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1.
Acta Psychiatr Scand ; 141(3): 254-264, 2020 03.
Article in English | MEDLINE | ID: mdl-31797353

ABSTRACT

OBJECTIVE: To identify factors associated with a history of suicide attempt in medical students. METHODS: A Web-based survey was sent out to a sample of medical students. A multi-predictor Poisson regression was performed to identify factors associated with a history of suicide attempt. In addition, an elastic net regularization was used to build a risk calculator to identify students at risk for attempted suicide. RESULTS: A total of 4,840 participants were included in the study. Prevalence of suicide attempts in the sample was 8.94%. Risk factors associated with past suicide attempt in the multi-predictor Poisson regression were as follows: female gender (P < 0.001); homosexuality (P < 0.001); low income (P = 0.026); bullying by university peers (P = 0.006); childhood (P = 0.001) or adult (P = 0.001) trauma; family history of suicide (P = 0.005); suicidal ideation within the last month (P < 0.001); daily tobacco use (P = 0.037); and being at severe risk for alcohol abuse (P = 0.023). Our elastic net model performed well with an AUC of 0.83. CONCLUSIONS: This study identifies a number of key factors associated with a history of suicide attempts among medical students. Future longitudinal studies should assess the causal relationship between these factors and suicide attempts. Additionally, these results demonstrate that current available data on suicide attempts among medical students can be used to develop an accurate risk algorithm.


Subject(s)
Students, Medical/psychology , Suicidal Ideation , Suicide, Attempted/statistics & numerical data , Adolescent , Adult , Brazil/epidemiology , Bullying/statistics & numerical data , Female , Humans , Male , Prevalence , Risk Factors , Sex Factors , Surveys and Questionnaires , Young Adult
2.
Vet Immunol Immunopathol ; 187: 48-54, 2017 May.
Article in English | MEDLINE | ID: mdl-28494929

ABSTRACT

Immune mediated keratitis (IMMK) is primarily a non-ulcerative keratitis in horses causing intermittent ocular pain, eventually resulting in visual impairment. Affected horses typically respond to immunomodulatory treatment. However, the underlying cause of the disease remains enigmatic. The current study was undertaken to investigate the presence of autoantibodies in horses with immune mediated keratitis. Using 28 horses with IMMK and 27 healthy controls screening for serum autoantibodies against the corneal proteome using indirect immunofluorescence, one-dimensional (1DE) and two-dimensional electrophoresis (2DE) with subsequent western blot analysis was performed followed by mass spectrometric identification of bands or spots of interest. Indirect immunofluorescence did not reveal a difference in immune response towards corneal proteins between healthy horses and those with IMMK. Using western blot analysis some horses affected by IMMK (4/28) showed a single band (1D) or a single spot (2DE) (5/28) not detected in healthy controls. The corresponding spot was identified as maspin (SERPINB5), a protein responsible for the inhibition of corneal vascularisation, cell migration and cell adhesion to the extracellular matrix. Tests with a recombinant human protein commercially available did not verify blot findings, but the human protein may not be fully cross-reactive. Still, maspin might play a role in some cases of equine IMMK. Further research is needed to clarify the etiology of this disease.


Subject(s)
Autoantibodies/immunology , Autoimmune Diseases/veterinary , Cornea/immunology , Horse Diseases/immunology , Keratitis/veterinary , Animals , Autoimmune Diseases/immunology , Autoimmune Diseases/pathology , Case-Control Studies , Cornea/pathology , Fluorescent Antibody Technique, Indirect/veterinary , Horse Diseases/pathology , Horses/immunology , Keratitis/immunology , Keratitis/pathology
3.
Eur J Trauma Emerg Surg ; 43(1): 9-17, 2017 Feb.
Article in English | MEDLINE | ID: mdl-28101655

ABSTRACT

INTRODUCTION: There is still no general consensus about the management of osteoporotic vertebral fractures. Recommendations depend on type of fracture, grade of instability, bone quality, and general conditions of the patient. Spontaneous fractures may be considered to be treated different compared to cases with high-velocity trauma. METHODS: According to the DVO, patients without trauma should first be treated conservatively. However, there is no more strict time protocol of 3 or 6 week conservative treatment before operations may be indicated. Surgical criteria are not yet distinctly defined. For highly unstable fractures (type B and C according to the AO Spine Classification), posterior instrumentation with cement augmented screws and as long construct, respectively, is adequate. Current literature has been analysed for diagnostic and therapeutic protocols. RESULTS: There is no clear operative concept for burst fractures and classic osteoporotic fractures with dynamic ongoing sintering. Percutaneous vertebral augmentation showed to prevent the fractures from ongoing kyphotic deformity and the patients from painful immobilization. Indications and results of classical vertebroplasty and kyphoplasty have been discussed intensively in the literature. Further development included special injection techniques, cements with different viscosities and stenting systems to reach more stable constructs and avoid typical complications, such as cement extrusion. CONCLUSIONS: This review reports upon indications and limitations of percutaneous vertebral augmentation and the potential development of classifications and therapeutic algorithms.


