Linkage of atopic dermatitis to chromosomes 4q22, 3p24 and 3q21

Atopic dermatitis (AD) is a common, itchy skin disease of complex inheritance characterized by dermal and epidermal inflammation. The heritability is considerable and well documented. To date, four genome scans have examined the AD phenotype, showing replicated linkage at 3p26-22, 3q13-21 and 18q11-21. Our previous AD scan showed evidence of linkage to loci at 3p and 18q, and furthermore at 4p15-14. In order to further investigate the genetic basis of AD, we collected and analysed a new Danish family sample consisting of 130 AD sib pair families (555 individuals including 295 children with AD). AD was diagnosed after clinical examination, AD severity was scored and specific IgE was determined. A linkage scan of chromosome 3, 4 and 18 was performed using 91 microsatellite markers. Linkage analyses were performed of dichotomous phenotypes and semi-quantitative traits including the AD severity score. We analysed the novel AD sample alone and together with the previously examined sample. AD severity showed a maximum Z-score of 3.7 at 4q22.1 suggesting the localization of a novel gene for AD severity. A maximum MOD score of 4.6 was obtained at 3p24 for the AD phenotype, providing the first significant linkage of AD at this locus. A maximum MLS score of 3.3 was obtained at 3q21 for IgE-associated AD, and evidence of linkage was also obtained at 3p22.2-21.31, 3q13, 4q35, and 18q12. The results presented should provide a firm basis for gene-targeting studies of AD and related disorders.     

Estimating plant competition coefficients and predicting community dynamics from non-destructive pin-point data: a case study with Calluna?vulgaris and Deschampsia?flexuosa

A method is proposed for estimating plant competition coefficients and predicting the dynamics of herb and grassland plant communities from non-destructive pin-point measurements. The method is applied to inter-specific competition in a natural heathland community with relatively few interacting species. The study shows that the dynamics of the heathland plant community may be thought of as essentially a two-species system of Calluna vulgaris and Deschampsia flexuosa. There were significant competitive interactions between C. vulgaris and D. flexuosa. D. flexuosa affected both the cover and compactness of C. vulgaris individuals as a function of the compactness the previous year, whereas C. vulgaris significantly affected only the compactness of D. flexuosa. There was a significant negative effect of drought on the compactness of both C. vulgaris and D. flexuosa individuals, whereas night warming had no significant effects on either species. The predicted long-term outcome of the competitive interaction between C. vulgaris and D. flexuosa was that of unstable equilibrium, where the more dominant of the two will outcompete the other. However, when both species are found at relatively high plant covers the two species are predicted to co-exist for a long time period relatively to the time scale of the ageing process of C. vulgaris. Direct analyses of the inter-specific competitive interactions in natural plant communities with non-destructive measurements can provide important new insight into the processes that determine the composition of plant communities.     

Impaired Lysosomal Trimming of N-Linked Oligosaccharides Leads to Hyperglycosylation of Native Lysosomal Proteins in Mice with α-Mannosidosis

