Evaluation of a prototype array for daily quality assurance in spot scanning proton therapy.

BACKGROUND: Quality assurance (QA) on a daily basis is an essential task in radiotherapy. In pencil beam scanning proton therapy (PBS), there is a lack of available practical QA devices for routine daily QA in comparison to conventional radiotherapy. PURPOSE: The aim was to characterize and evaluate a prototype for the task of daily QA routine for PBS with parameters recommended by the AAPM TG 224, that is, the dose output constancy, the spot position and the distal range verification. Furthermore, a time efficient calibration method for fast and reliable daily QA routine was established for the prototype. METHODS: First, a calibration routine was designed and evaluated, which characterizes the array response and allows for a conversion of the measured signal to clinically needed QA parameters. Finally, a time and resource efficient daily QA routine was developed and tested. RESULTS: The prototype array can distinguish spot position deviations with sub-millimeter accuracy, as well as changes in the spot size in terms of FWHM with a 2 % $\%$ sensitivity. The range and thus the energy can be evaluated at different depths also with sub-millimeter precision. After some training, the setup of the prototype device took roughly two minutes and the total beamtime was about one minute on cyclotron site and five minutes for synchrotrons. CONCLUSIONS: A prototype for daily QA in spot scanning proton therapy was evaluated, which features a fast and easy setup and allows for measuring relevant beam parameters, typically within less than a minute of beam time. All QA parameters as recommended by the AAPM TG 224 report can be analyzed with sufficient accuracy.

Structure Elucidation and Mitigation of Endogenous Interferences in LC-MS-Based Metabolic Profiling of Urine.

Liquid chromatography-mass spectrometry (LC-MS) is the main workhorse of metabolomics owing to its high degree of analytical sensitivity and specificity when measuring diverse chemistry in complex biological samples. LC-MS-based metabolic profiling of human urine, a biofluid of primary interest for clinical and biobank studies, is not widely considered to be compromised by the presence of endogenous interferences and is often accomplished using a simple "dilute-and-shoot" approach. Yet, it is our experience that broad obscuring signals are routinely observed in LC-MS metabolic profiles and represent interferences that lack consideration in the relevant metabolomics literature. In this work, we chromatographically isolated the interfering metabolites from human urine and unambiguously identified them via de novo structure elucidation as two separate proline-containing dipeptides: N,N,N-trimethyl-l-alanine-l-proline betaine (l,l-TMAP) and N,N-dimethyl-l-proline-l-proline betaine (l,l-DMPP), the latter reported here for the first time. Offline LC-MS/MS, magnetic resonance mass spectrometry (MRMS), and nuclear magnetic resonance (NMR) spectroscopy were essential components of this workflow for the full chemical and spectroscopic characterization of these metabolites and for establishing the coexistence of cis and trans isomers of both dipeptides in solution. Analysis of these definitive structures highlighted intramolecular ionic interactions as responsible for slow interconversion between these isomeric forms resulting in their unusually broad elution profiles. Proposed mitigation strategies, aimed at increasing the quality of LC-MS-based urine metabolomics data, include modification of column temperature and mobile-phase pH to reduce the chromatographic footprint of these dipeptides, thereby reducing their interfering effect on the underlying metabolic profiles. Alternatively, sample dilution and internal standardization methods may be employed to reduce or account for the observed effects of ionization suppression on the metabolic profile.

Revealing the Reactivity of Individual Chemical Entities in Complex Mixtures: the Chemistry Behind Bio-Oil Upgrading.

Bio-oils are precursors for biofuels but are highly corrosive necessitating further upgrading. Furthermore, bio-oil samples are highly complex and represent a broad range of chemistries. They are complex mixtures not simply because of the large number of poly-oxygenated compounds but because each composition can comprise many isomers with multiple functional groups. The use of hyphenated ultrahigh-resolution mass spectrometry affords the ability to separate isomeric species of complex mixtures. Here, we present for the first time, the use of this powerful analytical technique combined with chemical reactivity to gain greater insights into the reactivity of the individual isomeric species of bio-oils. A pyrolysis bio-oils and its esterified bio-oil were analyzed using gas chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry, and in-house software (KairosMS) was used for fast comparison of the hyphenated data sets. The data revealed a total of 10,368 isomers in the pyrolysis bio-oil and an increase to 18,827 isomers after esterification conditions. Furthermore, the comparison of the isomeric distribution before and after esterification provide new light on the reactivities within these complex mixtures; these reactivities would be expected to correspond with carboxylic acid, aldehyde, and ketone functional groups. Using this approach, it was possible to reveal the increased chemical complexity of bio-oils after upgrading and target detection of valuable compounds within the bio-oils. The combination of chemical reactions alongside with in-depth molecular characterization opens a new window for the understanding of the chemistry and reactivity of complex mixtures.

