progression of disease is, slowly but surely, being unveiled. Recent strides in the design and refinement of analytical techniques-sugar profiling, glyco-arrays and functional studies-have helped us gain a better understanding of the complexity and richness of diversity that bestow sugars with an unsurpassed, biospecific coding capacity. Cracking this 'sugar code', and unravelling the structural frameworks and recognition strategies of sugar-based interactions in biological systems that relate to both health and disease, holds tremendous promise for deciphering disease mechanisms. It will also provide a cutting edge potential for the development of novel diagnostic and therapeutic interventions.

Seven genomic subtypes of chronic fatigue syndrome/myalgic encephalomyelitis: a detailed analysis of gene networks and clinical phenotypes.

AIM: Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is a multisystem disease, the pathogenesis of which remains undetermined. The authors have recently reported a study of gene expression that identified differential expression of 88 human genes in patients with CFS/ME. Clustering of quantitative PCR (qPCR) data from patients with CFS/ME revealed seven distinct subtypes with distinct differences in Medical Outcomes Survey Short Form-36 scores, clinical phenotypes and severity. METHODS: In this study, for each CFS/ME subtype, those genes whose expression differed significantly from that of normal blood donors were identified, and then gene interactions, disease associations and molecular and cellular functions of those gene sets were determined. Genomic analysis was then related to clinical data for each CFS/ME subtype. RESULTS: Genomic analysis revealed some common (neurological, haematological, cancer) and some distinct (metabolic, endocrine, cardiovascular, immunological, inflammatory) disease associations among the subtypes. Subtypes 1, 2 and 7 were the most severe, and subtype 3 was the mildest. Clinical features of each subtype were as follows: subtype 1 (cognitive, musculoskeletal, sleep, anxiety/depression); subtype 2 (musculoskeletal, pain, anxiety/depression); subtype 3 (mild); subtype 4 (cognitive); subtype 5 (musculoskeletal, gastrointestinal); subtype 6 (postexertional); subtype 7 (pain, infectious, musculoskeletal, sleep, neurological, gastrointestinal, neurocognitive, anxiety/depression). CONCLUSION: It was particularly interesting that in the seven genomically derived subtypes there were distinct clinical syndromes, and that those which were most severe were also those with anxiety/depression, as would be expected in a disease with a biological basis.

The potential role of human endogenous retrovirus K10 in the pathogenesis of rheumatoid arthritis: a preliminary study.

OBJECTIVE: To examine whether human endogenous retrovirus K10 is associated with autoimmune rheumatic disease. DESIGN: A novel multiplex reverse transcription polymerase chain reaction (RT-PCR) system was developed to investigate HERV-K10 mRNA expression in patients with rheumatoid arthritis. METHODS: 40 patients with rheumatoid arthritis, 17 with osteoarthritis, and 27 healthy individuals were recruited and total RNA was extracted from peripheral blood mononuclear cells (PBMCs) and analysed using multiplex RT-PCR for the level of HERV-K10 gag mRNA expression. Southern blot and DNA sequencing confirmed the authenticity of the PCR products. RESULTS: Using the histidyl tRNA synthetase (HtRNAS) gene as a housekeeping gene in the optimised multiplex RT-PCR, a significantly higher level of HERV-K10 gag mRNA expression was found in rheumatoid arthritis than in osteoarthritis (p = 0.01) or in the healthy controls (p = 0.02). CONCLUSION: There is enhanced mRNA expression of the HERV-K10 gag region in rheumatoid arthritis compared with osteoarthritis or healthy controls. This could contribute to the pathogenesis of rheumatoid arthritis.

Serum cortisol reduction and abnormal prolactin and CD4+/CD8+ T-cell response as a result of controlled exercise in patients with rheumatoid arthritis and systemic lupus erythematosus despite unaltered muscle energetics.