α-Mannosidosis is caused by the genetic defect of the lysosomal α-d-mannosidase (LAMAN), which is involved in the breakdown of free α-linked mannose-containing oligosaccharides originating from glycoproteins with N-linked glycans, and thus manifests itself in an extensive storage of mannose-containing oligosaccharides. Here we demonstrate in a model of mice with α-mannosidosis that native lysosomal proteins exhibit elongated N-linked oligosaccharides as shown by two-dimensional difference gel electrophoresis, deglycosylation assays, and mass spectrometry. The analysis of cathepsin B-derived oligosaccharides revealed a hypermannosylation of glycoproteins in mice with α-mannosidosis as indicated by the predominance of extended Man3GlcNAc2 oligosaccharides. Treatment with recombinant human α-mannosidase partially corrected the hyperglycosylation of lysosomal proteins in vivo and in vitro. These data clearly demonstrate that LAMAN is involved not only in the lysosomal catabolism of free oligosaccharides but also in the trimming of asparagine-linked oligosaccharides on native lysosomal proteins.The lysosomal α-d-mannosidase (LAMAN; EC 3.2.1.24) belongs to the group of at least seven lysosomal exoglycosidases which sequentially degrade oligosaccharides derived from glycoproteins (2, 31). These glycoproteins enter the lysosomal compartment by either endocytic pathways (extracellular and plasma membrane proteins) or autophagic processes (intracellular proteins). In addition, free oligosaccharides originating from lipid-linked oligosaccharides in the endoplasmic reticulum and from glycoproteins by the endoplasmic reticulum-associated protein degradation (ERAD) pathway are transported into the lysosome, where these oligosaccharides are subsequently degraded (9, 45). Inside the lysosome, the degradation of the glycoproteins is described as a bidirectional process in which on the one hand the polypeptide is hydrolyzed by a cohort of lysosomal endo- and exoproteases with partially overlapping specificities like cathepsins and other peptidases (like DPP II and TPP-I [19, 40, 52). On the other hand, the sugar moiety is stepwise hydrolyzed into its monosaccharides by exoglycosidases. The precise order of the bidirectional breakdown of glycoproteins is unclear, although assumptions can be made based on the analysis of the storage products of the different glycoproteinoses (31). Therefore, it is assumed that an efficient degradation of the oligosaccharide chain is highly dependent on the cleavage of the protein-oligosaccharide linkage by the glycosylasparaginase (2, 31). In contrast, the proteolysis of the polypeptide backbone is mainly unaffected by intact oligosaccharide structures on the glycoproteins (1).LAMAN has a broad substrate specificity, cleaving nonreducing terminal α1,2-, α1,3-, and α1,6-mannosyl linkages found in complex-type, hybrid-type, and high-mannose-type asparagine-linked glycans (30, 60). Additionally, a second lysosomal mannosidase (MAN2B2) specific for the core α1,6 branch was characterized and found to be dependent on the prior enzymatic activity of lysosomal glycosylasparaginase or chitobiase, releasing Man3GlcNAc2 and Man3GlcNAc oligosaccharides, respectively (21, 36). The cooperation of this novel core-specific α1,6-mannosidase with chitobiase is also reflected by their similar tissue-specific expression patterns in humans and rodents and their simultaneous absence in cattle and cats (2, 14).LAMAN deficiency results in the rare lysosomal storage disorder (LSD) α-mannosidosis, which is clinically characterized by progressive mental retardation, dysostosis multiplex, impaired hearing, immune defects, and mild hepatosplenomegaly. However, the onset of symptoms varies greatly and the clinical severity of α-mannosidosis patients ranges from mildly affected to severely affected, lacking a genotype-phenotype correlation (29). Patients also show elevated serum and urine oligosaccharide levels and an enlargement of the lysosomal compartment which is considered to be caused by the accumulation of undegraded oligosaccharides. The major lysosomal storage product is the trisaccharide Man2GlcNAc, although oligosaccharides with up to eight mannosyl residues were detected in the urine and serum of patients, indicating their lysosomal accumulation as well (4, 33). From these findings, one can draw the conclusion that beside metabolic intermediates of the glycoprotein degradation, a considerable number of oligosaccharides originate from dolichol-linked oligosaccharides or from glycoproteins that failed quality control in the endoplasmic reticulum and thus are degraded by the proteasome, leaving behind highly mannosylated glycans (23, 32, 41). It is assumed that 70% of the stored oligosaccharides derive from complex- and hybrid-type glycans, 10% derive from high-mannose-type glycans, and 20% derive from biosynthetic intermediates, e.g., lipid-linked oligosaccharides (61).Naturally occurring animal models for α-mannosidosis have been described for cats (8, 55), cattle (6, 24), and guinea pigs (12). The animal models have been the subjects of various studies dealing with neuropathological, behavioral, and therapeutic aspects of α-mannosidosis (3, 13, 38). It was shown with guinea pigs and cats that enzyme replacement therapy (ERT) and bone marrow transplantation, respectively, provided a benefit concerning clinical manifestations and remarkable success in the central nervous system of cats after bone marrow transplantation (13, 56).Aside from the naturally occurring models, a mouse model for α-mannosidosis was generated in which the LAMAN gene was disrupted by gene targeting. This mouse model phenotypically resembled a mild variant of the human disease (46). We exploited this mouse model to develop an ERT approach as already clinically established for other LSDs like Gaucher disease, Hunter disease, or Pompe disease. For this purpose, LAMAN preparations from different species were proven to be efficacious for visceral organs, and most remarkably, we demonstrated that high-dose administration of recombinant human LAMAN (rhLAMAN) affected the central neural storage (39). Very recently, Blanz et al. confirmed the influence of high-dosage ERT on the peripheral as well as the central nervous system in the same mouse model and showed clearance of storage material in hippocampal neurons in particular (5). Here, we report on structural alterations of lysosomal proteins in mice with α-mannosidosis due to hyperglycosylation and the reversibility by ERT.     

