Highly sensitive therapeutic drug monitoring of infliximab in serum by targeted mass spectrometry in comparison to ELISA data.

BACKGROUND: Presently, antibody concentration measurements for patients undergoing treatment are predominantly determined by ELISA, which still comes with known disadvantages. Therefore, our aim was to establish a targeted mass-spectrometric assay enabling the reproducible absolute quantification of peptides from the hypervariable and interaction regions of infliximab. METHODS: Peptides of infliximab were measured post-trypsin digestion and subsequent separation on a Vanquish Horizon UHPLC coupled to a TSQ Altis Triple-Quad mass spectrometer. Normalization and absolute quantification were conducted using stable isotope-synthesized peptides. Calibration curves covering a range of 0.25-50 µg/ml were employed for quantitation. RESULTS: We demonstrated the substantial influence of peptide selection, choice of hydrolase for digestion, and digestion time on absolute peptide yield (28-44% for peptide 1 and 64-97% for peptide 2). Furthermore, we showed that the generated calibration curves for absolute quantification were highly reproducible and robust (LLOQ1 0.72 µg/ml and LLOQ2 1.00 µg/ml) over several months. In comparison to ELISA values, the absolute values obtained by mass spectrometry often yielded lower results for both targeted peptides. CONCLUSIONS: In this study, a semi-automated workflow was employed and tested with 8 patients and corresponding replicates (n = 3-4). We demonstrated the robust implementation of calibration curves for the absolute quantification of infliximab in patient samples, with coefficients of variation ranging from 0.5 to 9%. Taken together, we have developed a platform enabling the rapid (2 days of sample preparation and 30 min of measurement time per sample) and robust quantification of Infliximab antibody concentration in patients. The use of mass spectrometry also facilitates the straightforward expansion of the method to include additional antibody peptides.

Proteomic analysis identifies argininosuccinate synthetase 1 and special AT-rich sequence binding protein 1 as reliable markers for the immunohistochemical distinction between WHO types A and B3 thymomas.

AIMS: The reliable classification of type A versus type B3 thymomas has prognostic and therapeutic relevance, but can be problematic due to considerably overlapping morphology. No immunohistochemical markers aiding in this distinction have been published so far. METHODS AND RESULTS: We identified and quantified numerous differentially expressed proteins using an unbiased proteomic screen by mass spectrometry in pooled protein lysates from three type A and three type B3 thymomas. From these, candidates were validated in a larger series of paraffin-embedded type A and B3 thymomas. We identified argininosuccinate synthetase 1 (ASS1) and special AT-rich sequence binding protein 1 (SATB1) as highly discriminatory between 34 type A and 20 type B3 thymomas (94% sensitivity, 98% specificity and 96% accuracy). Although not the focus of this study, the same markers also proved helpful in the diagnosis of type AB (n = 14), B1 (n = 4) and B2 thymomas (n = 10). CONCLUSIONS: Mutually exclusive epithelial expression of ASS1 in 100% of type B3 thymomas and ectopic nuclear expression of SATB1 in 92% of type A thymomas support the distinction between type A and type B3 thymomas with 94% sensitivity, 98% specificity and 96% accuracy.

Rapid Detection of Lithium Concentrations in Oral Fluid.

BACKGROUND: Lithium medication is considered to be the first-line treatment for bipolar disorder as a monotherapy, and for treatment-resistant depression with lithium augmentation. However, because of potential toxicity, lithium levels must be monitored frequently. Recent studies have demonstrated a significant correlation between lithium levels measured in serum and those detected in oral fluid, suggesting that oral fluid analysis may represent an easy, noninvasive means to monitor lithium levels. The aim of this study was to evaluate the analytical performance of rapid assays for lithium measurements in oral fluid. METHODS: Levels of lithium in oral fluid from psychiatric patients (n = 108 in total) taking lithium medications were quantified using 2 rapid techniques: an automated clinical chemistry analyzer and a novel, commercially available colorimetric lithium assay. These results were compared with those obtained using inductively coupled plasma optical emission spectrometry (ICP-OES). RESULTS: The mean and median oral fluid lithium levels in this cohort were 1.43-1.61 mM and 1.32-1.52 mM, respectively, depending on the method, with the overall range, across all methods, being 0.213-4.42 mM. Linear regression analysis showed excellent agreement between the oral fluid values measured using ICP-OES and the colorimetric method (r 2 value = 0.926; P < 0.0001; slope = 1.084 ± 0.038). Similarly, excellent agreement was observed between ICP-OES and the automated method (r 2 = 0.872; P < 0.0001; slope = 1.019 ± 0.057). CONCLUSIONS: These results demonstrate that lithium levels in oral fluid can be rapidly and reliably quantified using colorimetric approaches. These findings may facilitate the development of point-of-care lithium monitoring systems for use in oral fluid.

