Antibodies to mutated citrullinated vimentin and disease activity score in early arthritis: a cohort study

Introduction  

The aim of our study was to investigate the association between arthritic disease activity and antibodies to mutated citrullinated vimentin (anti-MCV), because such a relation has been suggested.     

Effect of the environment on the physiology of the sow during late pregnancy, farrowing and early lactation

We investigated the effect of two types of housing on the duration of farrowing and the physiology of sows before and after farrowing. We assigned 20 sows (PEN) to farrowing pens (210 cm x 335 cm) enriched with straw bedding and 18 sows (CRATE) were placed in farrowing crates (80 cm x 210 cm) with no bedding material. We sampled the animals during period A (from day -5 before farrowing to day +1 of lactation) and during period B (from days +2 to +5 of lactation). We took daily venous blood samples for progesterone measurements and four salivary samples per day (at 09:00, 11:00, 13:00 and 15:00) for cortisol determination. In addition, intensive blood sampling was performed in 18 catheterised sows (9 PEN and 9 CRATE) to determine the level of oxytocin during farrowing. The treatment had no effect on litter size, piglet mortality at birth and weaning, growth of the piglets between days 1 and 5 of life. We found no significant difference in the cortisol concentration between the two groups during period A (p=0.36). Significant difference was found in period B, where the CRATE group had a higher concentration of cortisol than the PEN group (p=0.03). Progesterone concentration did not differ between the two groups (p=0.80). The duration of farrowing was on average 93 min longer in the CRATE sows (n=15), with a mean of 311+/-35 min (mean+/-S.D.), than in the PEN sows (n=19), with a mean of 218+/-24 min (p=0.03). In addition, the mean interval between each piglet expulsion was longer in the CRATE group (n=11), with a mean of 25+/-4 min, than in the PEN group (n=15) with a mean of 16+/-2 min (p=0.05). During farrowing, the post-expulsion oxytocin pulses average tended to be higher in the PEN group (77.6+/-47.6 ng/ml) than in the CRATE group (38.1+/-24.6 pg/ml) (p=0.08). The concentration of oxytocin strongly affected the duration of farrowing (p<0.001). In conclusion, this study showed that the environment influences the physiology of the sow during farrowing and early lactation.     

Strain persistence of invasive Candida albicans in chronic hyperplastic candidosis that underwent malignant change

Objectives: The aim of this study was to assess persistence and tissue invasion of Candida albicans strains isolated from a 65 year‐old patient with chronic hyperplastic candidosis (CHC), that subsequently developed into squamous cell carcinoma (SCC). Materials and Methods: C. albicans (n=7) were recovered from the oral cavity of the patient over seven years. Confirmation of CHC and SCC in this patient was achieved by histopathological examination of incisional biopsy tissue. DNA fingerprinting was performed on the seven isolates from the CHC patient together with a further eight isolates from patients with normal oral mucosa (n=2), chronic atrophic candidosis (n=1), SCC (n=1) and CHC (n=4). Genotyping involved the use of inter‐repeat PCR using the eukaryotic repeat primer 1251. Characterisation of the tissue invasive abilities of the isolates was achieved by infecting a commercially available reconstituted human oral epithelium (RHE; SkinEthic, Nice, France). After 24 h. C. albicans tissue invasion was assessed by histopathological examination. Results: DNA fingerprinting demonstrated strain persistence of C. albicans in the CHC patient over a seven year period despite provision of systemic antifungal therapy. The strain of C. albicans isolated from this patient was categorised as a high invader within the RHE compared to other isolates. Conclusions: Candidal strain persistence was evident in a patient with CHC over seven years. This persistence may be due to incomplete eradication from the oral cavity following antifungal therapy or subsequent recolonisation from other body sites or separate exogenous sources. The demonstration of enhanced in vitro tissue invasion by this particular strain may, in part, explain the progression to carcinoma.     