Subject(s)
Fracture Fixation, Internal/methods , Osteoporotic Fractures/surgery , Spinal Fractures/surgery , Vertebroplasty/methods , Bone Cements , Bone Screws , Humans
4.
Unfallchirurg ; 119(10): 817-24, 2016 Oct.
Article in German | MEDLINE | ID: mdl-27646696

ABSTRACT

BACKGROUND: Intraoperative imaging during spinal interventions has experienced significant developments over the last two decades. By the introduction of flat screen detectors, 3D imaging has been made possible and easier and by developing compact and mobile systems computed tomography can even be used in the operating theater. OBJECTIVE: Presentation of modern intraoperative 3D imaging and navigation in spinal surgery. MATERIAL AND METHODS: The techniques of intraoperative 3D imaging and navigation during spinal procedures are presented based on the currently available literature and own experiences at a German national spine and trauma center. RESULTS: The use of flat panel detectors and the possibility of 3D visualization nowadays substantially facilitate the use of navigation and allow certain control of surgical results even during the intervention. Radiation exposure of the whole team in the operating theater can be significantly reduced by the new techniques. CONCLUSION: The advantages of intraoperative 3D imaging with a clear improvement of visualization for spinal surgeons and the certain control of materials at the end of the operation are obvious. Even the use of navigation has been greatly simplified and can therefore lead to an even greater precision and less radiation exposure. There are even more sophisticated developments, such as operation suites and intraoperative computed tomography but these are initially reserved for selected centers.


Subject(s)
Imaging, Three-Dimensional/methods , Spinal Diseases/diagnostic imaging , Spinal Diseases/surgery , Spinal Fractures/diagnostic imaging , Spinal Fractures/surgery , Surgery, Computer-Assisted/methods , Evidence-Based Medicine , Humans , Laminectomy/methods , Monitoring, Intraoperative/methods , Tomography, X-Ray Computed/methods , Treatment Outcome
5.
Oncogene ; 35(32): 4269-81, 2016 08 11.
Article in English | MEDLINE | ID: mdl-26776161

ABSTRACT

Constitutive activation of the antiapoptotic nuclear factor-κB (NF-κB) signaling pathway is a hallmark of the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphomas (DLBCL). Recurrent oncogenic mutations are found in the scaffold protein CARMA1 (CARD11) that connects B-cell receptor (BCR) signaling to the canonical NF-κB pathway. We asked how far additional downstream processes are activated and contribute to the oncogenic potential of DLBCL-derived CARMA1 mutants. To this end, we expressed oncogenic CARMA1 in the NF-κB negative DLBCL lymphoma cell line BJAB. By a proteomic approach we identified recruitment of ß-catenin and its destruction complex consisting of APC, AXIN1, CK1α and GSK3ß to oncogenic CARMA1. Recruitment of the ß-catenin destruction complex was independent of CARMA1-BCL10-MALT1 complex formation or constitutive NF-κB activation and promoted the stabilization of ß-catenin. The ß-catenin destruction complex was also recruited to CARMA1 in ABC DLBCL cell lines, which coincided with elevated ß-catenin expression. In line, ß-catenin was frequently detected in non-GCB DLBCL biopsies that rely on chronic BCR signaling. Increased ß-catenin amounts alone were not sufficient to induce classical WNT target gene signatures, but could augment TCF/LEF-dependent transcriptional activation in response to WNT signaling. In conjunction with NF-κB, ß-catenin enhanced expression of immunosuppressive interleukin-10 and suppressed antitumoral CCL3, indicating that ß-catenin can induce a favorable tumor microenvironment. Thus, parallel activation of NF-κB and ß-catenin signaling by gain-of-function mutations in CARMA1 augments WNT stimulation and is required for regulating the expression of distinct NF-κB target genes to trigger cell-intrinsic and extrinsic processes that promote DLBCL lymphomagenesis.


Subject(s)
CARD Signaling Adaptor Proteins/genetics , CARD Signaling Adaptor Proteins/metabolism , Carcinogenesis , Guanylate Cyclase/genetics , Guanylate Cyclase/metabolism , Lymphoma, Large B-Cell, Diffuse/metabolism , NF-kappa B/metabolism , Signal Transduction , beta Catenin/metabolism , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Humans , Lymphoma, Large B-Cell, Diffuse/pathology , Mutation , Protein Stability , TCF Transcription Factors/metabolism , rho GTP-Binding Proteins/metabolism
6.
Chirurg ; 86(9): 901-14; quiz 915-6, 2015 Sep.
Article in German | MEDLINE | ID: mdl-26307631

ABSTRACT

Fractures of the thoracic and lumbar spine result from high velocity trauma, assuming bone density is normal. The main location of fractures is the thoracolumbar junction. Most injuries can be treated conservatively; however, patients transferred to hospitals and spine centers represent a preselection with more severe trauma and a higher incidence of operative treatment. There is a large variety of operative techniques that can be used, which can be principally differentiated by the approach: posterior or anterior. Dorsal approaches are differentiated by the instrumentation for spondylodesis as open or percutaneous techniques. Minimally invasive options are favored more and more. For osteoporotic bone, cement augmented solutions may be used. Correct reduction of mainly kyphotic malalignment is crucial for the long-term outcome. Biomechanically stable reconstruction of the anterior spinal column is important mainly for the thoracolumbar junction.