Krebs von den Lungen-6 as a Potential Predictive Biomarker in Fibrosing Interstitial Lung Diseases.

BACKGROUND: As fibrosing interstitial lung diseases (fILDs) are associated with high mortality, monitoring of disease activity under treatment is highly relevant. Krebs von den Lungen-6 (KL-6) is associated with the presence and severity of different fILDs, mainly in Asian patient populations. OBJECTIVES: Our aim was to evaluate KL-6 as a predictive biomarker in fILDs in Caucasian patients. METHODS: Consecutive patients with fILDs were recruited prospectively and serum concentrations of KL-6 were measured at baseline (BL), after 6 and 12 months (6 Months, 12 Months). Clinical characteristics including pulmonary function tests were assessed at 6-monthly visits and correlated with KL-6 BL levels as well as with KL-6 level changes. RESULTS: A total of 47 fILD patients were included (mean age: 65 years, 68% male). KL-6 levels at BL were significantly higher in fILD patients than in healthy controls (n = 44, mean age: 45, 23% male) (ILD: 1,757 ± 1960 U/mL vs. control: 265 ± 107 U/mL, p < 0.0001). However, no differences were noted between ILD subgroups. KL-6 decreased significantly under therapy (6M∆BL-KL6: -486 ± 1,505 mean U/mL, p = 0.032; 12M∆BL-KL6: -547 ± 1,782 mean U/mL, p = 0.041) and KL-6 level changes were negatively correlated with changes in pulmonary function parameters (forced vital capacity [FVC]: r = -0.562, p < 0.0001; DLCOSB: r = -0.405, p = 0.013). While neither absolute KL-6 levels at BL nor KL-6 level changes were associated with ILD progression (FVC decline ≥10%, DLCOSB decline ≥15% or death), patients with a stable FVC showed significantly decreasing KL-6 levels (p = 0.022). CONCLUSIONS: A decline of KL-6 under therapy correlated with a clinically relevant stabilization of lung function. Thus, KL-6 might serve as a predictive biomarker, which however must be determined by larger prospective cohorts.

A multicenter performance evaluation of the new Elecsys Vitamin D total III assay versus reference isotope dilution liquid chromatography tandem mass spectrometry and commercially available comparators.

BACKGROUND: Vitamin D deficiency/insufficiency and toxicity are worldwide issues; thus, accurate diagnostic assays are required to measure vitamin D. We evaluated the performance of the new Elecsys® Vitamin D total III assay (Roche Diagnostics International Ltd). METHODS: Repeatability and intermediate precision of the Elecsys Vitamin D total III assay (cobas e 601 analyzer) were evaluated at three sites using five human serum pools (HSPs) and two PreciControls (five-day model, one reagent lot [CLSI-EP05-A3]) and compared against prespecified acceptance criteria. A serum verification panel, with reference isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) values, was used for comparator assay/concordance studies at two sites, assessed using unweighted Deming regression. Testing of serum vs. plasma on the Elecsys assay was conducted at one site using samples from healthy adults; assessed using Passing-Bablok regression. RESULTS: Repeatability (HSP1 [16.8-18.4 ng/ml], SD 0.87-1.07; HSP5 [94.5-98.0 ng/ml], CV 1.58%-2.76%) and intermediate precision (HSP1, SD 1.14-1.77; HSP5, CV 2.00%-4.13%) met acceptance criteria across sites. Agreement was observed between the Elecsys assay and (i) the ID-LC-MS/MS verification panel (slope, 0.936-1.01; Pearson's r, 0.960-0.986) and (ii) comparator assays (slope, 0.921-1.15; Pearson's r, 0.958-0.982). The Elecsys assay correctly assigned the highest combined percentage of samples to deficient (100%) and insufficient (89.5%) vitamin D categories vs. comparator assays and demonstrated comparable performance in serum and plasma (y = 0.103 + 0.984x). CONCLUSIONS: The Elecsys Vitamin D total III assay demonstrated good analytical performance and compared favorably with other assays, supporting its use in clinical practice.