Assessment of the Potential Role of Muscle Spindle Mechanoreceptor Afferents in Chronic Muscle Pain in the Rat Masseter Muscle

Background

The phenotype of large diameter sensory afferent neurons changes in several models of neuropathic pain. We asked if similar changes also occur in “functional” pain syndromes.

Methodology/Principal Findings

Acidic saline (AS, pH 4.0) injections into the masseter muscle were used to induce persistent myalgia. Controls received saline at pH 7.2. Nocifensive responses of Experimental rats to applications of Von Frey Filaments to the masseters were above control levels 1–38 days post-injection. This effect was bilateral. Expression of c-Fos in the Trigeminal Mesencephalic Nucleus (NVmes), which contains the somata of masseter muscle spindle afferents (MSA), was above baseline levels 1 and 4 days after AS. The resting membrane potentials of neurons exposed to AS (n = 167) were hyperpolarized when compared to their control counterparts (n = 141), as were their thresholds for firing, high frequency membrane oscillations (HFMO), bursting, inward and outward rectification. The amplitude of HFMO was increased and spontaneous ectopic firing occurred in 10% of acid-exposed neurons, but never in Controls. These changes appeared within the same time frame as the observed nocifensive behaviour. Ectopic action potentials can travel centrally, but also antidromically to the peripheral terminals of MSA where they could cause neurotransmitter release and activation of adjacent fibre terminals. Using immunohistochemistry, we confirmed that annulospiral endings of masseter MSA express the glutamate vesicular transporter VGLUT1, indicating that they can release glutamate. Many capsules also contained fine fibers that were labelled by markers associated with nociceptors (calcitonin gene-related peptide, Substance P, P2X3 receptors and TRPV1 receptors) and that expressed the metabotropic glutamate receptor, mGluR5. Antagonists of glutamatergic receptors given together with the 2^nd injection of AS prevented the hypersensitivity observed bilaterally but were ineffective if given contralaterally.

Conclusions/Significance

Low pH leads to changes in several electrical properties of MSA, including initiation of ectopic action potentials which could propagate centrally but could also invade the peripheral endings causing glutamate release and activation of nearby nociceptors within the spindle capsule. This peripheral drive could contribute both to the transition to, and maintenance of, persistent muscle pain as seen in some “functional” pain syndromes.     

Dissecting complex phenotypes using the genomics of twins

Genetics in the post-genomic period is shifting from structural to functional genetics or genomics. Meanwhile, the use of twins is largely expanding from traditional heritability estimation for disease phenotypes to the study of both diseases and various molecular phenotypes, such as the regulatory phenotypes in functional genomics concerning gene expression and regulation, by engaging both classical twin design and marker-based gene mapping techniques in genetic epidemiology. New research designs have been proposed for making novel uses of twins in studying the molecular basis in the epigenetics of human diseases. Besides, twins not only serve as ideal samples for disease gene mapping using conventional genetic markers but also represent an excellent model for associating DNA copy number variations, a structural genetic marker, with human diseases. It is believed that, with the rapid development in biotechniques and new advances in bioinformatics, the unique samples of twins will make new contributions to our understanding of the nature and nurture in complex disease development and in human health. This paper aims at summarizing the new uses of twins in current genetic studies and suggesting novel proposes together with useful design and analytical strategies.     

