Circulating surfactant protein -D is low and correlates negatively with systemic inflammation in early, untreated rheumatoid arthritis

Introduction

Surfactant protein D (SP-D) is a collectin with immuno-regulatory functions, which may depend on oligomerization. Anti-microbial and anti-inflammatory properties have been attributed to multimeric SP-D variants, while trimeric subunits per se have been suggested to enhance inflammation. Previously, we reported low circulating SP-D in early rheumatoid arthritis (RA), and the present investigation aims to extend these data by serial SP-D serum measurements, studies on synovial fluid, SP-D size distribution and genotyping in patients with early RA.

Methods

One-hundred-and-sixty disease-modifying antirheumatic drug (DMARD) naïve RA patients with disease duration less than six months were studied prospectively for four years (CIMESTRA (Ciclosporine, Methotrexate, Steroid in RA) trial) including disease activity measures (C-reactive protein, joint counts and Health Assessment Questionnaire (HAQ) score), autoantibodies, x-ray findings and SP-D. SP-D was quantified by enzyme-linked immunosorbent assay (ELISA) and molecular size distribution was assessed by gel filtration chromatography. Further, SP-D Met11Thr single nucleotide polymorphism (SNP) analysis was performed.

Results

Serum SP-D was significantly lower in RA patients at baseline compared with healthy controls (P < 0.001). SP-D increased slightly during follow-up (P < 0.001), but was still subnormal at four years after adjustment for confounders (P < 0.001). SP-D in synovial fluid was up to 2.5-fold lower than in serum. While multimeric variants were detected in serum, SP-D in synovial fluid comprised trimeric subunits only. There were no significant associations between genotype distribution and SP-D. Baseline SP-D was inversely associated to CRP and HAQ score. A similar relationship was observed regarding temporal changes in SP-D and CRP (zero to four years). SP-D was not associated to x-ray findings.

Conclusions

This study confirms that circulating SP-D is persistently subnormal in early and untreated RA despite a favourable therapeutic response obtained during four years of follow-up. SP-D correlated negatively to disease activity measures, but was not correlated with x-ray progression or SP-D genotype. These observations suggest that SP-D is implicated in RA pathogenesis at the protein level. The exclusive presence of trimeric SP-D in affected joints may contribute to the maintenance of joint inflammation.

Trial registration

(j.nr NCT00209859).     

Therapeutically optimized rates of drug release can be achieved by varying the drug-to-lipid ratio in liposomal vincristine formulations

The anti-tumor efficacy of liposomal formulations of cell cycle dependent anticancer drugs is critically dependent on the rates at which the drugs are released from the liposomes. Previous work on liposomal formulations of vincristine have shown increasing efficacy for formulations with progressively slower release rates. Recent work has also shown that liposomal formulations of vincristine with higher drug-to-lipid (D/L) ratios exhibit reduced release rates. In this work, the effects of very high D/L ratios on vincristine release rates are investigated, and the antitumor efficacy of these formulations characterized in human xenograft tumor models. It is shown that the half-times (T(1/2)) for vincristine release from egg sphingomyelin/cholesterol liposomes in vivo can be adjusted from T(1/2) = 6.1 h for a formulation with a D/L of 0.025 (wt/wt) to T(1/2) = 117 h (extrapolated) for a formulation with a D/L ratio of 0.6 (wt/wt). The increase in drug retention at the higher D/L ratios appears to be related to the presence of drug precipitates in the liposomes. Variations in the D/L ratio did not affect the circulation lifetimes of the liposomal vincristine formulations. The relationship between drug release rates and anti-tumor efficacy was evaluated using a MX-1 human mammary tumor model. It was found that the antitumor activity of the liposomal vincristine formulations increased as D/L ratio increased from 0.025 to 0.1 (wt/wt) (T(1/2) = 6.1-15.6 h respectively) but decreased at higher D/L ratios (D/L = 0.6, wt/wt) (T(1/2) = 117 h). Free vincristine exhibited the lowest activity of all formulations examined. These results demonstrate that varying the D/L ratio provides a powerful method for regulating drug release and allows the generation of liposomal formulations of vincristine with therapeutically optimized drug release rates.     