Fluoxetine Enhances Synaptic Vesicle Trafficking and Energy Metabolism in the Hippocampus of Socially Isolated Rats.

Chronic social isolation (CSIS)-induced alternation in synaptic and mitochondrial function of specific brain regions is associated with major depressive disorder (MDD). Despite the wide number of available medications, treating MDD remains an important challenge. Although fluoxetine (Flx) is the most frequently prescribed antidepressant, its mode of action is still unknown. To delineate affected molecular pathways of depressive-like behavior and identify potential targets upon Flx treatment, we performed a comparative proteomic analysis of hippocampal purified synaptic terminals (synaptosomes) of rats exposed to six weeks of CSIS, an animal model of depression, and/or followed by Flx treatment (lasting three weeks of six-week CSIS) to explore synaptic protein profile changes. Results showed that Flx in controls mainly induced decreased expression of proteins involved in energy metabolism and the redox system. CSIS led to increased expression of proteins that mainly participate in Ca2+/calmodulin-dependent protein kinase II (Camk2)-related neurotransmission, vesicle transport, and ubiquitination. Flx treatment of CSIS rats predominantly increased expression of proteins involved in synaptic vesicle trafficking (exocytosis and endocytosis), and energy metabolism (glycolytic and mitochondrial respiration). Overall, these Flx-regulated changes in synaptic and mitochondrial proteins of CSIS rats might be critical targets for new therapeutic development for the treatment of MDD.

Intraocular epidermal growth factor concentration, axial length, and high axial myopia.

PURPOSE: Various molecules such as dopamine have been found to be associated with axial elongation in experimental studies. Here, we examined whether intraocular EGF is associated with axial length in myopic patients. METHODS: The hospital-based investigation included patients of European descent without optic nerve, retinal, or macular diseases except for myopic maculopathy. Using aqueous humor samples collected during surgery, the EGF concentration was examined applying a cytometric bead array. High myopia was defined by an axial length of ≥ 27.0 mm. RESULTS: The study included a non-highly myopic group of 11 patients (mean age, 72.9 ± 10.8 years; mean axial length, 24.3 ± 1.1 mm) and a highly myopic group of three patients (age, 81.11 ± 12.3 years; axial length, 29.5 ± 1.3 mm), with one of them having pathologic myopic maculopathy. In multivariable linear regression analysis, higher EGF concentration was correlated with the highly myopic versus non-highly myopic group (beta, 1.24; non-standardized correlation coefficient B, 6.24; 95% confidence interval (CI), 0.10,12.4;P = 0.047) after adjusting for axial length. The amount of intraocular EGF was significantly higher in the highly myopic group than in the non-highly myopic group (89.1 ± 40.8 pg versus 34.1 ± 13.2 pg; P = 0.005), and it was highest in the eye with myopic maculopathy (135 pg). CONCLUSIONS: The intraocular amount of EGF is higher in highly myopic versus non-highly myopic eyes.

Spatial Distribution of Endogenous Tissue Protease Activity in Gastric Carcinoma Mapped by MALDI Mass Spectrometry Imaging.

Aberrant protease activity has been implicated in the etiology of various prevalent diseases including neurodegeneration and cancer, in particular metastasis. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has recently been established as a key technology for bioanalysis of multiple biomolecular classes such as proteins, lipids, and glycans. However, it has not yet been systematically explored for investigation of a tissue's endogenous protease activity. In this study, we demonstrate that different tissues, spray-coated with substance P as a tracer, digest this peptide with different time-course profiles. Furthermore, we reveal that distinct cleavage products originating from substance P are generated transiently and that proteolysis can be attenuated by protease inhibitors in a concentration-dependent manner. To show the translational potential of the method, we analyzed protease activity of gastric carcinoma in mice. Our MSI and quantitative proteomics results reveal differential distribution of protease activity - with strongest activity being observed in mouse tumor tissue, suggesting the general applicability of the workflow in animal pharmacology and clinical studies.

Development of a Mass Spectrometry-Based Method for Quantification of Ustekinumab in Serum Specimens.

