Peter Findeisen

Cytokine concentration in aqueous humour of eyes with exudative age-related macular degeneration

Purpose:  To measure the concentration of cytokines in the aqueous humour of eyes with exudative age‐related macular degeneration (AMD).

Cytokine concentration in aqueous humor of eyes with diabetic macular edema.

Purpose: To measure cytokine concentrations in aqueous humor of eyes with diffuse diabetic macular edema. Methods: The interventional clinical comparative study included a study group of 23 patients with diffuse diabetic macular edema and a control group of 22 patients undergoing cataract surgery. Cytokine concentrations were measured in aqueous humor samples using a Luminex xMAP suspension array technology. Results: In the study group as compared with the control group, significantly higher concentrations were measured for epidermal growth factor (P < 0.001), human growth factor (P < 0.001), intercellular adhesion molecule-1 (ICAM-1; P < 0.001), interleukin (IL)-1a2 (P = 0.04), IL-6 (P = 0.001), IL-8 (P < 0.001), interferon gamma–induced protein (P = 0.004), monocyte chemoattractant protein-1 (P < 0.001), monokine induced by interferon gamma (P < 0.001), matrix metalloproteinase 1 (P = 0.02), matrix metalloproteinase 9 (P < 0.001), plasminogen activator inhibitor 1 (P < 0.001), placenta growth factor (P < 0.001), tissue growth factor beta (P = 0.003), vascular cell adhesion molecule (P < 0.001), and vascular endothelial growth factor (P < 0.001). Retinal macula thickness was significantly associated with the concentrations of the epidermal growth factor (P = 0.005; &rgr; = 0.45), ICAM-1 (P < 0.001; &rgr; = 0.65), IL-3 (P = 0.002; &rgr; = 0.48), IL-6 (P = 0.003; &rgr; = 0.47), IL-8 (P < 0.001; &rgr; = 0.71), monocyte chemoattractant protein-1 (P = 0.001; &rgr; = 0.53), monokine induced by interferon gamma (P < 0.001; &rgr; = 0.57), matrix metalloproteinase 9 (P < 0.001; &rgr; = 0.61), tissue growth factor beta (P = 0.01; &rgr; = 0.42), placenta growth factor (P = 0.004; &rgr; = 0.46), vascular cell adhesion molecule (P = 0.006; &rgr; = 0.44), and vascular endothelial growth factor (P = 0.01; &rgr; = 0.42). In multivariate analysis, macular thickness remained to be significantly associated with the concentration of ICAM-1 (P = 0.03; r = 0.30). Vascular endothelial growth factor concentrations were correlated with concentration of placenta growth factor (P < 0.001; &rgr; = 0.78), plasminogen activator inhibitor 1 (P = 0.001; &rgr; = 0.54), ICAM-1 (P < 0.001; &rgr; = 0.47), monokine induced by interferon gamma (P = 0.004; &rgr; = 0.44), monocyte chemoattractant protein-1 (P = 0.003; &rgr; = 0.43), vascular cell adhesion molecule (P = 0.01; &rgr; = 0.38), IL-6 (P = 0.02; &rgr; = 0.35), IL-8 (P = 0.02; &rgr; = 0.37), epidermal growth factor (P = 0.01; &rgr; = 0.39), and macrophage migration inhibitory factor (P = 0.01; &rgr; = 0.37). Conclusion: Numerous cytokines are associated with the presence and the amount of diabetic macular edema. Among these cytokines, ICAM-1 was the most significantly associated with the disease parameters.

ACBD5 and VAPB mediate membrane associations between peroxisomes and the ER

Peroxisomes (POs) and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism and form tight structural associations, which were first observed in ultrastructural studies decades ago. PO–ER associations have been suggested to impact on a diverse number of physiological processes, including lipid metabolism, phospholipid exchange, metabolite transport, signaling, and PO biogenesis. Despite their fundamental importance to cell metabolism, the mechanisms by which regions of the ER become tethered to POs are unknown, in particular in mammalian cells. Here, we identify the PO membrane protein acyl-coenzyme A–binding domain protein 5 (ACBD5) as a binding partner for the resident ER protein vesicle-associated membrane protein-associated protein B (VAPB). We show that ACBD5–VAPB interaction regulates PO–ER associations. Moreover, we demonstrate that loss of PO–ER association perturbs PO membrane expansion and increases PO movement. Our findings reveal the first molecular mechanism for establishing PO–ER associations in mammalian cells and report a new function for ACBD5 in PO–ER tethering.