Subject(s)
Lumbar Vertebrae/injuries , Spinal Fractures/surgery , Thoracic Vertebrae/injuries , Accidents, Traffic , Adult , Female , Fracture Fixation/methods , Humans , Imaging, Three-Dimensional , Magnetic Resonance Imaging , Male , Middle Aged , Minimally Invasive Surgical Procedures/methods , Multiple Trauma/diagnosis , Multiple Trauma/surgery , Spinal Fractures/diagnosis , Spinal Fusion/methods , Tomography, X-Ray Computed , Trauma Centers
7.
Theriogenology ; 84(9): 1490-8, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26296522

ABSTRACT

Membrane vesicles (MVs) in the ejaculate have been identified in various species and are considered to affect membrane fluidity due to their characteristic molecular composition. Addition of MV to human frozen semen has been shown to improve post-thaw motility. Similarly, a beneficial effect has been suggested for frozen equine semen. As post-thaw canine semen quality varies widely between dogs, the aim of our study was to test for the effect of addition of canine MV on post-thaw semen quality in dogs. Semen samples from 10 male dogs were purified from MV and prepared for freezing. In experiment 1, three groups were compared: sperm frozen (1) with MV (S1); (2) without MV, but MV added immediately after thawing (S2); and (3) without MV (C). Semen analysis included computer-assisted sperm analysis of motility parameters immediately after thawing (t0), after 10 (t10) and 30 minutes (t30), % living sperm, % membrane intact, % morphologically normal sperm (all t0 and t30). Computer-assisted sperm analysis motility distance and velocity parameters (all P < 0.05) and % living sperm (P < 0.001) were significantly affected by treatment with a temporary increase of distance and velocity parameters at t0 to t10, but a significant decrease of the aforementioned parameters at t30 in samples with MV. In experiment 2, different MV protein concentrations added after thawing were compared: 0.05 mg, 0.1 mg, and 0.2 mg/mL. Computer-assisted sperm motility analysis was performed at t0, t10, and t30. No differences between MV concentrations were identified, only a significant interaction between effect of treatment and time for progressive motility (P < 0.01). Our study identified a short-term beneficial effect of canine MV on post-thaw distance and velocity parameters, whereas at t30 progressive motility, motility parameters and % living sperm were reduced in samples with MV compared to C. The results point to species-specific differences regarding the MV effect on frozen semen and indicate the need for further studies using different semen and MV purification protocols and more frequent analyses. At the moment, addition of MV is not an option to improve post-thaw semen quality in dogs.


Subject(s)
Freezing , Semen Preservation/veterinary , Semen/physiology , Animals , Dogs , Male , Semen Preservation/methods
8.
Micron ; 77: 66-73, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26142562

ABSTRACT

Membrane vesicles (MV) have been identified in seminal plasma from various species and they are thought to have a significant impact on semen quality and fertilisation. Although recently presence of MV has been also described in the canine ejaculate, detailed knowledge on their morphology is missing by now. This is, however, needed to provide a basis for detailed biochemical and functional studies as it is generally assumed that different MV populations are responsible for distinct tasks. MV were prepared for light (LM) and transmission electron microscopy (TEM) analysis using samples from normospermic dogs (n=15), hypokinozoospermic dogs (n=2, h) and one castrated azoospermic dog (a). For TEM, a new preparation protocol was used resulting in a higher MV retrieval rate. Using fractionated semen samples, most MV were identified in the second (sperm-rich) fraction in LM. Using pooled ejaculates, three different MV types could be identified in LM: (1) large MV with a marginal accumulation of opaque, granulated material, (2) medium- to small size MV with dense, opaque content and (3) small MV with no further defined contents. No direct contact between sperm and MV could be visualised. In TEM, 11 different MV types were identified based on diameter, structure, contents and electron density of contents as well as presence, number and size of smaller MV inside the MV itself. In normospermic males, secondary vesicles (type i, H, K1/2) included smaller vesicles and had a weighted mean diameter of 409.46 nm; hereof types i, H and K1 were smaller (mean: 287.55 nm, range: 51.25-994.86 nm) and type K2 was larger (mean: 1746.43 nm, range: 1003.66-3289.34 nm). Primary vesicles (mean diameter: 135.29 nm) - without vesicles inside - were differentiated into larger MV (A, B, C1/2) with a mean diameter of 219.63 nm (range: 39.08-1300.13 nm) and small primary MV (F, G) with a mean diameter of 66.12 nm (range: 24.62-99.84 nm). Whereas all mentioned MV were round to oval and mostly double-, rarely multiple-membrane surrounded, one longish primary MV type (L) was identified. In general, small primary vesicles were most common independent of semen quality, but distribution frequency of vesicle types differed between normospermic, pathospermic dogs and the castrated male. Mean weighted diameter of MV was 195.14 nm (range: 24.62-3289.34 nm) in normospermic males with the maximum diameter being smaller in the other dogs (h: 2096.78 nm; a: 1314.06 nm). Our results provide new information about ultrastructure and distribution frequency of canine MV in normospermic males and point to possible differences in MVs depending on semen quality. They provide the basis for further detailed functional analysis of MV subpopulations. Furthermore, the presence of MV in the castrated azoospermic male confirms an at least partly prostatic origin of canine MV.