BACKGROUND: Ustekinumab (UST) is a human monoclonal antibody used to treat moderate-to-severe Crohn disease by blocking the interleukin-12/23 pathway. Although an optimized therapeutic concentration of UST is associated with clinical response and improved prognosis, the availability of clinical laboratory methods for UST monitoring is limited. Furthermore, the commercially available methods are immunoassays that are prone to interference of antidrug antibodies. This study aimed to develop a liquid chromatography-tandem mass spectrometry method for quantification of UST in human serum specimens. METHODS: A tryptic peptide that is specific to the heavy chain variable region of UST was selected. Quantification of UST was performed by selective reaction monitoring on a quadrupole TQ-XS with an internal standard. After digestion with trypsin, peptides were separated by reverse-phase C18 liquid chromatography; peptides were detected by MS/MS, and analyte to internal standard peak area ratios were used for the quantification. Finally, serum samples from patients treated with UST were collected at trough levels (n = 66). RESULTS: The assay showed a broad dynamic range with linearity between 0.4 and 20 mg/L (R = 0.995). The lower limit of quantification was found to be 0.4 mg/L. The reproducibility was tested with 3 different UST concentrations (2, 8, and 16 mg/L). The coefficients of intra-assay and interassay variations were 2.2%-4.0% and 2.7%-5.3%, respectively. UST serum concentrations of 2-16 mg/L were stable for up to 14 days when specimens were left at room temperature (20°C). CONCLUSIONS: The newly developed LC/MS-based method was shown to be feasible for UST quantification. This analytical approach may lead to individualized dosing and improved patient care.

Mass spectrometry based analytical quality assessment of serum and plasma specimens with patterns of endo- and exogenous peptides.

Background Inappropriate preanalytical sample handling is a major threat for any biomarker discovery approach. Blood specimens have a genuine proteolytic activity that leads to a time dependent decay of peptidic quality control markers (QCMs). The aim of this study was to identify QCMs for direct assessment of sample quality (DASQ) of serum and plasma specimens. Methods Serum and plasma specimens of healthy volunteers and tumor patients were spiked with two synthetic reporter peptides (exogenous QCMs) and aged under controlled conditions for up to 24 h. The proteolytic fragments of endogenous and exogenous QCMs were monitored for each time point by mass spectrometry (MS). The decay pattern of peptides was used for supervised classification of samples according to their respective preanalytical quality. Results The classification accuracy for fresh specimens (1 h) was 96% and 99% for serum and plasma specimens, respectively, when endo- and exogenous QCMs were used for the calculations. However, classification of older specimens was more difficult and overall classification accuracy decreased to 79%. Conclusions MALDI-TOF MS is a simple and robust method that can be used for DASQ of serum and plasma specimens in a high throughput manner. We propose DASQ as a fast and simple step that can be included in multicentric large-scale projects to ensure the homogeneity of sample quality.

Rapid susceptibility testing of multi-drug resistant Escherichia coli and Klebsiella by glucose metabolization monitoring.

Background The increasing number of multi-drug resistant (MDR) bacteria provides enormous challenges for choosing an appropriate antibiotic therapy in the early phase of sepsis. While bacterial identification has been greatly accelerated by the introduction of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), the antibiotic susceptibility testing (AST) remains time-consuming. Here, we present a rapid susceptibility testing method for testing Gram-negative bacteria, exemplarily validated for Escherichia coli and Klebsiella spp. Methods Gram-negative isolates (E. coli and Klebsiella spp.) were either taken as single colonies from agar plates (n=136) or directly extracted and identified from positive blood cultures (n=42) using MALDI-TOF MS. Bacteria were incubated in glucose-supplemented Luria broths (LBs) each containing one antibiotic (ceftazidime, piperacillin, imipenem and ciprofloxacin), routinely used to classify Gram-negative bacteria in Germany. To determine susceptibility the dynamics of glucose utilization in bacterial suspensions were quantitatively measured in the presence or absence of antibiotics designated liquid-AST (L-AST). Results The L-AST can be run on clinical-chemistry analyzers and integrated into laboratory routines. It yields critical resistance information within 90-150 min downstream of a MS-based identification. The results showed a high concordance with routine susceptibility testing, with less than 1% very major errors (VME) and 3.51% major errors (ME) for 178 assessed isolates. Analysis of turnaround time (TAT) for 42 clinical samples indicated that L-AST results could be obtained 34 h earlier than the routine results. Conclusions As exemplified for E. coli and Klebsiella spp., L-AST provides substantial acceleration of susceptibility testing following MALDI-TOF MS identification. The assay is a simple and low-cost method that can be integrated into clinical laboratory to allow for 24/7 AST. This approach could improve antibiotic therapy.