Mass spectrometry based proteomics profiling as diagnostic tool in oncology: current status and future perspective.

Abstract Proteomics analysis has been heralded as a novel tool for identifying new and specific biomarkers that may improve diagnosis and monitoring of various disease states. Recent years have brought a number of proteomics profiling technologies. Although proteomics profiling has resulted in the detection of disease-associated differences and modification of proteins, current proteomics technologies display certain limitations that are hampering the introduction of these new technologies into clinical laboratory diagnostics and routine applications. In this review, we summarize current advances in mass spectrometry based biomarker discovery. The promises and challenges of this new technology are discussed with particular emphasis on diagnostic perspectives of mass-spectrometry based proteomics profiling for malignant diseases. Clin Chem Lab Med 2009;47:666–84.

Transmigration of polymorphnuclear neutrophils and monocytes through the human blood-cerebrospinal fluid barrier after bacterial infection in vitro

BackgroundBacterial invasion through the blood-cerebrospinal fluid barrier (BCSFB) during bacterial meningitis causes secretion of proinflammatory cytokines/chemokines followed by the recruitment of leukocytes into the CNS. In this study, we analyzed the cellular and molecular mechanisms of polymorphonuclear neutrophil (PMN) and monocyte transepithelial transmigration (TM) across the BCSFB after bacterial infection.MethodsUsing an inverted transwell filter system of human choroid plexus papilloma cells (HIBCPP), we studied leukocyte TM rates, the migration route by immunofluorescence, transmission electron microscopy and focused ion beam/scanning electron microscopy, the secretion of cytokines/chemokines by cytokine bead array and posttranslational modification of the signal regulatory protein (SIRP) α via western blot.ResultsPMNs showed a significantly increased TM across HIBCPP after infection with wild-type Neisseria meningitidis (MC58). In contrast, a significantly decreased monocyte transmigration rate after bacterial infection of HIBCPP could be observed. Interestingly, in co-culture experiments with PMNs and monocytes, TM of monocytes was significantly enhanced. Analysis of paracellular permeability and transepithelial electrical resistance confirmed an intact barrier function during leukocyte TM. With the help of the different imaging techniques we could provide evidence for para- as well as for transcellular migrating leukocytes. Further analysis of secreted cytokines/chemokines showed a distinct pattern after stimulation and transmigration of PMNs and monocytes. Moreover, the transmembrane glycoprotein SIRPα was deglycosylated in monocytes, but not in PMNs, after bacterial infection.ConclusionsOur findings demonstrate that PMNs and monoctyes differentially migrate in a human BCSFB model after bacterial infection. Cytokines and chemokines as well as transmembrane proteins such as SIRPα may be involved in this process.

Functional protease profiling for diagnosis of malignant disease.

Clinical proteomic profiling by mass spectrometry (MS) aims at uncovering specific alterations within mass profiles of clinical specimens that are of diagnostic value for the detection and classification of various diseases including cancer. However, despite substantial progress in the field, the clinical proteomic profiling approaches have not matured into routine diagnostic applications so far. Their limitations are mainly related to high‐abundance proteins and their complex processing by a multitude of endogenous proteases thus making rigorous standardization difficult. MS is biased towards the detection of low‐molecular‐weight peptides. Specifically, in serum specimens, the particular fragments of proteolytically degraded proteins are amenable to MS analysis. Proteases are known to be involved in tumour progression and tumour‐specific proteases are released into the blood stream presumably as a result of invasive progression and metastasis. Thus, the determination of protease activity in clinical specimens from patients with malignant disease can offer diagnostic and also therapeutic options. The identification of specific substrates for tumour proteases in complex biological samples is challenging, but proteomic screens for proteases/substrate interactions are currently experiencing impressive progress. Such proteomic screens include peptide‐based libraries, differential isotope labelling in combination with MS, quantitative degradomic analysis of proteolytically generated neo‐N‐termini, monitoring the degradation of exogenous reporter peptides with MS, and activity‐based protein profiling. In the present article, we summarize and discuss the current status of proteomic techniques to identify tumour‐specific protease‐substrate interactions for functional protease profiling. Thereby, we focus on the potential diagnostic use of the respective approaches.