Forensic Discrimination of Differentially Sourced Animal Blood Using a Bottom-Up Proteomics Based MALDI MS Approach.

Recently published work has reported the development and application of a bottom-up proteomic approach to distinguish between human and animal blood (down to animal species level), by rapid screening using Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI MS). In that study, it was additionally observed that intravenous animal blood exhibits different spectral profiles from blood collected within the animal chest cavity as well as from the diluted blood collected within packets of meat. In this follow-up study we explored the resulting hypothesis that, depending on how blood is shed or collected, protein biomarker profiles vary to the extent of systematically permitting a distinction between possible sources of blood (for example, flesh wound versus packaged meat). This intelligence may be important in reconstructing the dynamics of the crime. The combination of statistical analysis and tandem mass spectrometry has yielded additional animal blood markers as well as confirming the ability to correctly determine the animal species from which blood derived, regardless of the retailer selling it (amongst the five investigated). These data confirm the initial hypothesis and demonstrate the opportunity for the proteomics-MALDI combined approach to provide additional intelligence to the investigation of violent crimes when examining blood evidence.

Detection and mapping of haemoglobin variants in blood fingermarks by MALDI MS for suspect "profiling".

Over the past seven years Matrix Assisted Laser Desorption Ionisation Mass Spectrometry Profiling (MALDI MSP) and Imaging (MALDI MSI) have proven to be feasible tools for the detection of blood and its provenance in stains and fingermarks. However, whilst this capability as a confirmatory test addresses the primary questions at the scene of a violent crime, additional intelligence recoverable from blood can also prove important for investigations. A DNA profile is the most obvious and important example of such intelligence; however, it is not always suitable for identification purposes, depending on quantity, age and environmental conditions. Proteins are much more stable and determining the presence of haemoglobin variants in blood recovered at a crime scene may provide associative and possibly corroborating evidence on the presence of an individual at a particular location. This evidence gains more incriminatory value, the lower the incidence of the variant in a certain geographical area or population and may contribute to narrowing down the pool of suspects. In this study, a MALDI based mass spectrometric method has been developed and tested on six haemoglobin variants for their fast and reliable identification and mapping in blood fingermarks.

Gas-Phase Transformation of Fluorinated Benzoporphyrins to Porphyrin-Embedded Conical Nanocarbons.

Geodesic nitrogen-containing graphene fragments are interesting candidates for various material applications, but the available synthetic protocols, which need to overcome intrinsic strain energy during the formation of the bowl-shaped skeletons, are often incompatible with heteroatom-embedded structures. Through this mass spectrometry-based gas-phase study, we show by means of collision-induced dissociation experiments and supported by density functional theory calculations, the first evidence for the formation of a porphyrin-embedded conical nanocarbon. The influences of metalation and functionalization of the used tetrabenzoporphyrins have been investigated, which revealed different cyclization efficiencies, different ionization possibilities, and a variation of the dissociation pathway. Our results suggest a stepwise process for HF elimination from the fjord region, which supports a selective pathway towards bent nitrogen-containing graphene fragments.

3D black-blood 3T-MRI for the diagnosis of abdominal large vessel vasculitis.