In vitro zygotic embryo culture of wild Musa acuminata ssp.malaccensis and factors affecting germination and seedling growth

In vitro zygotic embryo culture of wild banana significantly increased the germination compared to greenhouse grown seeds. Embryo orientation and BAP concentration significantly affected germination rate. These factors together with gelling agent, dark and light conditions and coconut water, also showed variable effects on the number of roots per plant, root length, shoot length, number of days to root emergence and number of days to shoot emergence.     

Expression of six peptidases from Lactobacillus helveticus in Lactococcus lactis

For development of novel starter strains with improved proteolytic properties, the ability of Lactococcus lactis to produce Lactobacillus helveticus aminopeptidase N (PepN), aminopeptidase C (PepC), X-prolyl dipeptidyl aminopeptidase (PepX), proline iminopeptidase (PepI), prolinase (PepR), and dipeptidase (PepD) was studied by introducing the genes encoding these enzymes into L. lactis MG1363 and its derivatives. According to Northern analyses and enzyme activity measurements, the L. helveticus aminopeptidase genes pepN, pepC, and pepX are expressed under the control of their own promoters in L. lactis. The highest expression level, using a low-copy-number vector, was obtained with the L. helveticus pepN gene, which resulted in a 25-fold increase in PepN activity compared to that of wild-type L. lactis. The L. helveticus pepI gene, residing as a third gene in an operon in its host, was expressed in L. lactis under the control of the L. helveticus pepX promoter. The genetic background of the L. lactis derivatives tested did not affect the expression level of any of the L. helveticus peptidases studied. However, the growth medium used affected both the recombinant peptidase profiles in transformant strains and the resident peptidase activities. The levels of expression of the L. helveticus pepD and pepR clones under the control of their own promoters were below the detection limit in L. lactis. However, substantial amounts of recombinant pepD and PepR activities were obtained in L. lactis when pepD and pepR were expressed under the control of the inducible lactococcal nisA promoter at an optimized nisin concentration.     

Further phenotypic delineation of subtelomeric (terminal) 4q deletion with emphasis on intracranial and reproductive anatomy

Hypoplastic left heart syndrome(HLHS) refers to the abnormal development of the left-sided cardiac structures, resulting in obstruction to blood flow from the left ventricular outflow tract. In addition, the syndrome includes underdevelopment of the left ventricle, aorta, and aortic arch, as well as mitral atresia or stenosis. HLHS has been reported to occur in approximately 0.016 to 0.036% of all live births. Newborn infants with the condition generally are born at full term and initially appear healthy. As the arterial duct closes, the systemic perfusion becomes decreased, resulting in hypoxemia, acidosis, and shock. Usually, no heart murmur, or a non-specific heart murmur, may be detected. The second heart sound is loud and single because of aortic atresia. Often the liver is enlarged secondary to congestive heart failure. The embryologic cause of the disease, as in the case of most congenital cardiac defects, is not fully known. The most useful diagnostic modality is the echocardiogram. The syndrome can be diagnosed by fetal echocardiography between 18 and 22 weeks of gestation. Differential diagnosis includes other left-sided obstructive lesions where the systemic circulation is dependent on ductal flow (critical aortic stenosis, coarctation of the aorta, interrupted aortic arch). Children with the syndrome require surgery as neonates, as they have duct-dependent systemic circulation. Currently, there are two major modalities, primary cardiac transplantation or a series of staged functionally univentricular palliations. The treatment chosen is dependent on the preference of the institution, its experience, and also preference. Although survival following initial surgical intervention has improved significantly over the last 20 years, significant mortality and morbidity are present for both surgical strategies. As a result pediatric cardiologists continue to be challenged by discussions with families regarding initial decision relative to treatment, and long-term prognosis as information on long-term survival and quality of life for those born with the syndrome is limited.     