Transforming Growth Factor-ß Drives the Transendothelial Migration of Hepatocellular Carcinoma Cells.

The entry of malignant hepatocytes into blood vessels is a key step in the dissemination and metastasis of hepatocellular carcinoma (HCC). The identification of molecular mechanisms involved in the transmigration of malignant hepatocytes through the endothelial barrier is of high relevance for therapeutic intervention and metastasis prevention. In this study, we employed a model of hepatocellular transmigration that mimics vascular invasion using hepatic sinusoidal endothelial cells and malignant hepatocytes evincing a mesenchymal-like, invasive phenotype by transforming growth factor (TGF)-ß. Labelling of respective cell populations with various stable isotopes and subsequent mass spectrometry analyses allowed the "real-time" detection of molecular changes in both transmigrating hepatocytes and endothelial cells. Interestingly, the proteome profiling revealed 36 and 559 regulated proteins in hepatocytes and endothelial cells, respectively, indicating significant changes during active transmigration that mostly depends on cell-cell interaction rather than on TGF-ß alone. Importantly, matching these in vitro findings with HCC patient data revealed a panel of common molecular alterations including peroxiredoxin-3, epoxide hydrolase, transgelin-2 and collectin 12 that are clinically relevant for the patient's survival. We conclude that hepatocellular plasticity induced by TGF-ß is crucially involved in blood vessel invasion of HCC cells.

Neisseria meningitidis elicits a pro-inflammatory response involving IκBζ in a human blood-cerebrospinal fluid barrier model.

BACKGROUND: The human-specific, Gram-negative bacterium Neisseria meningitidis (Nm) is a leading cause of bacterial meningitis worldwide. The blood-cerebrospinal fluid barrier (BCSFB), which is constituted by the epithelial cells of the choroid plexus (CP), has been suggested as one of the potential entry sites of Nm into the CSF and can contribute to the inflammatory response during infectious diseases of the brain. Toll-like receptors (TLRs) are involved in mediating signal transduction caused by the pathogens. METHODS: Using a recently established in vitro model of the human BCSFB based on human malignant CP papilloma (HIBCPP) cells we investigated the cellular response of HIBCPP cells challenged with the meningitis-causing Nm strain, MC58, employing transcriptome and RT-PCR analysis, cytokine bead array, and enzyme-linked immunosorbent assay (ELISA). In comparison, we analyzed the answer to the closely related unencapsulated carrier isolate Nm α14. The presence of TLRs in HIBCPP and their role during signal transduction caused by Nm was studied by RT-PCR and the use of specific agonists and mutant bacteria. RESULTS: We observed a stronger transcriptional response after infection with strain MC58, in particular with its capsule-deficient mutant MC58siaD-, which correlated with bacterial invasion levels. Expression evaluation and Gene Set Enrichment Analysis pointed to a NFκB-mediated pro-inflammatory immune response involving up-regulation of the transcription factor IκBζ. Infected cells secreted significant levels of pro-inflammatory chemokines and cytokines, including, among others, IL8, CXCL1-3, and the IκBζ target gene product IL6. The expression profile of pattern recognition receptors in HIBCPP cells and the response to specific agonists indicates that TLR2/TLR6, rather than TLR4 or TLR2/TLR1, is involved in the cellular reaction following Nm infection. CONCLUSIONS: Our data show that Nm can initiate a pro-inflammatory response in human CP epithelial cells probably involving TLR2/TLR6 signaling and the transcriptional regulator IκBζ.

Morphology and function: MR pineal volume and melatonin level in human saliva are correlated.