OBJECTIVES: To assess the value of a T1-3D black-blood turbo spin echo (TSE) sequence for the diagnosis of abdominal large vessel vasculitis (LVV). MATERIALS AND METHODS: The study included 20 patients with abdominal LVV and 17 controls, who underwent a 3T-MRI scan using a modified T1-3D volumetric isotropic TSE acquisition and a segmented T1-3D turbo field echo sequence (T1-mVISTA/T1-eTHRIVE). Two radiologists independently analyzed the aorta for concentric contrast enhancement, concentric wall thickening, image quality, and flow artifact intensity (CCE/CWT/IQ/FAI; 4-point scales). The mean aortic wall thickness (MAWT) in post-contrast T1-mVISTA was compared between patients and controls. RESULTS: IQ of T1-mVISTA was rated good to excellent in 91.5% of 282 evaluated vessel segments with no or minor FAI present in 85.5%. The inter-observer reproducibility for the identification of CCE/CWT on T1-mVISTA was 0.92 and 0.93 (p < 0.001). The distribution of segmental inflammation in T1-mVISTA significantly correlated with T1-eTHRIVE (CCE, κ = 0.768; CWT, κ = 0.715; p < 0.001), resulting in a sensitivity, specificity, and positive predictive value of 100%, 81.3%, and 83.3%. The MAWT significantly differed between patients and controls (3.29 ± 0.81 vs. 2.24 ± 0.45 mm; p < 0.001). CONCLUSIONS: T1-mVISTA enables the evaluation of the MAWT and allows the detection of abdominal LVV. KEY POINTS: • 3D T1w-mVISTA accurately depicted the large abdominal vessels. • 3D T1w-mVISTA enables accurate measurements of the abdominal aortic wall thickness. • 3D T1w-mVISTA is useful for the detection of abdominal LVV.

Systematic Isolation and Structure Elucidation of Urinary Metabolites Optimized for the Analytical-Scale Molecular Profiling Laboratory.

Annotation and identification of metabolite biomarkers is critical for their biological interpretation in metabolic phenotyping studies, presenting a significant bottleneck in the successful implementation of untargeted metabolomics. Here, a systematic multistep protocol was developed for the purification and de novo structural elucidation of urinary metabolites. The protocol is most suited for instances where structure elucidation and metabolite annotation are critical for the downstream biological interpretation of metabolic phenotyping studies. First, a bulk urine pool was desalted using ion-exchange resins enabling large-scale fractionation using precise iterations of analytical scale chromatography. Primary urine fractions were collected and assembled into a "fraction bank" suitable for long-term laboratory storage. Secondary and tertiary fractionations exploited differences in selectivity across a range of reversed-phase chemistries, achieving the purification of metabolites of interest yielding an amount of material suitable for chemical characterization. To exemplify the application of the systematic workflow in a diverse set of cases, four metabolites with a range of physicochemical properties were selected and purified from urine and subjected to chemical formula and structure elucidation by respective magnetic resonance mass spectrometry (MRMS) and NMR analyses. Their structures were fully assigned as tetrahydropentoxyline, indole-3-acetic-acid-O-glucuronide, p-cresol glucuronide, and pregnanediol-3-glucuronide. Unused effluent was collected, dried, and returned to the fraction bank, demonstrating the viability of the system for repeat use in metabolite annotation with a high degree of efficiency.

A complex pattern of autonomic dysfunction in familial Mediterranean fever. Results from a controlled cross-sectional study.

OBJECTIVES: Autonomic dysfunction (AD) has been described in various chronic inflammatory diseases. Studies of AD in patients with familial Mediterranean fever (FMF) are inconclusive. We aimed to assess AD in a cohort of FMF patients. METHODS: Signs and symptoms of AD were investigated in patients with FMF and compared to age and gender matched healthy controls. Symptoms of AD were assessed by COMPASS-31, a validated questionnaire to evaluate orthostatic, vasomotor, secretomotor, gastrointestinal, pupillomotor and bladder function domains. Assessment of objective AD comprised heart rate variability during deep breathing, skin conductance changes during mental arithmetic, blood pressure response to pain and dynamic infrared pupillometry. RESULTS: 25 patients and 25 healthy controls were included and evaluated by COMPASS-31 and objective testing of AD. FMF patients had higher median COMPASS-31 total scores than controls (23.7 vs. 1.6, p=0.024). Significant differences were also found in the secretomotor and gastrointestinal sub-domains (4.2 vs. 0.0; p<0.001 and 8.0 vs. 0.0; p=0.004, respectively). Symptoms of autonomic dysfunction were correlated with patient reported global disease activity (r=0.71; p<0.001) and pain level (r=0.68; p<0.001). There were no differences in heart rate variability (HRV), skin conductance, blood pressure response to pain or sympathetic pupillomotor function between patients and controls. FMF patients revealed impaired parasympathetic pupillomotor function that was not associated with clinical parameters. However, patients that were on IL-1-blocking therapy had better parasympathetic pupillary function than patients on conventional treatment. CONCLUSIONS: FMF patients have AD in terms of symptoms and parasympathetic pupillomotor function. Dynamic pupillometry can provide additional information on autonomic regulation in patients with FMF.