OBJECTIVE: To investigate muscle energetics in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) and measure serum cortisol, prolactin and CD4+/CD8+ T-cell levels during and after controlled exhaustive exercise. METHODS: Patients with RA (n = 7), patients with SLE (n = 6) and healthy individuals (HI) (n = 10) performed incremental cycle ergometry to the limit of tolerance. Ventilation, oxygen uptake (VO2) and carbon dioxide output were measured and the lactate threshold (LT) was estimated. Serum cortisol, prolactin, CD4+ and CD8+ lymphocyte subset levels were determined at baseline, peak exercise and 1 h after exercise. RESULTS: Exercise tolerance was reduced in patients with RA and patients with SLE, as reflected by peak VO2 and LT, but muscle energetics were not altered. In RA and SLE, there was significant reduction in cortisol levels at peak (-10%; P = 0.03) and post-exercise times (-36%; P = 0.05). Prolactin varied significantly at peak exercise in HI only (+60%; P = 0.05). There was a significant reduction in CD4+ T cells at peak exercise in RA (-15%; P = 0.02) and SLE patients (-8%; P = 0.04) and an increase after exercise in SLE patients (+11%; P = 0.03). In HI, CD8+ T cells increased significantly (+47%; P = 0.01) at peak exercise, but this was not found in RA and SLE patients. A significant reduction in CD8+ T cells was noted after exercise in SLE patients (-6%; P = 0.05). CONCLUSION: RA and lupus patients do not have significantly altered muscle energetics, but have abnormal cortisol, prolactin and CD4+/CD8+ T-cell responses to exercise. Further studies need to be carried out to evaluate whether short bouts of strenuous exercise have detrimental clinical effects.

Quantitative magnetic resonance imaging in neuropsychiatric systemic lupus erythematosus.

Neuropsychiatric symptoms are common in systemic lupus erythematosus (SLE) but are poorly understood. Although there is a wide spectrum of clinical manifestations, brain histology often simply shows a bland vasculopathy. Magnetic resonance techniques such as magnetic resonance spectroscopy, magnetization transfer imaging and diffusion weighted imaging have been used to try to improve our understanding of the pathophysiological mechanisms involved in neuropsychiatric lupus (NPSLE). This article reviews the current literature on the use of these techniques and their possible future role as diagnostic tools in NPSLE.

Increased apoptosis of bone marrow CD34(+) cells and impaired function of bone marrow stromal cells in patients with systemic lupus erythematosus.

The changes in bone marrow (BM) stem cell reserve and function and stromal cell function in patients with active systemic lupus erythematosus (SLE) were investigated. The study was carried out on seven SLE patients and 28 healthy controls using flow cytometry and in vitro cell culture assays. We found that patients had low CD34(+) cells, compared with the control group, reflecting the decrease of both CD34(+)/CD38(-) and CD34(+)/CD38(+) cells. Patient CD34(+)/Fas(+) but not CD34(-)/Fas(+) cells were significantly increased. Apoptotic (7AAD(dim)) cells were higher among CD34(+)/Fas(+) than among CD34(+)/Fas(-) cells, and individual values of apoptotic CD34+ cells strongly correlated with the number of CD34(+)/Fas(+) cells. These findings are suggestive of a Fas-mediated apoptosis accounting for the low CD34(+) cells in SLE patients. Moreover, we found that patients had low numbers of granulocyte-macrophage colony-forming units (CFU-GM) and erythroid burst-forming units (BFU-E), compared with the control group, and that the generation of colony-forming cells in long-term BM cultures was significantly reduced. Patient BM stroma failed to support allogeneic progenitor cell growth. In one patient, CD34(+) cells were increased, apoptotic CD34(+)/Fas(+) cells were normalized and defective stromal cell function was restored after autologous stem cell transplantation. We concluded that defective haemopoiesis in SLE patients is probably caused, at least in part, to the presence of autoreactive lymphocytes in BM.

Characterization of changes in IgG associated oligosaccharide profiles in rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis using fluorophore linked carbohydrate electrophoresis.