Diversity and host specificity of the Verminephrobacter-earthworm symbiosis

Symbiotic bacteria of the genus Verminephrobacter (Betaproteobacteria) were detected in the nephridia of 19 out of 23 investigated earthworm species (Oligochaeta: Lumbricidae) by 16S rRNA gene sequence analysis and fluorescence in situ hybridization (FISH). While all four Lumbricus species and three out of five Aporrectodea species were densely colonized by a mono-species culture of Verminephrobacter, other earthworm species contained mixed bacterial populations with varying proportions of Verminephrobacter; four species did not contain Verminephrobacter at all. The Verminephrobacter symbionts could be grouped into earthworm species-specific sequence clusters based on their 16S rRNA and RNA polymerase subunit B (rpoB) genes. Closely related host species harboured more closely related symbionts than did distantly related hosts. Co-diversification of the symbiotic partners could not be demonstrated unambiguously due to the poor resolution of the host phylogeny [based on histone H3 and cytochrome c oxidase subunit I (COI) gene sequence analyses]. However, there was a pattern of symbiont diversification within four groups of closely related hosts. The mean rate of symbiont 16S rRNA gene evolution was determined using a relaxed clock model, and the rate was calibrated with paleogeographical estimates of the time of origin of Lumbricid earthworms. The calibrated rates of symbiont 16S rRNA gene evolution are 0.012-0.026 substitutions per site per 50 million years and thus similar to rates reported from other symbiotic bacteria.     

Reaction engineering studies for the production of 2-hydroxyisobutyric acid with recombinant Cupriavidus necator H 16

Recombinant Cupriavidus necator H 16 with a novel metabolic pathway using a cobalamin-dependent mutase was exploited to produce 2-hydroxyisobutyric acid (2-HIBA) from renewable resources through microbial fermentation. 2-HIBA production capacities of different strains of C. necator H 16 deficient in the PHB synthase gene and genetically engineered to enable the production of 2-HIBA from the intracellular PHB precursor (R)-3-hydroxybutyryl-CoA were evaluated in 48 parallel milliliter-scale stirred tank bioreactors (V = 11 mL). The effects of media composition, limitations, pH, and feed rate were studied with respect to the overall process performances of the different recombinant strains. 2-HIBA production was at a maximum at nitrogen limiting conditions and if the pH was controlled between 6.8 and 7.2 under fed-batch operating conditions (intermittent fructose addition). The final concentration of 2-HIBA was 7.4 g L⁻¹ on a milliliter scale. Best reaction conditions identified on the milliliter scale were transferred to a laboratory-scale fed-batch process in a stirred tank bioreactor (V = 2 L). Two different process modes for the production of 2-HIBA, a single-phase and a dual-phase fermentation procedure, were evaluated and compared on a liter scale. The final concentration of 2-HIBA was 6.4 g L⁻¹ on a liter scale after 2 days of cultivation.     

Vaccination with autologous dendritic cells pulsed with multiple tumor antigens for treatment of patients with malignant melanoma: results from a phase I/II trial

Background aimsDendritic cells are regarded as the most effective antigen presenting cells and coordinators of the immune response and therefore suitable as vaccine basis. Here we present results from a clinical study in which patients with malignant melanoma (MM) with verified progressive disease received vaccination with autologous monocyte-derived mature dendritic cells (DC) pulsed with p53, survivin and telomerase-derived peptides (HLA-A2⁺ patients) or with autologous/allogeneic tumor lysate (HLA-A2^? patients) in combination with low-dose interleukin (IL)-2 and interferon (IFN)-α2b.ResultsOf 46 patients who initiated treatment, 10 stopped treatment within 1–4 weeks because of rapid disease progression and deterioration. After 8 weeks, 36 patients were evaluable: no patient had an objective response, 11 patients had stable disease (SD); six had continued SD after 4 months, and three patients had prolonged SD for more than 6 months. The mean overall survival time was 9 months, with a significantly longer survival (18.4 months) of patients who attained SD compared with patients with progressive disease (PD) (5 months). Induction of antigen-specific T-cell responses was analyzed by multidimensional encoding of T cells using HLA-A2 major histocompatibility complex (MHC) multimers. Immune responses against five high-affinity vaccine peptides were detectable in the peripheral blood of six out of 10 analyzed HLA-A2⁺ patients. There was no observed correlation between the induction of immune responses and disease stabilization. A significant lower blood level of regulatory T cells (CD25^high CD4 T cells) was demonstrable after six vaccinations in patients with SD compared with PD.ConclusionsVaccination was feasible and safe. Treatment-associated SD was observed in 24% of the patients. SD correlated with prolonged survival suggesting a clinical benefit. Differences in the level of regulatory T cells among SD and PD patients could indicate a significant role of these immune suppressive cells.     

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).