Intracranial arterial calcifications as a prognostic factor for subsequent major adverse cardiovascular events (MACE).

BACKGROUND: Intracranial arterial calcifications (ICAC) are often detected on unenhanced CT of patients with an age > 60. However, association with the subsequent occurrence of major adverse cardiovascular events (MACE) has not yet been evaluated. PURPOSE: This study aimed at evaluating the association of ICAC with subsequent MACE and overall mortality. METHODS: In this retrospective, IRB approved study, we included 175 consecutive patients (89 males, mean age 78.3 ± 8.5 years) of age > 60 years who underwent an unenhanced CT of the head due to minor trauma or neurological disorders. Presence of ICAC was determined in seven intracranial arteries using a semi-quantitative scale, which resulted in the calcified plaque score (CPS). Clinical follow-up information was obtained by questionnaires and telephone interviews. MACE was defined as myocardial infarction or revascularization, stroke or death due to cardiovascular event. RESULTS: Mean follow-up time was 39.8 ± 7.8 months, resulting in 579.7 patient-years of follow-up. Overall, 36 MACE occurred during follow-up (annual event rate = 6.2%/year). Mean CPS was significantly higher in subjects with MACE during follow-up compared to subjects without MACE (p < 0.01). In 15 patients CPS was 0; in none of these patients MACE was registered. Kaplan-Meier-analysis revealed that patients with a low plaque burden (CPS < 5) had a significant longer MACE-free and overall survival than patients with a high plaque burden (CPS ≥ 5) (p < 0.01). CONCLUSION: Patients with ICAC have an increased risk for future cardio- or cerebrovascular events. Therefore, ICAC might be a prognostic factor to determine the risk for these events in older patients.

Application of FT-ICR MS Equipped with Quadrupole Detection for Analysis of Crude Oil.

Resolving power is a critical factor determining the quality of ultrahigh-resolving power mass spectra of crude oil. In this study, 7T Fourier-transform ion cyclotron mass spectrometry (FT-ICR MS), equipped with quadrupole detection, was applied and evaluated for crude oil analysis for the first time. Four spectra were obtained from two oil samples using two ionization methods. Resolving power of 1500000 was observed at m/z 400 with 4 s transient signal. Comparison with literature reports revealed that the achieved resolving power was comparable with or superior to those obtained from instruments using higher magnetic fields but without quadrupole detection. A total of 6000-10000 peaks with an S/N ratio of 3 or higher were observed from the obtained spectra and over 97% of the peaks could be assigned to appropriate chemical formulas with an error within 1 ppm. Double bond equivalents vs carbon number plots generated from the obtained data agreed well with those previously reported without quadrupole detection. Mass accuracy values of the assigned elemental formulas were examined and the average root-mean-square error was calculated to be only 160 ppb. Low unassignment rate of the observed peaks and strong agreement with previously reported results suggests that unwanted harmonics of reduced frequency are not significant for the data obtained with quadrupole detection. Overall, the data presented in this study show that FT-ICR MS equipped with quadrupole detection can be a powerful tool to examine complex mixtures like crude oil. To the best of our knowledge, this is the first paper reporting application of FT-ICR MS equipped with quadrupole detection for the oil analysis.

Nonuniform Sampling Acquisition of Two-Dimensional Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Increased Mass Resolution of Tandem Mass Spectrometry Precursor Ions.

Obtaining the full MS/MS map for fragments and precursors of complex mixtures without hyphenation with chromatographic separation by a data-independent acquisition is a challenge in mass spectrometry which is solved by two-dimensional (2D) Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). Until now 2D FTICR MS afforded only a moderate resolution for precursor ion since this resolution is limited by the number of evolution interval steps to which the number of scans, the acquisition time, and the sample consumption are proportional. An overnight acquisition is already required to reach a quadrupole mass filter-like unit mass resolution. Here, we report that 2D FTICR MS using nonuniform sampling (NUS) obtained by randomly skipping points in the first dimension corresponding to the precursor selection gives access, after data processing, to the same structural information contained in a complex mixture. The resolution increases roughly as the inverse of the NUS ratio, up to 26 times at NUS 1/32, leading to an acquisition time reduced in the same ratio compared to a classical acquisition at the same resolution. As an example, the analysis of a natural oil is presented.