OBJECTIVE: To investigate fluorophore linked carbohydrate electrophoresis (FCE) as a method of analyzing serum immunoglobulin G (IgG) oligosaccharides in healthy individuals and those with rheumatic disease and compare with lectin binding assays of carbohydrate composition. METHODS: IgG was isolated from patients with rheumatoid arthritis (RA) (n = 21), ankylosing spondylitis (AS) (n = 20), psoriatic arthritis (PsA) (n = 20), and healthy adults (n = 36). IgG oligosaccharides were released enzymatically, fluorescently labelled using 8 aminonaphthalene-136 trisulfonic acid; and identification of the oligosaccharide bands was by stepwise enzymatic degradation. Comparison of FCE was made with lectin binding analysis in which the lectins Ricinus communis (RCA1) and Bandeiraea simplicifolia (BSII) were used to detect galactose (Gal) and N-acetylglucosamine (GlcNAc), respectively. RESULTS: Each disease could be differentiated from healthy adults on the basis of Band 1 asialodigalacto core fucosylated oligosaccharide (gf2) intensity (p = 0.001), but not from each other. Reduced levels of different sugars were associated with specific diseases: reduced gf2 with RA (p < 0.001), PsA (p < 0.001) and AS (p < 0.02), reduced Band 5 disialo-digalacto core fucosylated (a2f) oligosaccharide with AS (p < 0.001), reduced Band 6 disialo-digalacto (a2) oligosaccharide with AS (p < 0.001) and PsA (p = 0.021). All diseases were associated with a significant increase in Band 4 asialo-agalacto core fucosylated oligosaccharide (g0f) (p < 0.001). In RA, FCE band intensities correlated with sugar quantity when identified using lectin binding analysis (p < 0.003). In contrast, there was no correlation between the same bands in healthy individuals. CONCLUSION: FCE is an accurate method of analyzing IgG associated oligosaccharides and reveals unique band patterns or sugar prints associated with healthy adults and patients with RA, PsA, and AS, and comparison with lectin binding analysis suggests undetected RA glycoprotein structural differences. FCE has potential in the early diagnosis and differentiation of rheumatic diseases.

The impact of glycobiology on medicine.

Proteins essential to normal cell physiology are usually glycosylated and variation in their glycosylation patterns often leads to changes in their function. Changes in glycosylation pattern can also be associated with disease. It is now becoming increasingly clear how important it is to understand these changes, to gain insight into their involvement in disease mechanisms and the potential for novel therapeutic interventions.

Sensitivity of quantitative (1)H magnetic resonance spectroscopy of the brain in detecting early neuronal damage in systemic lupus erythematosus.

OBJECTIVE: To quantify N-acetylaspartate (NAA), total creatines (tCr), total cholines (tCho), and myo-inositol (mI) levels in normal and abnormal appearing white matter of patients with neuropsychiatric systemic lupus erythematosus (NPSLE) in order to determine the specific changes in metabolite concentrations. METHODS: Axial proton density and T(2) weighted magnetic resonance images, and short echo time (TE 30 ms) (1)H spectra were acquired with a GE SIGNA 1.5 T magnetic resonance system. Concentrations of NAA, tCr, tCho, and mI were determined, using brain tissue water as a reference, from nine patients (seven female, mean age 40.3 years, range 16-65) with NPSLE and eight healthy women (mean age 43 years, range 31-65). RESULTS: A significant rise of tCho (12.4%, p<0.05) and mI (31.4%, p<0.005) and a significant reduction in NAA (-12%, p<0.05) was found in normal appearing white matter compared with controls. Analysis according to severity of the clinical NPSLE features (subgrouped as major or minor) showed that SLE major had reduced NAA compared with SLE minor (-18.4%, p<0.05) and controls (-20%, p<0.005). The SLE major group showed a significant rise of mI (32%, p<0.01) and the SLE minor group a significant increase in tCho (18.6%, p<0.05) compared with controls. Longitudinal analysis of brain metabolites in normal appearing white matter showed consistent abnormalities in NAA, tCho, and mI in a patient with stable clinical features and a constant rise of tCho, but transient rise of mI was seen during a flare of disease in another patient. CONCLUSION: Quantitative (1)H magnetic resonance spectroscopy (MRS) suggests a particular course of neurometabolite changes that precedes irreversible reductions in NAA and permanent neuronal loss. Initially, in patients with SLE minor, there is a significant rise in tCho and a trend (reversible) for mI also to be raised. In patients with SLE major the NAA is significantly and permanently reduced and mI is significantly raised, whereas the tCho levels are near normal. Further investigations are needed to determine how specific MRS is as a clinical marker for brain disturbance in SLE.

Primary care-based evaluation of patient education for osteoarthritis of the knee.

