Antibodies of IgG,IgA and IgM isotypes against cyclic citrullinated peptide precede the development of rheumatoid arthritis

Introduction  

We and others have previously shown that antibodies against cyclic citrullinated proteins (anti-CCP) precede the development of rheumatoid arthritis (RA) and in a more recent study we reported that individuals who subsequently developed RA had increased concentrations of several cytokines and chemokines years before the onset of symptoms of joint disease. Here we aimed to evaluate the prevalence and predictive values of anti-CCP antibodies of IgG, IgM and IgA isotype in individuals who subsequently developed RA and also to relate these to cytokines and chemokines, smoking, genetic factors and radiographic score.     

Increased expression of bradykinin type-1 receptors in endothelium of intramyocardial coronary vessels in human failing hearts

In experimental animals, bradykinin type-1 receptors (BK-1Rs) are induced during inflammation and ischemia, and, by exerting either cardioprotective or cardiotoxic effects, they may contribute to the pathogenesis of heart failure. Nothing is known about the expression of BK-1Rs in human heart failure. Human heart tissue was obtained from excised hearts of patients undergoing cardiac transplantation (n = 13), due to idiopathic dilated cardiomyopathy (IDC; n = 7) or to coronary heart disease (CHD; n = 6), and from normal hearts (n = 6). The expression of BK-1Rs was analyzed by means of competitive RT-PCR, Western blot analysis, and immunohistochemistry. Expression of BK-1R mRNA was increased in both IDC (2.8-fold) and CHD (2.1-fold) hearts compared with normal hearts. The observed changes were verified at the protein level. Expression of BK-1Rs in failing hearts localized to the endothelium of intramyocardial coronary vessels and correlated with an increased expression of TNF-alpha in the vessel wall. Treatment of human coronary artery endothelial cells with TNF-alpha increases their BK-1R expression. These novel results show that BK-1Rs are induced in the endothelium of intramyocardial coronary vessels in failing human hearts and so may participate in the pathogenesis of heart failure.     

Fas/Fas Ligand�Cmediated Apoptosis in Different Cell Lineages and Functional Compartments of Human Lymph Nodes

We have optimized an immunohistochemical double-staining method combining immunohistochemical lymphocyte lineage marker detection and apoptosis detection with terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling. The method was used to trace Fas-mediated apoptosis in human reactive lymph nodes according to cell lineage and anatomical location. In addition to Fas, we also studied the expression of Fas ligand (FasL), CD3, CD20, CD19, CD23, and CD68 of apoptotic cells. The presence of simultaneous Fas and FasL positivity indicated involvement of activation-induced death in the induction of paracortical apoptosis. FasL expression in the high endothelial venules might be an inductor of apoptosis of Fas-positive lymphoid cells. In addition to B-lymphocyte apoptosis in the germinal centers, there was often a high apoptosis rate of CD23-expressing follicular dendritic cells. In summary, our double-staining method provides valuable new information about the occurrence and mechanisms of apoptosis of different immune cell types in the lymph node compartments. Among other things, we present support for the importance of Fas/FasL–mediated apoptosis in lymph node homeostasis. (J Histochem Cytochem 58:131–140, 2010)     

Effect of Concentrate Crude Protein Level on Grass Silage Intake,Milk Yield and Nutrient Utilisation by Dairy Cows in Early Lactation

Twenty-one multiparous dairy cows were fed concentrates containing three levels (119, 154 and 191g/kg DM) of crude protein (CP) during the first ten weeks of lactation. Part of the grain and molassed sugar beat pulp was substituted with 0% (RSM0), 15% (RSM15) or 30% (RSM30) rapeseed meal. Wilted grass silage was fed ad libitum after calving. The average response between RSM0 and RSM15 was +1.66kg milk/d per percentage unit change in concentrate CP content. No further response occurred between RSM15 and RSM30. The positive effect of RSM inclusion was seen throughout the experimental period and was associated with increased plasma non esterified fatty acids (NEFA) and decreased plasma insulin concentration one week after calving, and higher efficiency of metabolisable energy utilisation for milk production. Digestibility of the diet remained unaffected. Milk and plasma urea tended to increase with RSM30 indicating excessive supply of rumen degradable protein. Because of the limited potential of cows to compensate for a deficit in feed protein supply by mobilising tissue protein, a substantial milk yield response can be achieved with a moderate level of protein supplementation during early lactation.     

D2 Dopamine Receptors Stimulate Mitogenesis Through Pertussis Toxin-Sensitive G Proteins and Ras-Involved ERK and SAP/JNK Pathways in Rat C6-D2L Glioma Cells