Paradoxical antidepressant effects of alcohol are related to acid sphingomyelinase and its control of sphingolipid homeostasis.

Alcohol is a widely consumed drug that can lead to addiction and severe brain damage. However, alcohol is also used as self-medication for psychiatric problems, such as depression, frequently resulting in depression-alcoholism comorbidity. Here, we identify the first molecular mechanism for alcohol use with the goal to self-medicate and ameliorate the behavioral symptoms of a genetically induced innate depression. An induced over-expression of acid sphingomyelinase (ASM), as was observed in depressed patients, enhanced the consumption of alcohol in a mouse model of depression. ASM hyperactivity facilitates the establishment of the conditioned behavioral effects of alcohol, and thus drug memories. Opposite effects on drinking and alcohol reward learning were observed in animals with reduced ASM function. Importantly, free-choice alcohol drinking-but not forced alcohol exposure-reduces depression-like behavior selectively in depressed animals through the normalization of brain ASM activity. No such effects were observed in normal mice. ASM hyperactivity caused sphingolipid and subsequent monoamine transmitter hypo-activity in the brain. Free-choice alcohol drinking restores nucleus accumbens sphingolipid- and monoamine homeostasis selectively in depressed mice. A gene expression analysis suggested strong control of ASM on the expression of genes related to the regulation of pH, ion transmembrane transport, behavioral fear response, neuroprotection and neuropeptide signaling pathways. These findings suggest that the paradoxical antidepressant effects of alcohol in depressed organisms are mediated by ASM and its control of sphingolipid homeostasis. Both emerge as a new treatment target specifically for depression-induced alcoholism.

LC-MS/MS Detection of Karlotoxins Reveals New Variants in Strains of the Marine Dinoflagellate Karlodinium veneficum from the Ebro Delta (NW Mediterranean).

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the detection and quantitation of karlotoxins in the selected reaction monitoring (SRM) mode. This novel method was based upon the analysis of purified karlotoxins (KcTx-1, KmTx-2, 44-oxo-KmTx-2, KmTx-5), one amphidinol (AM-18), and unpurified extracts of bulk cultures of the marine dinoflagellate Karlodinium veneficum strain CCMP2936 from Delaware (Eastern USA), which produces KmTx-1 and KmTx-3. The limit of detection of the SRM method for KmTx-2 was determined as 2.5 ng on-column. Collision induced dissociation (CID) spectra of all putative karlotoxins were recorded to present fragmentation patterns of each compound for their unambiguous identification. Bulk cultures of K. veneficum strain K10 isolated from an embayment of the Ebro Delta, NW Mediterranean, yielded five previously unreported putative karlotoxins with molecular masses 1280, 1298, 1332, 1356, and 1400 Da, and similar fragments to KmTx-5. Analysis of several isolates of K. veneficum from the Ebro Delta revealed small-scale diversity in the karlotoxin spectrum in that one isolate from Fangar Bay produced KmTx-5, whereas the five putative novel karlotoxins were found among several isolates from nearby, but hydrographically distinct Alfacs Bay. Application of this LC-MS/MS method represents an incremental advance in the determination of putative karlotoxins, particularly in the absence of a complete spectrum of purified analytical standards of known specific potency.

Clinical experience with the European Ankylosing Spondylitis Infliximab Cohort (EASIC): long-term extension over 7 years with focus on clinical efficacy and safety.