Primary healthcare research in the UK has expanded in conjunction with the wider agenda of a primary care led National Health Service (NHS). This reflects international reconsideration of the place of primary healthcare within national healthcare systems. However the role and standing of research in primary care tends to be marginal and less influential than that originating in research establishments or medical schools pursuing biomedical investigations. There are many reasons for this, some of which this paper attempts to elucidate. We address the perspectives and relationships between 'stakeholder' groups in the field of primary care and highlight the ways in which such relationships affect research in this area. The development of a primary care research project concerned with osteoarthritis of the knee (the OAK project) is used as an exemplar of these stresses and we suggest ways in which the difficulties encountered may be overcome.

Immunoglobulin G glycosylation and clinical outcome in rheumatoid arthritis during pregnancy.

OBJECTIVE: To determine whether clinical outcome during pregnancy in rheumatoid arthritis (RA) is associated with changes in the levels of exposed immunoglobulin G (IgG) terminal sugars. METHODS: Serum IgG glycosylation from 23 pregnant patients with RA was analyzed during the prenatal, antenatal, and post-partum periods. Patients were randomly selected on the basis of whether they achieved spontaneous remission (n = 11) or did not remit (n = 12); of the latter group 6 patients experienced a relapse in disease activity. Levels of exposed terminal IgG sugars, galactose (Gal), N-acetylglucosamine (GlcNAc), and sialic acid (SA), were estimated in a lectin binding assay using Ricinis (communis, Bandeiraea simplicifolia II, and Sambucus nigra, respectively. RESULTS: Exposed Gal levels increased (p<0.02) and GlcNAc levels decreased (p<0.05) in the antenatal period, and returned to preconception levels during post-partum. GlcNAc rebound was instantaneous (p<0.005), whereas Gal remained high for a further 10 weeks. SA did not undergo any major changes. Remission was associated with an earlier and significantly greater antenatal reduction in GlcNAc (2nd and 3rd trimester; p<0.02) in comparison to the groups that did not experience a decrease in disease activity. Analysis of individual IgG samples during the first trimester revealed a significant negative correlation between Gal and GlcNAc in the remission group (r = -0.80; p<0.05), which was opposite to that found in the relapse group (r = +0.87; p<0.03). There was no significant difference between the groups with regard to the timing and/or incidence of a post-partum flare of disease. CONCLUSION: Temporal changes in the levels of IgG terminal sugars, in particular exposed GlcNAc, are integrally associated with the clinical manifestation of RA in pregnancy. Generation of IgG sugar micro-heterogeneity is complex and understanding it may help unravel pathogenic features associated with RA.

Increased IgA rheumatoid factor and V(H)1 associated cross reactive idiotype expression in patients with Lyme arthritis and neuroborreliosis.

OBJECTIVE: To investigate whether autoreactive mechanisms occur in Lyme disease (LD) by determining IgA, IgG and IgM rheumatoid factor (RF) concentrations and RF associated cross reactive idiotype (CRI) expression in the serum of LD patients, with comparison to patients with rheumatoid arthritis (RA). METHODS: The RF isotype profiles were determined in 59 patients with LD; erythema migrans (EM) (n=19), neuroborreliosis (NB) (n=20) and Lyme arthritis (LA) (n=20). Mouse monoclonal antibodies (mAbs) G6 and G8 (V(H)1 gene associated), D12 (V(H)3 gene associated) and C7 (V(kappa)III gene associated) were then used to determine the RF associated CRI expression on IgM antibodies in 16 of these LD patients (eight seropositive for RF); (EM (n=3), NB (n=6), LA (n=7)). RESULTS: Seven (18%) patients with either NB or LA had increased concentrations of IgA RF compared with none with EM. Significant differences in the number of patients with raised concentrations of IgG RF or IgM RF were not found between the LD patient groups. Five (3NB, 1LA and 1 EM) (31%) and three (2NB and 1LA) (19%) of LD patients had raised concentrations of the CRIs recognised by mAbs G6 and G8, respectively. These CRIs were detected in LD sera both with and without raised concentrations of RF and were not demonstrated on anti-Borrelia burgdorferi antibodies using ELISA. No LD sera tested had raised concentrations of the determinants recognised by mAbs C7 or D12. CONCLUSION: Significantly raised concentrations of IgA RF and increased use of V(H)1 germline gene associated CRIs are found on IgM antibodies in the serum of LD patients. These data indicate the recruitment of autoreactive B lymphocytes in some patients with the later stages of LD.