Subject(s)
Extracellular Vesicles/ultrastructure , Membranes/ultrastructure , Semen/cytology , Animals , Dogs , Ejaculation , Male , Microscopy, Electron, Transmission , Semen Analysis/methods , Spermatozoa/cytology , Spermatozoa/ultrastructure
9.
Mol Metab ; 4(1): 39-50, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25685688

ABSTRACT

OBJECTIVE: Excess lipid intake has been implicated in the pathophysiology of hepatosteatosis and hepatic insulin resistance. Lipids constitute approximately 50% of the cell membrane mass, define membrane properties, and create microenvironments for membrane-proteins. In this study we aimed to resolve temporal alterations in membrane metabolite and protein signatures during high-fat diet (HF)-mediated development of hepatic insulin resistance. METHODS: We induced hepatosteatosis by feeding C3HeB/FeJ male mice an HF enriched with long-chain polyunsaturated C18:2n6 fatty acids for 7, 14, or 21 days. Longitudinal changes in hepatic insulin sensitivity were assessed via the euglycemic-hyperinsulinemic clamp, in membrane lipids via t-metabolomics- and membrane proteins via quantitative proteomics-analyses, and in hepatocyte morphology via electron microscopy. Data were compared to those of age- and litter-matched controls maintained on a low-fat diet. RESULTS: Excess long-chain polyunsaturated C18:2n6 intake for 7 days did not compromise hepatic insulin sensitivity, however, induced hepatosteatosis and modified major membrane lipid constituent signatures in liver, e.g. increased total unsaturated, long-chain fatty acid-containing acyl-carnitine or membrane-associated diacylglycerol moieties and decreased total short-chain acyl-carnitines, glycerophosphocholines, lysophosphatidylcholines, or sphingolipids. Hepatic insulin sensitivity tended to decrease within 14 days HF-exposure. Overt hepatic insulin resistance developed until day 21 of HF-intervention and was accompanied by morphological mitochondrial abnormalities and indications for oxidative stress in liver. HF-feeding progressively decreased the abundance of protein-components of all mitochondrial respiratory chain complexes, inner and outer mitochondrial membrane substrate transporters independent from the hepatocellular mitochondrial volume in liver. CONCLUSIONS: We assume HF-induced modifications in membrane lipid- and protein-signatures prior to and during changes in hepatic insulin action in liver alter membrane properties - in particular those of mitochondria which are highly abundant in hepatocytes. In turn, a progressive decrease in the abundance of mitochondrial membrane proteins throughout HF-exposure likely impacts on mitochondrial energy metabolism, substrate exchange across mitochondrial membranes, contributes to oxidative stress, mitochondrial damage, and the development of insulin resistance in liver.

10.
Br J Dermatol ; 172(4): 994-1001, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25244099

ABSTRACT

BACKGROUND: Chronic hand eczema (CHE) is a common skin disease with a high socioeconomic impact. While some light has been shed on the genetic factors that predispose individuals to the disease, little is known about its actual pathogenesis. OBJECTIVES: We aimed to carry out a systematic and comprehensive analysis of the differential protein expression in CHE using modern mass spectrometry. METHODS: We performed liquid chromatography with tandem mass spectrometry analyses and label-free quantification to analyse the proteomic profile of palmar skin from 12 individuals (six patients with hand eczema and six healthy volunteers). Immunohistochemistry of the palmar skin from seven different patients with hand eczema and seven different healthy volunteers was performed in a second step. RESULTS: With this method we were able to identify 185 candidate proteins with a significantly different abundance in the hand eczema samples. Among them we found several barrier proteins: filaggrin (FLG), FLG-2 and hornerin were all downregulated in the hand eczema samples, as were the desquamation-related enzymes kallikrein-related peptidase (KLK)5 and KLK7 and cystatin E/M. The antimicrobial peptides S100A7 and S100A8/A9 and the small proline-rich protein 2B and S100A11 were upregulated in the diseased skin. Immunohistochemistry confirmed these findings. CONCLUSIONS: Our results corroborate the assumption that skin barrier dysfunction plays an essential role in the pathogenesis of CHE.