OBJECTIVES: Knowledge on the long-term effects of anti-TNF therapy in patients with ankylosing spondylitis (AS) is still limited. Our objective was to study the long-term efficacy and safety of anti-TNF therapy in AS. METHODS: After having completed the first part of the EASIC trial a total of 71 patients were enrolled into this 96-week extension study. Patients were treated with the same dosages and dosing intervals of infliximab as in the EASIC core study. Efficacy was assessed by using standardised assessment tools such as BASDAI, BASFI, BASMI, patient global assessment, CRP levels and the proportion of patients without any sign of enthesitis or arthritis. Long-term safety was assessed by documenting adverse events (AE), serious adverse events (SAE) and reasons for dropping out. RESULTS: Of the 71 patients included, 64 (90.1%) completed the trial , and 7 discontinued: one was lost to follow-up, 3 withdrew informed consent and in 3 patients therapy was stopped for different reasons: secondary loss of response, recurrent infections and basal cell carcinoma of the skin. The completers showed rather stable low scores of BASDAI (mean 2.4, median 2.52), BASFI (mean 3.1, median 2.76) and BASMI (mean 3.2, median 3) as well as patients global assessment and CRP. The vast majority of patients did not have enthesitis or arthritis. A total of 476 AE were observed, 13 of which were SAE. The majority of these were infections and most of them affected the respiratory tract. Two malignancies occurred: one basal cell carcinoma and one malignant melanoma. These were the only SAE judged to be possibly related to the study drug. CONCLUSIONS: Anti-TNF treatment with infliximab is efficacious over long periods of time in patients with AS. The observation of two skin related malignancies, including one melanoma, during the whole study period of 7 years is in line with reports from previous large AS data sets.

The influence of long distance running on sonographic joint and tendon pathology: results from a prospective study with marathon runners.

BACKGROUND: The impact of physical exercise on joints and tendons is still a matter of debate. The aim of this study was to investigate with ultrasound the acute effects of extreme physical exercise on knee and ankle joints and their surrounding structures in trained athletes. METHODS: Participants of the Munich marathon were examined by arthrosonography before and after long distance running. Ultrasound assessment included grey scale and power Doppler examination of the knee and talocrural joints with surrounding tendons. Findings consistent with joint effusion, tendon and/or entheseal pathologies were documented. In addition to the ultrasound evaluation, information on training habits and past or present arthralgia or joint swelling was gathered. RESULTS: One Hundred Five runners completed both the pre- and post-excercise ultrasound assessments (baseline and follow-up), resulting in the sonographic evaluation of 420 knee and talocrural joints. At baseline, 105 knee (50) and 38 talocrural joints (18.1) showed effusions, compared to 100 knee (47.6) and 33 talocrural joints (15.7 %) at follow-up. The differences were not significant (p > 0.05 each). Effusion size did not correlate with the timepoint of ultrasound assessment and was independent of covariates such as gender, age or running distance. Hypervascularity of the patellar tendon was detected in 21 cases (10.0 %) at follow-up in contrast to one at baseline (p < 0.001). This observation was more frequent in male than in female participants (p < 0.05). CONCLUSIONS: Acute physical stress is significantly associated with hypervascularity of the patellar tendon. No significant changes of synovial effusion were detected in knee and talocrural joints.

Decreased expression of miR-146a and miR-155 contributes to an abnormal Treg phenotype in patients with rheumatoid arthritis.

OBJECTIVES: MicroRNAs (miRNAs) have been implicated in the pathogenesis of autoimmune diseases, not least for their critical role in the regulation of regulatory T cell (Treg) function. Deregulated expression of miR-146a and miR-155 has been associated with rheumatoid arthritis (RA). We therefore investigated miR-146a and miR-155 expression in Tregs of patients with RA and their possible impact on Treg function and disease activity. METHODS: Expression of miR-146a and miR-155 was assessed in RA patients and controls. MiRNA expression was correlated with disease activity and expression of target genes. Interference with biological activity of miRNAs was evaluated in functional Treg assays. RESULTS: Diminished upregulation of miR-146a and miR-155 in response to T cell stimulation was found in Tregs of RA patients. Diminution of miR-146a expression was observed in particular in patients with active disease, and correlated with joint inflammation. In patients with active RA, Tregs demonstrated a pro-inflammatory phenotype characterised by inflammatory cytokine expression. This was due to an augmented expression and activation of signal transducer and activator transcription 1 (STAT1), a direct target of miR-146a. CONCLUSIONS: Our results suggest that in RA miR-146a facilitates a pro-inflammatory phenotype of Tregs via increased STAT1 activation, and contributes thereby to RA pathogenesis.

Relevance of grade 1 gray-scale ultrasound findings in wrists and small joints to the assessment of subclinical synovitis in rheumatoid arthritis.