A TNF-Regulated Recombinatorial Macrophage Immune Receptor Implicated in Granuloma Formation in Tuberculosis

Macrophages play a central role in host defense against mycobacterial infection and anti- TNF therapy is associated with granuloma disorganization and reactivation of tuberculosis in humans. Here, we provide evidence for the presence of a T cell receptor (TCR) αβ based recombinatorial immune receptor in subpopulations of human and mouse monocytes and macrophages. In vitro, we find that the macrophage-TCRαβ induces the release of CCL2 and modulates phagocytosis. TNF blockade suppresses macrophage-TCRαβ expression. Infection of macrophages from healthy individuals with mycobacteria triggers formation of clusters that express restricted TCR Vβ repertoires. In vivo, TCRαβ bearing macrophages abundantly accumulate at the inner host-pathogen contact zone of caseous granulomas from patients with lung tuberculosis. In chimeric mouse models, deletion of the variable macrophage-TCRαβ or TNF is associated with structurally compromised granulomas of pulmonary tuberculosis even in the presence of intact T cells. These results uncover a TNF-regulated recombinatorial immune receptor in monocytes/macrophages and demonstrate its implication in granuloma formation in tuberculosis.

Work Flow Analysis of Around-the-clock Processing of Blood Culture Samples and Integrated MALDI-TOF Mass Spectrometry Analysis for the Diagnosis of Bloodstream Infections

BACKGROUND Because sepsis has a high mortality rate, rapid microbiological diagnosis is required to enable efficient therapy. The effectiveness of MALDI-TOF mass spectrometry (MALDI-TOF MS) analysis in reducing turnaround times (TATs) for blood culture (BC) pathogen identification when available in a 24-h hospital setting has not been determined. METHODS On the basis of data from a total number of 912 positive BCs collected within 140 consecutive days and work flow analyses of laboratory diagnostics, we evaluated different models to assess the TATs for batch-wise and for immediate response (real-time) MALDI-TOF MS pathogen identification of positive BC results during the night shifts. The results were compared to TATs from routine BC processing and biochemical identification performed during regular working hours. RESULTS Continuous BC incubation together with batch-wise MALDI-TOF MS analysis enabled significant reductions of up to 58.7 h in the mean TATs for the reporting of the bacterial species. The TAT of batch-wise MALDI-TOF MS analysis was inferior by a mean of 4.9 h when compared to the model of the immediate work flow under ideal conditions with no constraints in staff availability. CONCLUSIONS Together with continuous cultivation of BC, the 24-h availability of MALDI-TOF MS can reduce the TAT for microbial pathogen identification within a routine clinical laboratory setting. Batch-wise testing of positive BC loses a few hours compared to real-time identification but is still far superior to classical BC processing. Larger prospective studies are required to evaluate the contribution of rapid around-the-clock pathogen identification to medical decision-making for septicemic patients.

Rapid Detection of Ampicillin Resistance in Escherichia coli by Quantitative Mass Spectrometry

ABSTRACT Early targeted antimicrobial therapy helps decrease costs and prevents the spread of antimicrobial resistance, including in Escherichia coli, the most frequent Gram-negative bacterium that causes sepsis. Therefore, rapid susceptibility testing represents the major prerequisite for knowledge-based successful antimicrobial treatment. To accelerate testing for antibiotic susceptibility, we have developed a new mass spectrometry-based assay for antibiotic susceptibility testing (MAAST). For proof of principle, we present an ampicillin susceptibility test for E. coli with a turnaround time of 90 min upon growth detection.

Pineal volume and circadian melatonin profile in healthy volunteers: An interdisciplinary approach

To correlate pineal parenchyma volume (PP) to circadian melatonin profiles and to determine the 24‐hour melatonin per volume of pineal tissue (MLPV). Furthermore, we compared melatonin profiles of cystic and solid glands.