Glycoproteomics and Glycomics: Method Development and Clinical Applications

Wuhrer, M. [Promotor]Kolarich, D. [Copromotor

Development of a 96-well plate sample preparation method for integratedN- andO-glycomics using porous graphitized carbon liquid chromatography-mass spectrometry

Changes in glycosylation signatures of cells have been associated with pathological processes in cancer as well as infectious and autoimmune diseases. The current protocols for comprehensive analysis ofN-glycomics andO-glycomics derived from cells and tissues often require a large amount of biological material. They also only allow the processing of very limited numbers of samples at a time. Here we established a workflow for sequential release ofN-glycans andO-glycans based on PVDF membrane immobilization in 96-well format from 5 x 10(5)cells. Released glycans are reduced, desalted, purified, and reconstituted, all in 96-well format plates, without additional staining or derivatization. Glycans are then analyzed with porous graphitized carbon nano-liquid chromatography coupled to tandem mass spectrometry using negative-mode electrospray ionization, enabling the chromatographic resolution and structural elucidation of glycan species including many compositional isomers. The approach was demonstrated using glycoprotein standards and further applied to analyze the glycosylation of the murine mammary gland NMuMG cell line. The developed protocol allows the analysis ofN- andO-glycans from relatively large numbers of samples in a less time consuming way with high repeatability. Inter- and intraday repeatability of the fetuinN-glycan analysis showed two median intraday coefficients of variations (CVs) of 7.6% and 8.0%, and a median interday CV of 9.8%. Median CVs of 7.9% and 8.7% for the main peaks ofN- andO-glycans released from the NMuMG cell line indicate a very good repeatability. The method is applicable to purified glycoproteins as well as to biofluids and cell- or tissue-based samples.Cancer Signaling networks and Molecular Therapeutic

Predictors of outcome after 6 and 12 months following anthroposophic therapy for adult outpatients with chronic disease: a secondary analysis from a prospective observational study

<p>Abstract</p> <p>Background</p> <p>Anthroposophic medicine is a physician-provided complementary therapy system involving counselling, artistic and physical therapies, and special medications. The purpose of this analysis was to identify predictors of symptom improvement in patients receiving anthroposophic treatment for chronic diseases.</p> <p>Methods</p> <p>913 adult outpatients from Germany participated in a prospective cohort study. Patients were starting anthroposophic treatment for mental (30.4% of patients, n = 278/913), musculoskeletal (20.2%), neurological (7.6%), genitourinary (7.4%) or respiratory disorders (7.2%) or other chronic indications. Stepwise multiple linear regression analysis was performed with the improvement of Symptom Score (patients' assessment, 0: not present, 10: worst possible) after 6 and 12 months as dependent variables. 61 independent variables pertaining to socio-demographics, life style, disease status, co-morbidity, health status (SF-36), depression, and therapy factors were analysed.</p> <p>Results</p> <p>Compared to baseline, Symptom Score improved by average 2.53 points (95% confidence interval 2.39-2.68, p < 0.001) after six months and by 2.49 points (2.32-2.65, p < 0.001) after 12 months. The strongest predictor for improvement after six months was baseline Symptom Score, which alone accounted for 25% of the variance (total model 32%). Improvement after six months was also positively predicted by better physical function, better general health, shorter disease duration, higher education level, a diagnosis of respiratory disorders, and by a higher therapy goal documented by the physician at baseline; and negatively predicted by the number of physiotherapy sessions in the pre-study year and by a diagnosis of genitourinary disorders. Seven of these nine variables (not the two diagnoses) also predicted improvement after 12 months. When repeating the 0-6 month analysis on two random subsamples of the original sample, three variables (baseline Symptom Score, physical function, general health) remained significant predictors in both analyses, and three further variables (education level, respiratory disorders, therapy goal) were significant in one analysis.</p> <p>Conclusion</p> <p>In adult outpatients receiving anthroposophic treatment for chronic diseases, symptom improvement after 6 and 12 months was predicted by baseline symptoms, health status, disease duration, education, and therapy goal. Other variables were not associated with the outcome. This secondary predictor analysis of data from a pre-post study does not allow for causal conclusions; the results are hypothesis generating and need verification in subsequent studies.</p

Pitfalls and complications in the treatment of cervical spine fractures in patients with ankylosing spondylitis

Patients with ankylosing spondylitis are at significant risk for sustaining cervical spine injuries following trauma predisposed by kyphosis, stiffness and osteoporotic bone quality of the spine. The risk of sustaining neurological deficits in this patient population is higher than average. The present review article provides an outline on the specific injury patterns in the cervical spine, diagnostic algorithms and specific treatment modalities dictated by the underlying disease in patients with ankylosing spondylitis. An emphasis is placed on the risks and complication patterns in the treatment of these rare, but challenging injuries