Subject(s)
Calcium-Binding Proteins/metabolism , Eczema/etiology , Hand Dermatoses/etiology , Intermediate Filament Proteins/metabolism , Adult , Case-Control Studies , Chronic Disease , Cornified Envelope Proline-Rich Proteins/metabolism , Cystatins/metabolism , Down-Regulation/physiology , Epidermis/metabolism , Female , Filaggrin Proteins , Humans , Immunohistochemistry , Kallikreins/metabolism , Male , Mass Spectrometry , Middle Aged , Proteome/metabolism , S100 Proteins/metabolism , Up-Regulation/physiology
11.
Article in English | MEDLINE | ID: mdl-25293886

ABSTRACT

BACKGROUND: The German National Cohort (GNC) is designed to address research questions concerning a wide range of possible causes of major chronic diseases (e.g. cancer, diabetes, infectious, allergic, neurologic and cardiovascular diseases) as well as to identify risk factors and prognostic biomarkers for early diagnosis and prevention of these diseases. The collection of biomaterials in combination with extensive information from questionnaires and medical examinations represents one of the central study components. OBJECTIVES: In two pretest studies of the German National Cohort conducted between 2011 and 2013, a range of biomaterials from a defined number of participants was collected. Ten study centres were involved in pretest 1 and 18 study centres were involved in pretest 2. Standard operation procedures (SOP) were developed and evaluated to minimize pre-analytical artefacts during biosample collection. Within the pretest studies different aspects concerning feasibility of sample collection/preparation [pretest 1 (a)] and quality control of biomarkers and proteome analyses were investigated [pretest 1 (b), (c)]. Additionally, recruitment of study participants for specific projects and examination procedures of all study centres in a defined time period according to common standards as well as transportation and decentralized storage of biological samples were tested (pretest 2). These analyses will serve as the basis for the biomaterial collection in the main study of the GNC starting in 2014. MATERIALS AND METHODS: Participants, randomly chosen from the population (n = 1000 subjects recruited at ten study sites in pretest 1) were asked to donate blood, urine, saliva and stool samples. Additionally, nasal and oropharyngeal swabs were collected at the study sites and nasal swabs were collected by the participants at home. SOPs for sample collection, preparation, storage and transportation were developed and adopted for pretest 2. In pretest 2, 18 study sites (n = 599 subjects) collected biomaterials mostly identical to pretest 1. Biomarker analyses to test the quality of the biomaterials were performed. RESULTS: In pretest 1 and 2, it was feasible to collect all biomaterials from nearly all invited participants without major problems. The mean response rate of the subjects was 95 %. As one important result we found for example that after blood draw the cellular fraction should be separated from the plasma and serum fractions during the first hour with no significant variation for up to 6 h at 4 ℃ for all analysed biomarkers. Moreover, quality control of samples using a proteomics approach showed no significant clustering of proteins according to different storage conditions. All developed SOPs were validated for use in the main study after some adaptation and modification. Additionally, electronic and paper documentation sheets were developed and tested to record time stamps, volumes, freezing times, and aliquot numbers of the collected biomaterials. DISCUSSION: The collection of the biomaterials was feasible without major problems at all participating study sites. However, the processing times were in some cases too long. To avoid pre-analytical artefacts in sample collection, appropriate standardisation among the study sites is necessary. To achieve this, blood and urine collection will have to be adapted to specific conditions of usage of liquid handling robots, which will be available at all participating study centres in the main study of the GNC. Strict compliance with the SOPs, thorough training of the staff and accurate documentation are mandatory to obtain high sample quality for later analyses. The so obtained biomaterials represent a valuable resource for research on infectious and other common complex diseases in the GNC.


Subject(s)
Biomarkers/analysis , Chronic Disease/epidemiology , Cohort Studies , Population Surveillance/methods , Quality Assurance, Health Care/statistics & numerical data , Specimen Handling/statistics & numerical data , Specimen Handling/standards , Adult , Aged , Chronic Disease/prevention & control , Feasibility Studies , Female , Germany/epidemiology , Humans , Male , Middle Aged , Young Adult
12.
J Proteomics ; 109: 50-62, 2014 Sep 23.
Article in English | MEDLINE | ID: mdl-24998980

ABSTRACT

Retinal pigment epithelium (RPE) builds the outer blood-retinal barrier of the eye and plays an important role in pathogenesis of the sight threatening disease equine recurrent uveitis (ERU). ERU is a spontaneous autoimmune mediated inflammatory disease characterised by the breakdown of the outer blood-retinal barrier and an influx of autoaggressive T-cells into the inner eye. Therefore, identification of molecular mechanisms contributing to changed function of blood-retinal barrier in ERU is important for the understanding of pathophysiology. Cell surface proteins of RPE collected from healthy horses and horses with ERU were captured by in situ biotinylation and analysed with high resolution mass spectrometry coupled to liquid chromatography (LC-MS/MS) to identify differentially expressed proteins. With label free differential proteomics, a total of 27 differently expressed cell surface proteins in diseased RPE could be detected. Significant down-regulation of three very interesting proteins, synaptotagmin 1, basigin and collectrin was verified and further characterised. BIOLOGICAL SIGNIFICANCE: We applied an innovative and successful method to detect changes in the plasma cell surface proteome of RPE cells in a spontaneous inflammatory eye disease, serving as a valuable model for human autoimmune uveitis. We were able to identify 27 differentially expressed plasma cell membrane proteins, including synaptotagmin 1, basigin and collectrin, which play important roles in cell adhesion, transport and cell communication.