N-linked (N-) Glycoproteomics of Urimary Exosomes*

Epithelial cells lining the urinary tract secrete urinary exosomes (40–100 nm) that can be targeted to specific cells modulating their functionality. One potential targeting mechanism is adhesion between vesicle surface glycoproteins and target cells. This makes the glycopeptide analysis of exosomes important. Exosomes reflect the physiological state of the parent cells; therefore, they are a good source of biomarkers for urological and other diseases. Moreover, the urine collection is easy and noninvasive and urinary exosomes give information about renal and systemic organ systems. Accordingly, multiple studies on proteomic characterization of urinary exosomes in health and disease have been published. However, no systematic analysis of their glycoproteomic profile has been carried out to date, whereas a conserved glycan signature has been found for exosomes from urine and other sources including T cell lines and human milk.Here, we have enriched and identified the N-glycopeptides from these vesicles. These enriched N-glycopeptides were solved for their peptide sequence, glycan composition, structure, and glycosylation site using collision-induced dissociation MS/MS (CID-tandem MS) data interpreted by a publicly available software GlycopeptideId. Released glycans from the same sample was also analyzed with MALDI-MS.We have identified the N-glycoproteome of urinary exosomes. In total 126 N-glycopeptides from 51 N-glycosylation sites belonging to 37 glycoproteins were found in our results. The peptide sequences of these N-glycopeptides were identified unambiguously and their glycan composition (for 125 N-glycopeptides) and structures (for 87 N-glycopeptides) were proposed. A corresponding glycomic analysis with released N-glycans was also performed. We identified 66 unique nonmodified N-glycan compositions and in addition 13 sulfated/phosphorylated glycans were also found. This is the first systematic analysis of N-glycoproteome of urinary exosomes.Urine is a combination of plasma filtrate and the secretion profile of cells lining the urino-genital tract. This secretion profile, in addition to proteins and metabolites, also contains exosomes and larger microvesicles that have glycoproteins on their surface. In healthy individuals, ∼70% of the urinary proteome originates from kidneys and the rest represents plasma filtered by glomeruli (1). Proteins present in urine are a collection of proteins secreted by a number of tissues, which changes in disease states (2). Therefore, the urinary proteome may serve as a rich source of biomarkers for urogenital and systemic diseases, which have been reviewed previously (3). Moreover, urine collection is a noninvasive procedure, which makes it an ideal candidate for discovery of novel biomarkers. Only a few large scale studies on urinary proteome and glycoproteome have been published (4, 5). However, most of them have not focused on the glycopeptide characterization.Microvesicles, including exosomes are secreted by many cell types and involved in functions including antigen-presentation, cell-to-cell communication, and immunomodulation (6). These are specialized compartments of cells and they mirror the physiological state of cells secreting them while also providing information about the environment into which they are secreted. For instance, the immunosuppressive and pro-angiogenic environment of cancer may be mediated partially by exosomes (6). Exosomes and other types of microvesicles are abundantly found in urine and thought to be mainly secreted by epithelial cells lining the urinary system (7). These vesicles contain DNA, RNA, and proteins.Glycosylation is an important post-translational modification of proteins and lipids and appears to play many roles, e.g. in cell adhesion, cell-to-cell communication, and immune response (8). Glycosylation is also very important for targeting of proteins to various compartments of the cells. Accordingly, glycans of glycoproteins have important roles in protein sorting to membrane microdomains and furthermore in influencing their intracellular trafficking (9). Microvesicles have a glycan signature that is distinct from the parent cell, suggesting that they originate from specialized membrane microdomains implying a role of glycosylation in microvesicle protein sorting (10, 11). Changes in N-glycans of exosomes from expressed prostatic secretions correlate with disease severity (12). HIV-1 particles were found to have a glycome (the comprehensive glycan profile of a protein, cell, or tissue) largely shared with microvesicles, which is taken to imply that the virus hijacks the glycomachinery of infected cells and uses it systematically to infect additional cells or to deceive the normal immunodefence (13). Thus, the specific glycoproteins of exosomes may be of major impact in targeting exosomes to distinct cells and tissues.Exosome uptake in various cell types has been shown to occur through the mechanisms of clathrin-mediated endocytosis, phagocytosis, and micropinocytosis (14, 15). The uptake of exosomes by dendritic cells and macrophages has been shown to be inhibited by mannose, N-acetylglucosamine, and lactose residues, respectively. This uptake is mediated by a C-type lectin in dendritic cells and galectin-5 in macrophages (14, 15). All this points toward a system in which the exosome glycosylation pattern is kept specific by the cells secreting them to suit the target cell makeup and uptake pathways, and further downstream functions. Taken together, these findings suggest that better understanding of surface glycosylation patterns as well as the glycomics and glycoproteomics of exosomes might help in establishing the specificity of exosome uptake by target cells and activated downstream pathways. This information about exosome uptake might be utilized in therapeutics involving exosomes. Glycome and glycoproteome of urinary microvesicles will provide information not only about the functional state of constituent proteins, but it will also highlight the similarities and differences among proteins that are specifically targeted to exosomes.An N-glycopeptide analysis using collision induced dissociation (CID)-Tandem MS has been reported previously for different sample types (16, 17). This approach provides information about the composition of glycan, partial structure, glycosylation site, and peptide sequence from the same molecule compared with approaches where released glycans or peptides of N-glycopeptides are analyzed separately.We have published an algorithm for an automated analysis of N-glycopeptides (18, 19). A public, web-based software with some changes to the original was developed and named as GlycopeptideId (www.appliednumerics.com, GlycopeptideID version 28–02-28 0.91 beta). The major change was to analyze glycan structures against a database and not with an iterative de novo glycan structure analysis. The proposed structures were further manually validated by the presence of diagnostic ions, when they were available for the given structure.This software was utilized in this study and a comprehensive glycopeptide characterization of urinary exosomal glycoproteins was carried out. We report here the glycan structure determination of urinary exosomal glycoproteins. We have characterized 126 N-glycopeptides representing 51 N-glycosylation sites that belong to 37 glycoproteins. Additionally, glycomic analysis of released N-glycans was also performed and 66 unique modified and 13 sulfated and/or phosphorylated glycans were found. A third of total glycan compositions were common to both the analysis, whereas approximately a third were unique to both the analysis.     