Lethal Antibody Enhancement of Dengue Disease in Mice Is Prevented by Fc Modification

Immunity to one of the four dengue virus (DV) serotypes can increase disease severity in humans upon subsequent infection with another DV serotype. Serotype cross-reactive antibodies facilitate DV infection of myeloid cells in vitro by promoting virus entry via Fcγ receptors (FcγR), a process known as antibody-dependent enhancement (ADE). However, despite decades of investigation, no in vivo model for antibody enhancement of dengue disease severity has been described. Analogous to human infants who receive anti-DV antibodies by transplacental transfer and develop severe dengue disease during primary infection, we show here that passive administration of anti-DV antibodies is sufficient to enhance DV infection and disease in mice using both mouse-adapted and clinical DV isolates. Antibody-enhanced lethal disease featured many of the hallmarks of severe dengue disease in humans, including thrombocytopenia, vascular leakage, elevated serum cytokine levels, and increased systemic viral burden in serum and tissue phagocytes. Passive transfer of a high dose of serotype-specific antibodies eliminated viremia, but lower doses of these antibodies or cross-reactive polyclonal or monoclonal antibodies all enhanced disease in vivo even when antibody levels were neutralizing in vitro. In contrast, a genetically engineered antibody variant (E60-N297Q) that cannot bind FcγR exhibited prophylactic and therapeutic efficacy against ADE-induced lethal challenge. These observations provide insight into the pathogenesis of antibody-enhanced dengue disease and identify a novel strategy for the design of therapeutic antibodies against dengue

Anthroposophic therapy for chronic depression: a four-year prospective cohort study

BACKGROUND: Depressive disorders are common, cause considerable disability, and do not always respond to standard therapy (psychotherapy, antidepressants). Anthroposophic treatment for depression differs from ordinary treatment in the use of artistic and physical therapies and special medication. We studied clinical outcomes of anthroposophic therapy for depression. METHODS: 97 outpatients from 42 medical practices in Germany participated in a prospective cohort study. Patients were aged 20–69 years and were referred to anthroposophic therapies (art, eurythmy movement exercises, or rhythmical massage) or started physician-provided anthroposophic therapy (counselling, medication) for depression: depressed mood, at least two of six further depressive symptoms, minimum duration six months, Center for Epidemiological Studies Depression Scale, German version (CES-D, range 0–60 points) of at least 24 points. Outcomes were CES-D (primary outcome) and SF-36 after 3, 6, 12, 18, 24, and 48 months. Data were collected from July 1998 to March 2005. RESULTS: Median number of art/eurythmy/massage sessions was 14 (interquartile range 12–22), median therapy duration was 137 (91–212) days. All outcomes improved significantly between baseline and all subsequent follow-ups. Improvements from baseline to 12 months were: CES-D from mean (standard deviation) 34.77 (8.21) to 19.55 (13.12) (p < 0.001), SF-36 Mental Component Summary from 26.11 (7.98) to 39.15 (12.08) (p < 0.001), and SF-36 Physical Component Summary from 43.78 (9.46) to 48.79 (9.00) (p < 0.001). All these improvements were maintained until last follow-up. At 12-month follow-up and later, 52%–56% of evaluable patients (35%–42% of all patients) were improved by at least 50% of baseline CES-D scores. CES-D improved similarly in patients not using antidepressants or psychotherapy during the first six study months (55% of patients). CONCLUSION: In outpatients with chronic depression, anthroposophic therapies were followed by long-term clinical improvement. Although the pre-post design of the present study does not allow for conclusions about comparative effectiveness, study findings suggest that the anthroposophic approach, with its recourse to non-verbal and artistic exercising therapies can be useful for patients motivated for such therapies

Retrotransposons and the evolution of mammalian gene expression

Transposable elements, and retroviral-like elements in particular, are a rich potential source of genetic variation within a host's genome. Many mutations of endogenous genes in phylogenetically diverse organisms are due to insertion of elements that affect gene expression by altering the normal pattern of regulation. While few such associations are known to have been maintained over time, two recently elucidated examples suggest transposable elements may have a significant impact in evolution of gene expression. The first example, concerning the mouse sex-limited protein ( Slp ), clearly establishes that ancient retroviral enhancer sequences now confer hormonal dependence on the adjacent gene. The second example shows that within the human amylase gene family, salivary specific expression has arisen due to inserted sequences, deriving perhaps from a conjunction of two retrotransposable elements.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42800/1/10709_2004_Article_BF00133720.pd