Subject(s)
Autoimmune Diseases/metabolism , Eye Proteins/biosynthesis , Horse Diseases/metabolism , Proteomics , Retinal Pigment Epithelium , Uveitis , Animals , Autoimmune Diseases/pathology , Autoimmune Diseases/veterinary , Chromatography, Liquid , Female , Gene Expression Regulation , Horse Diseases/pathology , Horses , Humans , Male , Mass Spectrometry , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/pathology , Uveitis/metabolism , Uveitis/pathology , Uveitis/veterinary
13.
Histochem Cell Biol ; 142(4): 361-71, 2014 Oct.
Article in English | MEDLINE | ID: mdl-24824474

ABSTRACT

The aim of this study was to establish an ex vivo model for a faster optimisation of sample preparation procedures, for example matrix choice, in matrix-assisted laser desorption/ionisation (MALDI) drug imaging studies. The ionisation properties of four drugs, afatinib, erlotinib, irinotecan and pirfenidone, were determined in an ex vivo tissue experiment by spotting decreasing dilution series onto liver sections. Hereby, the drug signals were distinctly detectable using different matrix compounds, which allowed the selection of the optimal matrix for each drug. The analysis of afatinib and erlotinib yielded high drug signals with α-cyano-4-hydroxycinnamic acid matrix, whereas 2,3-dihydroxybenzoic acid was identified as optimal matrix for irinotecan and pirfenidone detection. Our method was validated by a MALDI drug imaging approach of in vivo treated mouse tissue resulting in corresponding findings, indicating the spotting method as an appropriate approach to determine the matrix of choice. The present study shows the accordance between the detection of ex vivo spotted drugs and in vivo administered drugs by MALDI-TOF and MALDI-FT-ICR imaging, which has not been demonstrated so far. Our data suggest the ex vivo tissue spotting method as an easy and reliable model to optimise MALDI imaging measurements and to predict drug detection in tissue sections derived from treated mice prior to the recruitment of laboratory animals, which helps to save animals, time and costs.


Subject(s)
Camptothecin/analogs & derivatives , Liver/chemistry , Models, Animal , Pyridones/analysis , Quinazolines/analysis , Administration, Intravenous , Administration, Oral , Afatinib , Animals , Camptothecin/administration & dosage , Camptothecin/analysis , Erlotinib Hydrochloride , In Vitro Techniques , Irinotecan , Mice , Mice, Inbred C57BL , Mice, Nude , Molecular Structure , Pyridones/administration & dosage , Quinazolines/administration & dosage , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
14.
Leukemia ; 28(12): 2355-66, 2014 Dec.
Article in English | MEDLINE | ID: mdl-24736212

ABSTRACT

T cells have been proven to be therapeutically effective in patients with relapsed leukemias, although target antigens on leukemic cells as well as T-cell receptors (TCRs), potentially recognizing those antigens, are mostly unknown. We have applied an immunopeptidomic approach and isolated human leukocyte antigen (HLA) ligands from primary leukemia cells. We identified a number of ligands derived from different genes that are restrictedly expressed in the hematopoietic system. We exemplarily selected myeloperoxidase (MPO) as a potential target and isolated a high-avidity TCR with specificity for a HLA-B*07:02-(HLA-B7)-restricted epitope of MPO in the single HLA-mismatched setting. T cells transgenic for this TCR demonstrated high peptide and antigen specificity as well as leukemia reactivity in vitro and in vivo. In contrast, no significant on- and off-target toxicity could be observed. In conclusion, we here demonstrate, exemplarily for MPO, that leukemia-derived HLA ligands can be selected for specific effector tool development to redirect T cells to be used for graft manipulation or adoptive T-cell therapies in diverse transplant settings. This approach can be extended to other HLA ligands and HLA molecules in order to provide better treatment options for this life-threatening disease.