Hip joint replacement surgery for idiopathic osteoarthritis aggregates in families

In order to determine whether there is a genetic component to hip or knee joint failure due to idiopathic osteoarthritis (OA), we invited patients (probands) undergoing hip or knee arthroplasty for management of idiopathic OA to provide detailed family histories regarding the prevalence of idiopathic OA requiring joint replacement in their siblings. We also invited their spouses to provide detailed family histories about their siblings to serve as a control group. In the probands, we confirmed the diagnosis of idiopathic OA using American College of Rheumatology criteria. The cohorts included the siblings of 635 probands undergoing total hip replacement, the siblings of 486 probands undergoing total knee replacement, and the siblings of 787 spouses. We compared the prevalence of arthroplasty for idiopathic OA among the siblings of the probands with that among the siblings of the spouses, and we used logistic regression to identify independent risk factors for hip and knee arthroplasty in the siblings. Familial aggregation for hip arthroplasty, but not for knee arthroplasty, was observed after controlling for age and sex, suggesting a genetic contribution to end-stage hip OA but not to end-stage knee OA. We conclude that attempts to identify genes that predispose to idiopathic OA resulting in joint failure are more likely to be successful in patients with hip OA than in those with knee OA.     

饲喂黄曲霉毒素B_1大鼠肝中AFB_1-DNA加合物的免疫组织化学研究

应用免疫组织化学ＳＰ法对４例饲喂黄曲霉毒素Ｂ１的Ｗｉｓｔａｒ大鼠肝组织中ＡＦＢ１－ＤＮＡ加合物的染色及常规ＨＥ染色发现,４例肝细胞核均出现异型性,但未见肝细胞坏死、增生灶及肝细胞癌;免疫组化揭示部分肝细胞核出现棕黄色、不均质状的ＡＦＢ１－ＤＮＡ加合物,阳性细胞数占１０～８５％。结果提示免疫组织化学方法可作为一种ＡＦＢ１的定位方法,ＡＦＢ１在动物致癌过程中ＡＦＢ１－ＤＮＡ加合物可能具有启动作用