Abstract: Dopamine D2 receptors are members of the G protein-coupled receptor superfamily and are expressed on both neurons and astrocytes. Using rat C6 glioma cells stably expressing the rat D2L receptor, we show here that dopamine (DA) can activate both the extracellular signal-regulated kinase (ERK) and c-Jun NH₂-terminal kinase (JNK) pathways through a mechanism involving D2 receptor-G protein complexes and the Ras GTP-binding protein. Agonist binding to D2 receptors rapidly activated both kinases within 5 min, reached a maximum between 10 and 15 min, and then gradually decreased by 60 min. Maximal activation of both kinases occurred with 100 nM DA, which produced a ninefold enhancement of ERK activity and a threefold enhancement of JNK activity. DA-induced kinase activation was prevented by either (+)-butaclamol, a selective D2 receptor antagonist, or pertussis toxin, an uncoupler of G proteins from receptors, but not by (?)-butaclamol, the inactive isomer of (+)-butaclamol. Cotransfection of RasN17, a dominant negative Ras mutant, prevented DA-induced activation of both ERK and JNK. PD098059, a specific MEK1 inhibitor, also blocked ERK activation by DA. Transfection of SEK1(K → R) vector, a dominant negative SEK1 mutant, specifically prevented DA-induced JNK activation and subsequent c-Jun phosphorylation without effect on ERK activation. Furthermore, stimulation of D2 receptors promoted [³H]thymidine incorporation with a pattern similar to that for kinase activation. DA mitogenesis was tightly linked to Ras-dependent mitogen-activated protein kinase (MAPK) and JNK pathways. Transfection with RasN17 and application of PD098059 blocked DA-induced DNA synthesis. Transfection with FlagΔ169, a dominant negative c-Jun mutant, also prevented stimulation of [³H]thymidine incorporation by DA. The demonstration of D2 receptor-stimulated MAPK pathways may help to understand dopaminergic physiological functions in the CNS.     

Comparison of sensitivity between gas chromatography–low-resolution mass spectrometry and gas chromatography–high-resolution mass spectrometry for determining metandienone metabolites in urine

In doping control laboratories the misuse of anabolic androgenic steroids is commonly investigated in urine by gas chromatography–low-resolution mass spectrometry with selected ion monitoring (GC–LRMS–SIM). By using high-resolution mass spectrometry (HRMS) detection sensitivity is improved due to reduction of biological background. In our study HRMS and LRMS methods were compared to each other. Two different sets were measured both with HRMS and LRMS. In the first set metandienone (I) metabolites 17α-methyl-5β-androstan-3α,17β-diol (II), 17-epimetandienone (III), 17β-methyl-5β-androst-1-ene-3α,17α-diol (IV) and 6β-hydroxymetandienone (V) were spiked in urine extract prepared by solid-phase extraction, hydrolysis with β-glucuronidase from Escherichia coli and liquid–liquid extraction. In the second set the metabolites were first spiked in blank urine samples of four male persons before pretreatment. Concentration range of the spiked metabolites was 0.1–10 ng/ml in both sets. With HRMS (resolution of 5000) detection limits were 2–10 times lower than with LRMS. However, also with the HRMS method the biological background hampered detection and compounds from matrix were coeluted with some metabolites. For this reason the S/N values of the metabolites spiked had to be first compared to S/N values of coeluted matrix compounds to get any idea of detection limits. At trace concentrations selective isolation procedures should be implemented in order to confirm a positive result. The results suggest that metandienone misuse can be detected by HRMS for a prolonged period after stopping the intake of metandienone.     

Molecular mechanism of angelman syndrome in two large families involves an imprinting mutation.

Patients with Angelman syndrome (AS) and Prader-Willi syndrome with mutations in the imprinting process have biparental inheritance but uniparental DNA methylation and gene expression throughout band 15q11-q13. In several of these patients, microdeletions upstream of the SNRPN gene have been identified, defining an imprinting center (IC) that has been hypothesized to control the imprint switch process in the female and male germlines. We have now identified two large families (AS-O and AS-F) segregating an AS imprinting mutation, including one family originally described in the first genetic linkage of AS to 15q11-q13. This demonstrates that this original linkage is for the 15q11-q13 IC. Affected patients in the AS families have either a 5.5- or a 15-kb microdeletion, one of which narrowed the shortest region of deletion overlap to 1.15 kb in all eight cases. This small region defines a component of the IC involved in AS (ie., the paternal-to-maternal switch element). The presence of an inherited imprinting mutation in multiple unaffected members of these two families, who are at risk for transmitting the mutation to affected children or children of their daughters, raises important genetic counseling issues.     

Characterization of a New Epidemic Necrotic Pyoderma in Fur Animals and Its Association with Arcanobacterium phocae Infection

A new type of pyoderma was detected in Finnish fur animals in 2007. The disease continues to spread within and between farms, with severe and potentially fatal symptoms. It compromises animal welfare and causes considerable economic losses to farmers. A case-control study was performed in 2010–2011 to describe the entity and to identify the causative agent. Altogether 99 fur animals were necropsied followed by pathological and microbiological examination. The data indicated that the disease clinically manifests in mink (Neovison vison) by necrotic dermatitis of the feet and facial skin. In finnraccoons (Nyctereutes procyonoides), it causes painful abscesses in the paws. Foxes (Vulpes lagopus) are affected by severe conjunctivitis and the infection rapidly spreads to the eyelids and facial skin. A common finding at necropsy was necrotic pyoderma. Microbiological analysis revealed the presence of a number of potential causative agents, including a novel Streptococcus sp. The common finding from all diseased animals of all species was Arcanobacterium phocae. This bacterium has previously been isolated from marine mammals with skin lesions but this is the first report of A. phocae isolated in fur animals with pyoderma. The results obtained from this study implicate A. phocae as a potential causative pathogen of fur animal epidemic necrotic pyoderma (FENP) and support observations that the epidemic may have originated in a species -shift of the causative agent from marine mammals. The variable disease pattern and the presence of other infectious agents (in particular the novel Streptococcus sp.) suggest a multifactorial etiology for FENP, and further studies are needed to determine the environmental, immunological and infectious factors contributing to the disease.