Subject(s)
HLA Antigens/immunology , Leukemia, Myeloid/genetics , Leukemia, Myeloid/immunology , Peptides/immunology , Peroxidase/immunology , Receptors, Antigen, T-Cell/genetics , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Animals , Antigen Presentation/immunology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Line , Cell Survival/genetics , Cell Survival/immunology , Disease Models, Animal , Epitope Mapping , Epitopes, T-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/immunology , HLA Antigens/metabolism , HLA-B7 Antigen/immunology , HLA-B7 Antigen/metabolism , Heterografts , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class I/metabolism , Humans , Leukemia, Myeloid/metabolism , Leukemia, Myeloid/mortality , Ligands , Mice , Peptides/metabolism , Peroxidase/chemistry , Peroxidase/genetics , Receptors, Antigen, T-Cell/metabolism , T-Cell Antigen Receptor Specificity/immunology , Transduction, Genetic
15.
Allergy ; 69(5): 632-42, 2014 May.
Article in English | MEDLINE | ID: mdl-24661001

ABSTRACT

BACKGROUND: Type 2 immune responses directed by Th2 cells and characterized by the signature cytokines IL4, IL5, and IL13 play major pathogenic roles in atopic diseases. Single nucleotide polymorphisms in the human Th2 cytokine locus in particular in a locus control region within the DNA repair gene RAD50, containing several RAD50 DNase1-hypersensitive sites (RHS), have been robustly associated with atopic traits in genome-wide association studies (GWAS). Functional variants in IL13 have been intensely studied, whereas no causative variants for the IL13-independent RAD50 signal have been identified yet. This study aimed to characterize the functional impact of the atopy-associated polymorphism rs2240032 located in the human RHS7 on cis-regulatory activity and differential binding of transcription factors. METHODS: Differential transcription factor binding was analyzed by electrophoretic mobility shift assays (EMSAs) with Jurkat T-cell nuclear extracts. Identification of differentially binding factors was performed using mass spectrometry (LC-MS/MS). Reporter vector constructs carrying either the major or minor allele of rs2240032 were tested for regulating transcriptional activity in Jurkat and HeLa cells. RESULTS: The variant rs2240032 impacts transcriptional activity and allele-specific binding of SMAD3, SP1, and additional putative protein complex partners. We further demonstrate that rs2240032 is located in an RHS7 subunit which itself encompasses repressor activity and might be important for the fine-tuning of transcription regulation within this region. CONCLUSION: The human RHS7 critically contributes to the regulation of gene transcription, and the common atopy-associated polymorphism rs2240032 impacts transcriptional activity and transcription factor binding.


Subject(s)
Cytokines/genetics , Gene Expression Regulation , Hypersensitivity, Immediate/genetics , Hypersensitivity, Immediate/metabolism , Locus Control Region , Smad3 Protein/metabolism , Sp1 Transcription Factor/metabolism , Th2 Cells/metabolism , Transcription, Genetic , Alleles , Binding Sites , Gene Order , Humans , Hypersensitivity, Immediate/immunology , Linkage Disequilibrium , Nucleotide Motifs , Polymorphism, Single Nucleotide , Position-Specific Scoring Matrices , Promoter Regions, Genetic , Protein Binding , Regulatory Sequences, Nucleic Acid
16.
Unfallchirurg ; 117(8): 703-9, 2014 Aug.
Article in German | MEDLINE | ID: mdl-23732614

ABSTRACT

INTRODUCTION: Ventral thoracoscopic spondylodesis of the thoracolumbar spine is an elegant treatment strategy. MATERIAL AND METHODS: In the years 2002 and 2003 a total of 16 patients with incomplete cranial burst fractures were treated by ventral thoracoscopic monosegmental spondylodesis and were included in this study prospectively. The data acquisition was done preoperatively, postoperatively and after 3, 6, 12 and 18 months. After 6 years a follow-up examination was performed in 13 of these patients (5 men and 8 women, average age 36.3 years, follow-up rate 81%) and 8 patients were treated ventrally only whereas 5 patients were treated dorsoventrally. RESULTS: The operative reduction of the kyphotic malalignment was superior in the dorsoventrally treated patients. The persistent gain of monosegmental correction after 6 years seemed to be higher in the patient group treated dorsoventrally. The average physical component summary (PSC) scores were comparable to a control group of the same age and revision surgery was performed in two patients both related to the iliac crest bone graft. CONCLUSIONS: The ventral and dorsoventral therapy strategies showed good and very good functional outcomes, respectively. The dorsoventral treatment concept secured a persistent gain of monosegmental correction which seemed to be superior compared to a ventral only therapy strategy.