OBJECTIVE: To investigate the clinical relevance of grade 1 findings on gray-scale ultrasound (GSUS) of the joints in patients with rheumatoid arthritis (RA). METHODS: We examined the wrists and small joints of 100 patients with early or established RA and 30 healthy controls, using GSUS and power Doppler ultrasound (PDUS). Independent clinical assessment of all joints for tenderness and swelling according to the European League Against Rheumatism examination technique was performed. Joints with grade 1 findings on GSUS were identified, and associations with swelling, pain, and findings on PDUS were assessed. Grade 1 findings on GSUS in patients with early RA were reassessed after 6 months of antirheumatic treatment. RESULTS: Grade 1 results represented the majority of all GSUS findings in patients with RA and were also frequently recorded in healthy controls. Grade 1 GSUS findings were not associated with tenderness, swelling, or positive results on PDUS. In comparison to joints with grade 2 and grade 3 findings on GSUS, joints with grade 1 findings were less likely to respond to treatment. CONCLUSION: The present results indicate that grade 1 findings on GSUS have limited clinical relevance.

Increased interleukin-26 in the peripheral joints of patients with axial spondyloarthritis and psoriatic arthritis, co-localizing with CD68-positive synoviocytes.

Objectives: IL26 levels are elevated in the blood and synovial fluid of patients with inflammatory arthritis. IL26 can be produced by Th17 cells and locally within joints by tissue-resident cells. IL26 induces osteoblast mineralization in vitro. As osteoproliferation and Th17 cells are important factors in the pathogenesis of axial spondyloarthritis (axSpA), we aimed to clarify the cellular sources of IL26 in spondyloarthritis. Methods: Serum, peripheral blood mononuclear cells (n = 15-35) and synovial tissue (n = 3-9) of adult patients with axSpA, psoriatic arthritis (PsA) and rheumatoid arthritis (RA) and healthy controls (HCs, n = 5) were evaluated by ELISA, flow cytometry including PrimeFlow assay, immunohistochemistry and immunofluorescence and quantitative PCR. Results: Synovial tissue of axSpA patients shows significantly more IL26-positive cells than that of HCs (p < 0.01), but numbers are also elevated in PsA and RA patients. Immunofluorescence shows co-localization of IL26 with CD68, but not with CD3, SMA, CD163, cadherin-11, or CD90. IL26 is elevated in the serum of RA and PsA (but not axSpA) patients compared with HCs (p < 0.001 and p < 0.01). However, peripheral blood CD4+ T cells from axSpA and PsA patients show higher positivity for IL26 in the PrimeFlow assay compared with HCs. CD4+ memory T cells from axSpA patients produce more IL26 under Th17-favoring conditions (IL-1ß and IL-23) than cells from PsA and RA patients or HCs. Conclusion: IL26 production is increased in the synovial tissue of SpA and can be localized to CD68+ macrophage-like synoviocytes, whereas circulating IL26+ Th17 cells are only modestly enriched. Considering the osteoproliferative properties of IL26, this offers new therapeutic options independent of Th17 pathways.

MALDI mass spectrometric imaging based identification of clinically relevant signals in prostate cancer using large-scale tissue microarrays.

To identify molecular features associated with clinico-pathological parameters and TMPRSS2-ERG fusion status in prostate cancer, we employed MALDI mass spectrometric imaging (MSI) to a prostate cancer tissue microarray (TMA) containing formalin-fixed, paraffin-embedded tissues samples from 1,044 patients for which clinical follow-up data were available. MSI analysis revealed 15 distinct mass per charge (m/z)-signals associated to epithelial structures. A comparison of these signals with clinico-pathological features revealed statistical association with favorable tumor phenotype such as low Gleason grade, early pT stage or low Ki67 labeling Index (LI) for four signals (m/z 700, m/z 1,502, m/z 1,199 and m/z 3,577), a link between high Ki67LI for one signal (m/z 1,013) and a relationship with prolonged time to PSA recurrence for one signal (m/z 1,502; p = 0.0145). Multiple signals were associated with the ERG-fusion status of our cancers. Two of 15 epithelium-associated signals including m/z 1,013 and m/z 1,502 were associated with detectable ERG expression and five signals (m/z 644, 678, 1,044, 3,086 and 3,577) were associated with ERG negativity. These observations are in line with substantial molecular differences between fusion-type and non-fusion type prostate cancer. The signals observed in this study may characterize molecules that play a role in the development of TMPRSS2-ERG fusions, or alternatively reflect pathways that are activated as a consequence of ERG-activation. The combination of MSI and large-scale TMAs reflects a powerful approach enabling immediate prioritization of MSI signals based on associations with clinico-pathological and molecular data.