Modeling the impact of a biocontrol agent, Puccinia lagenophorae, on interactions between a crop, Daucus carota, and a weed, Senecio vulgaris

The impact of a biocontrol agent spreading from a point source on crop–weed interactions was modeled. The model encompassed: (i) severity of crop–weed competition as affected by a rust pathogen, (ii) velocity of spread of the rust pathogen, and (iii) density of infected plants introduced in the weed population as starting points (inoculum sources) for an epidemic. The model was parameterized for a study system encompassing the crop Daucus carota (carrot), the weed Senecio vulgaris (common groundsel), and its antagonist Puccinia lagenophorae. The parameters of (i) were estimated in a greenhouse study using a response-surface design. Estimates of the parameter of (ii) were obtained from the literature. The density of infected plants (iii) was varied to simulate crop loss as function of density. Simulations were run for various densities of the weed and various velocities of rust pathogen spread. The results of the simulations indicated a crop-loss ranging from 5 to 10% at levels of relatively weak D. carota–S. vulgaris competition. Density of inoculum sources and velocity of P. lagenophorae spread had only minor effects on crop loss. In contrast, density of inoculum sources and velocity of spread had major effects on crop loss at levels of intermediate (range of 10–35% loss) and severe competition (range of 30–70% loss). The results are discussed both with respect to biological control of S. vulgaris using P. lagenophorae as biocontrol agent and as a general model describing the impact of the spatial dynamics of a pathogen (natural enemy) on plant competition.     

A role for mixed lineage kinases in granule cell apoptosis induced by cytoskeletal disruption

Microtubule disruption by colchicine induces apoptosis in selected neuronal populations. However, little is known about the upstream death signalling events mediating the neurotoxicity. We investigated first whether colchicine-induced granule cell apoptosis activates the c-Jun N-terminal kinase (JNK) pathway. Cultured murine cerebellar granule cells were exposed to 1 microm colchicine for 24 h. Activation of the JNK pathway was detected by western blotting as well as immunocytochemistry using antibodies against phospho-c-Jun (p-c-Jun). Next, adult male rats were injected intracerebroventricularly with colchicine (10 microg), and JNK pathway activation in dentate granule cells (DGCs) was detected by antibodies against p-c-Jun. The second part of the study tested the involvement of mixed lineage kinases (MLK) as upstream activators of the JNK pathway in colchicine toxicity, using CEP-1347, a potent MLK inhibitor. In vitro, significant inhibition of the JNK pathway, activated by colchicine, was achieved by 100-300 nm CEP-1347, which blocked both activation of cell death proteases and apoptosis. Moreover, CEP-1347 markedly delayed neurite fragmentation and cell degeneration. In vivo, CEP-1347 (1 mg/kg) significantly prevented p-c-jun increase following injection of colchicine, and enhanced survival of DGCs. We conclude that colchicine-induced neuronal apoptosis involves the JNK/MLK pathway, and that protection of granule cells can be achieved by MLK inhibition.     

The CWPS Rubik’s cube: Linking diversity of cell wall polysaccharide structures with the encoded biosynthetic machinery of selected Lactococcus lactis strains

The biosynthetic machinery for cell wall polysaccharide (CWPS) production in lactococci is encoded by a large gene cluster, designated cwps. This locus displays considerable variation among lactococcal genomes, previously prompting a classification into three distinct genotypes (A–C). In the present study, the cwps loci of 107 lactococcal strains were compared, revealing the presence of a fourth cwps genotype (type D). Lactococcal CWPSs are comprised of two saccharidic structures: a peptidoglycan-embedded rhamnan backbone polymer to which a surface-exposed, poly/oligosaccharidic side-chain is covalently linked. Chemical structures of the side-chain of seven lactococcal strains were elucidated, highlighting their diverse and strain-specific nature. Furthermore, a link between cwps genotype and chemical structure was derived based on the number of glycosyltransferase-encoding genes in the cwps cluster and the presence of conserved genes encoding the presumed priming glycosyltransferase. This facilitates predictions of several structural features of lactococcal CWPSs including (a) whether the CWPS possesses short oligo/polysaccharide side-chains, (b) the number of component monosaccharides in a given CWPS structure, (c) the order of monosaccharide incorporation into the repeating units of the side-chain (for C-type strains), (d) the presence of Galf and phosphodiester bonds in the side-chain, and (e) the presence of glycerol phosphate substituents in the side-chain.     

Variation in Inflammatory Cytokine/Growth-Factor Genes and Mammographic Density in Premenopausal Women Aged 50–55

Background

Mammographic density (MD) has been found to be an independent risk factor for breast cancer. Although data from twin studies suggest that MD has a strong genetic component, the exact genes involved remain to be identified. Alterations in stromal composition and the number of epithelial cells are the most predominant histopathological determinants of mammographic density. Interactions between the breast stroma and epithelium are critically important in the maturation and development of the mammary gland and the cross-talk between these cells are mediated by paracrine growth factors and cytokines. The potential impact of genetic variation in growth factors and cytokines on MD is largely unknown.