Subject(s)
Fractures, Compression/surgery , Skull Fractures/surgery , Spinal Fractures/surgery , Spinal Fusion/methods , Thoracic Vertebrae/injuries , Thoracic Vertebrae/surgery , Adult , Female , Fractures, Compression/diagnostic imaging , Humans , Longitudinal Studies , Male , Radiography , Skull Fractures/diagnostic imaging , Spinal Fractures/diagnostic imaging , Thoracoscopy/methods , Treatment Outcome
17.
Unfallchirurg ; 117(5): 428-36, 2014 May.
Article in German | MEDLINE | ID: mdl-23736969

ABSTRACT

BACKGROUND: The purpose of this investigation was to evaluate the options of percutaneous systems for reducing relevant posttraumatic kyphosis in spinal burst fractures. Clinical advantages of percutaneous techniques are evident from the literature and a disadvantage can be a lack of repositioning options in reducing the fracture kyphosis. Better results seem to be possible with new techniques and especially monoaxial percutaneous screws. PATIENTS AND METHODS: A total of 70 patients with burst fractures (AO type Magerl A3.1-A3.3) of the thoracolumbar spine were treated with a special percutaneous reduction technique in the Trauma Clinic in Murnau (BGU) Germany between July 2009 and March 2011. Posttraumatic, intraoperative and postoperative kyphosis was measured in computed tomography (CT) scans in monosegmental and bisegmental angles. Two different percutaneous fixation systems were compared for reduction. Statistical analyses were carried out with Student's t-test. RESULTS: We found a highly significant difference between preoperative and postoperative kyphosis angles but no differences in reduction between the two percutaneous systems. In 39 cases additional reconstruction of the anterior column was necessary because of a ventral defect. In comparison to the MCS 2 study of the German Society of Trauma Surgery (DGU) we found no differences in postoperative kyphosis angles (3°). CONCLUSION: A significant reduction of posttraumatic kyphosis of thoracolumbar burst fractures is possible with percutaneous techniques. Prerequisites are percutaneous monoaxial screws and tools and a special percutaneous technique as described.


Subject(s)
Fracture Fixation, Internal/instrumentation , Fracture Fixation, Internal/methods , Fractures, Compression/surgery , Kyphosis/surgery , Spinal Fractures/surgery , Thoracic Vertebrae/injuries , Thoracic Vertebrae/surgery , Bone Screws , Female , Fractures, Compression/complications , Humans , Kyphosis/diagnosis , Kyphosis/etiology , Male , Middle Aged , Reoperation/instrumentation , Reoperation/methods , Spinal Fractures/complications , Treatment Outcome
18.
J Anim Physiol Anim Nutr (Berl) ; 98(1): 32-42, 2014 Feb.
Article in English | MEDLINE | ID: mdl-23173557

ABSTRACT

Colour vision in animals is an interesting, fascinating subject. In this study, we examined a wide variety of species for expression of S-opsin (blue sensitive) and M-/L-opsin (green-red sensitive) in retinal cones using two novel monoclonal antibodies specific for peptides from human opsins. Mouse, rat and hare did not express one of the investigated epitopes, but we could clearly prove existence of cones through peanut agglutinin labelling. Retinas of guinea pig, dog, wolf, marten, cat, roe deer, pig and horse were positive for S-opsin, but not for M-/L-opsin. Nevertheless all these species are clearly at least dichromats, because we could detect further S-opsin negative cones by labelling with cone arrestin specific antibody. In contrast, pheasant and char had M-/L-opsin positive cones, but no S-opsin expressing cones. Sheep, cattle, monkey, men, pigeon, duck and chicken were positive for both opsins. Visual acuity analyzed through density of retinal ganglion cells revealed least visual discrimination by horses and highest resolution in pheasant and pigeon. Most mammals studied are dichromats with visual perception similar to red-green blind people.


Subject(s)
Color Vision/physiology , Cone Opsins/metabolism , Gene Expression Regulation/physiology , Mammals/metabolism , Opsins/metabolism , Animals , Cone Opsins/genetics , Humans , Opsins/genetics , Species Specificity
19.
Vet J ; 198(3): 625-30, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24257070

ABSTRACT

Feline idiopathic cystitis (FIC) is a common lower urinary tract disorder in cats, which often recurs. Published reports document increased urine fibronectin and thioredoxin concentrations in cats with FIC compared with healthy control cats. Therefore, these proteins might be of interest in the pathophysiology of FIC. The purpose of the present study was to evaluate variations in these urine proteins throughout the course of FIC by assessing their concentrations in urine specimens from cats with a history of obstructive FIC. Urine total protein (TP) was measured using the Bradford assay, while urine fibronectin and thioredoxin concentrations were determined by Western blot analysis. Urine TP was significantly higher in cats with obstructive FIC at presentation (day 0) than in healthy control cats (P<0.01). There were significant decreases in urine TP in cats with obstructive FIC after 3 months (P<0.01). Significantly higher urine fibronectin (P<0.01) and thioredoxin (P<0.05) concentrations were demonstrated in cats with FIC at day 0 compared to control cats, but there was no significant change over time (P>0.05). Increased concentrations of these proteins over time might reflect ongoing structural and pathological alterations to functional processes in the urinary bladders of cats with obstructive FIC.


Subject(s)
Cat Diseases/urine , Cystitis/veterinary , Fibronectins/urine , Thioredoxins/urine , Animals , Blotting, Western/veterinary , Cat Diseases/etiology , Cats , Cystitis/etiology , Cystitis/urine , Electrophoresis, Agar Gel/veterinary , Female , Germany , Male , Time Factors
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