PURPOSE: To investigate the relation between circadian saliva melatonin levels and pineal volume as determined by MRI. Plasma melatonin levels follow a circadian rhythm with a high interindividual variability. MATERIALS AND METHODS: In 103 healthy individuals saliva melatonin levels were determined at four time points within 24 h and MRI was performed once (3.0 Tesla, including three-dimensional T2 turbo spin echo [3D-T2-TSE], susceptibility-weighted imaging [SWI]). Pineal volume as well as cyst volume were assessed from multiplanar reconstructed 3D-T2-TSE images. Pineal calcification volume tissue was determined on SWI. To correct for hormonal inactive pineal tissue, cystic and calcified areas were excluded. Sleep quality was assessed with the Landeck Inventory for sleep quality disturbance. RESULTS: Solid and uncalcified pineal volume correlated to melatonin maximum (r = 0.28; P < 0.05) and area under the curve (r = 0.29; P < 0.05). Of interest, solid and uncalcified pineal volume correlated negatively with the sleep rhythm disturbances subscore (r = -0.17; P < 0.05) despite a very homogenous population. CONCLUSION: Uncalcified solid pineal tissue measured by 3D-T2-TSE and SWI is related to human saliva melatonin levels. The analysis of the sleep quality and pineal volume suggests a linkage between better sleep quality and hormonal active pineal tissue.

Analytical performance evaluation of a new integrated clinical chemistry and immunoassay analyzer.

Background: Clinical laboratories perform a wide range of tests that are used by healthcare professionals to guide medical decision making. Use of automated analyzers in the clinical laboratory can improve patient care by not only reducing the turn-around-time (TAT) of results but also improving accuracy of the reported results by reducing human error. The aim of this study was to evaluate the performance characteristics of a new automated laboratory instrument, the Atellica® CI Analyzer, Model 1900, over a 3-month period in a European laboratory setting. Methods: Analytical performance of 17 analytes (13 chemistry and four immunochemistry) was assessed by evaluating repeatability and within-laboratory precision using anonymized remnant serum samples. Method comparison studies were performed on the Atellica CI Analyzer and the Roche cobas® 6000. Results: Excellent precision was observed with coefficients of variation (CVs) less than 2 % for repeatability and less than 3 % within-laboratory imprecision for most analytes. Comparison of select assays with the cobas 6000 system resulted in correlation coefficients ranging from 0.980 to 1.000. Conclusion: This is the first reported evaluation of the Atellica CI Analyzer in a clinical laboratory setting. The strong analytical performance of the Atellica CI Analyzer demonstrates that this instrument is suitable for routine clinical use.

Feasibility of using real-world free thyroxine data from the US and Europe to enable fast and efficient transfer of reference intervals from one population to another.

Objectives: The direct approach for determining reference intervals (RIs) is not always practical. This study aimed to generate evidence that a real-world data (RWD) approach could be applied to transfer free thyroxine RIs determined in one population to a second population, presenting an alternative to performing multiple RI determinations. Design and methods: Two datasets (US, n = 10,000; Europe, n = 10,000) were created from existing RWD. Descriptive statistics, density plots and cumulative distributions were produced for each data set and comparisons made. Cumulative probabilities at the lower and upper limits of the RIs were identified using an empirical cumulative distribution function. According to these probabilities, estimated percentiles for each dataset and estimated differences between the two sets of percentiles were obtained by case resampling bootstrapping. The estimated differences were then evaluated against a pre-determined acceptance criterion of ≤7.8% (inter-individual biological variability). The direct approach was used to validate the RWD approach. Results: The RWD approach provided similar descriptive statistics for both populations (mean: US = 16.1 pmol/L, Europe = 16.4 pmol/L; median: US = 15.4 pmol/L, Europe = 15.8 pmol/L). Differences between the estimated percentiles at the upper and lower limits of the RIs fulfilled the pre-determined acceptance criterion and the density plots and cumulative distributions demonstrated population homogeneity. Similar RI distributions were observed using the direct approach. Conclusions: This study provides evidence that a RWD approach can be used to transfer RIs determined in one population to another.

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

BACKGROUND: Bacterial 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. METHODS: Using 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. RESULTS: PMNs 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. CONCLUSIONS: Our 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.

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) αß based recombinatorial immune receptor in subpopulations of human and mouse monocytes and macrophages. In vitro, we find that the macrophage-TCRαß induces the release of CCL2 and modulates phagocytosis. TNF blockade suppresses macrophage-TCRαß expression. Infection of macrophages from healthy individuals with mycobacteria triggers formation of clusters that express restricted TCR Vß repertoires. In vivo, TCRαß 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-TCRαß 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.