Methods

We investigated the association between 89 single nucleotide polymorphisms (SNPs) in 7 cytokine/growth-factor genes (FGFR2, IGFBP1, IGFBP3, TGFB1, TNF, VEGF, IL6) and percent MD in 301 premenopausal women (aged 50 to 55 years) participating in the Norwegian Breast Cancer Screening Program. We evaluated the suggestive associations in 216 premenopausal Singapore Chinese Women of the same age.

Results

We found statistically significant associations between 9 tagging SNPs in the IL6 gene and MD in Norwegian women; the effect ranged from 3–5% in MD per variant allele (p-values = 0.02 to 0.0002). One SNP in the IL6 (rs10242595) significantly influenced MD in Singapore Chinese women.

Conclusion

Genetic variations in IL6 may be associated with MD and therefore may be an indicator of breast cancer risk in premenopausal women.     

Nitric Oxide Exposure of CC531 Rat Colon Carcinoma Cells Induces γ-glutamyltransferase which May Counteract Glutathione Depletion and Cell Death

&#110 -Glutamyltransferase (GGT) has a central role in glutathione homeostasis by initiating the breakdown of extracellular GSH. We investigated in the present study whether nitric oxide exposure of CC531 rat colon carcinoma cells modulates GGT and how the activity of the enzyme affects the level of intracellular GSH. The data show that GGT activity was induced in a dose-related manner by two NO-donors (spermineNONOate and nitrosoglutathione) and that antioxidants partly inhibited the induction. SpermineNONOate lowered intracellular GSH and induced apoptosis. Cultivating the cells in cystine-depleted medium also resulted in a 50% lowering of GSH, but this was avoided when GSH was added to the medium. This effect was mediated by the activity of GGT and shown after inhibiting GGT activity with acivicin and cyst(e)ine transporters with alanine and homocysteic acid. This shows that the cells benefit from GGT in maintaining the intracellular GSH level. Cells with induced GGT activity obtained after NO incubation showed a higher uptake rate of cysteine (2-fold), measured by incubating the cells with 35 S-radiolabeled GSH. The enzyme was also induced by interferon- &#110 and tumor necrosis factor- &#102, but this induction was not connected to activation of the endogenous nitric oxide synthase, as the addition of aminoguanidine, a NO-synthase inhibitor, did not affect the induction. The present study shows that the activity of GGT is upregulated by NO-donors and that the colon carcinoma cells, when cultivated in cystine-depleted medium, benefit from the enzyme in maintaining the intracellular level of GSH. Thus, the enzyme will add to the protective measures of the tumor cells during nitrosative stress.     

Identification of 2 loci associated with development of myxomatous mitral valve disease in Cavalier King Charles Spaniels

Myxomatous mitral valve disease (MMVD) is the most common heart disease in dogs. It is characterized by chronic progressive degenerative lesions of the mitral valve. The valve leaflets become thickened and prolapse into the left atrium resulting in mitral regurgitation (MR). MMVD is most prevalent in small to medium sized dog breeds, Cavalier King Charles Spaniels (CKCS) in particular. The onset of MMVD is highly age dependent, and at the age of 10 years, nearly all CKCS are affected. The incidence of a similar disease in humans-mitral valve prolapse-is 1-5%. By defining CKCSs with an early onset of MMVD as cases and old dogs with no or mild signs of MMVD as controls, we conducted a genome-wide association study (GWAS) to identify loci associated with development of MMVD. We have identified a 1.58 Mb region on CFA13 (P(genome) = 4.0 × 10(-5)) and a 1.68 Mb region on CFA14 (P(genome) = 7.9 × 10(-4)) associated with development of MMVD. This confirms the power of using the dog as a model to uncover potential candidate regions involved in the molecular mechanisms behind complex traits.     

RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design

Gene silencing through RNA interference (RNAi) has revolutionized the study of gene function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) RNAi has many times proven to be difficult to achieve. Most of the negative results have been anecdotal and the positive experiments have not been collected in such a way that they are possible to analyze. In this review, we have collected detailed data from more than 150 experiments including all to date published and many unpublished experiments. Despite a large variation in the data, trends that are found are that RNAi is particularly successful in the family Saturniidae and in genes involved in immunity. On the contrary, gene expression in epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding dsRNA requires high concentrations for success. Possible causes for the variability of success in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the innate immune response. Our general understanding of RNAi in Lepidoptera will be further aided in the future as our public database at http://insectacentral.org/RNAi will continue to gather information on RNAi experiments.