Structure-activity relationships of actively FhuE transported rifabutin derivatives with potent activity against Acinetobacter baumannii.

Hospital-acquired infections are on the rise and represent both, a clinical and financial burden. With resistance emerging and an ever-dwindling armamentarium at hand, infections caused by Acinetobacter baumannii are particularly problematic, since these bacteria have a high level of resistance and resilience to traditional and even last-resort antibiotics. The antibiotic rifabutin was recently found to show potent in vitro and in vivo activity against extensively drug resistant A. baumannii. Building on this discovery, we report on the synthesis and activity of rifabutin analogs, with a focus on N-functionalization of the piperidine ring. The antimicrobial testing uncovered structure activity relationships (SAR) for A. baumannii that were not reflected in Staphylococcus aureus. The cellular activity did not correlate with cell-free transcription inhibition, but with bacterial intracellular compound accumulation. Mass spectrometry-based accumulation studies confirmed the involvement of the siderophore receptor FhuE in active compound translocation at low concentrations, and they showed a strong impact of the culture medium on the accumulation of rifabutin. Overall, the study underlines the structural feature required for strong accumulation of rifabutin in A. baumannii and identifies analogs as or more potent than rifabutin against A. baumannii.

[Autoantibodies and the autoreactive immune response : There is more to ACPA than ACPA]. / Autoantikörper und die autoreaktive Immunantwort : ACPA sind mehr als nur ACPA.

The immune response against posttranslationally modified (PTM) antigens, in particular the generation of anti-citrullinated protein antibodies (ACPA), is a very specific hallmark of rheumatoid arthritis. The factors that initiate this immune response and the triggers that stimulate the transition from asymptomatic autoimmunity to autoimmune disease are so far unknown. Genetic risk factors and the maturation of the ACPA response prior to the onset of arthritis indicate an important role for helper T cells in this process. Antigens that trigger this process, however, remain to be defined. Notably, recent data demonstrate that ACPA do not only recognize citrullinated protein antigens. Other posttranslational protein modifications such as homocitrulline and acetyllysine are also recognized. This cross-reactivity towards different PTM antigens was found for various monoclonal ACPA and broadens the spectrum of antigens that can stimulate and activate ACPA-expressing B cells. Also, it suggests that such B cells could receive help from autoreactive but also from non-autoreactive T cells. This review summarizes these recent findings and provides insight into their potential relevance for the disease rheumatoid arthritis.

The next generation of current measurement for ionization chambers.

Re-entrant ionization chambers (ICs) are essential to radionuclide metrology and nuclear medicine for maintaining standards and measuring half-lives. The requirements of top-level metrology demand that systems must be precise and stable to 0.1 % over many years, and linear from 10-14 A to 10-8 A. Thus, laboratories depend on bespoke current measurement systems and often rely on sealed sources to generate reference currents. To maintain and improve present capabilities, metrologists need to overcome two looming challenges: ageing electronics and decreasing availability of sealed sources. Possible solutions using Ultrastable Low-Noise Current Amplifiers (ULCAs), resistive-feedback electrometers, and (quantum) single-electron pumps are reviewed. Broader discussions of IC design and methodology are discussed. ULCAs show promise and resistive-feedback systems which take advantage of standard resistor calibrations offer an alternative.

Antibodies and B cells recognising citrullinated proteins display a broad cross-reactivity towards other post-translational modifications.

OBJECTIVE: Autoantibodies against antigens carrying distinct post-translational modifications (PTMs), such as citrulline, homocitrulline or acetyllysine, are hallmarks of rheumatoid arthritis (RA). The relation between these anti-modified protein antibody (AMPA)-classes is poorly understood as is the ability of different PTM-antigens to activate B-cell receptors (BCRs) directed against citrullinated proteins (CP). Insights into the nature of PTMs able to activate such B cells are pivotal to understand the 'evolution' of the autoimmune response conceivable underlying the disease. Here, we investigated the cross-reactivity of monoclonal AMPA and the ability of different types of PTM-antigens to activate CP-reactive BCRs. METHODS: BCR sequences from B cells isolated using citrullinated or acetylated antigens were used to produce monoclonal antibodies (mAb) followed by a detailed analysis of their cross-reactivity towards PTM-antigens. Ramos B-cell transfectants expressing CP-reactive IgG BCRs were generated and their activation on stimulation with PTM-antigens investigated. RESULTS: Most mAbs were highly cross-reactive towards multiple PTMs, while no reactivity was observed to the unmodified controls. B cells carrying CP-reactive BCRs showed activation on stimulation with various types of PTM-antigens. CONCLUSIONS: Our study illustrates that AMPA exhibit a high cross-reactivity towards at least two PTMs indicating that their recognition pattern is not confined to one type of modification. Furthermore, our data show that CP-reactive B cells are not only activated by citrullinated, but also by carbamylated and/or acetylated antigens. These data are vital for the understanding of the breach of B-cell tolerance against PTM-antigens and the possible contribution of these antigens to RA-pathogenesis.

Noise-optimized ultrastable low-noise current amplifier.

We have developed a noise-optimized ultrastable low-noise current amplifier (ULCA) aimed at reducing the uncertainty at low currents. It involves a thin-film resistor network with 6.75 GΩ at the high-ohmic path which reduces the noise level to 1.6 fA/Hz. Noise investigations as well as short-term and long-term stability studies were carried out. The stability of the input current gain was measured using a cryogenic current comparator at ±6.1 nA. Methods for investigating the measurement accuracy at low input currents of about 100 pA at a level of below one part in 107 are introduced and experimentally verified. The performance of the noise-optimized ULCA is compared with that of the standard variant introduced in 2014. It is shown that the reduced noise floor is achieved without impairing the stability of the transresistance.

Measurement of sub-picoampere direct currents with uncertainties below ten attoamperes.

A new type of the ultrastable low-noise current amplifier (ULCA) is presented. It involves thick-film resistors to achieve a high feedback resistance of 185 GΩ at the input amplifier. An improved noise level of 0.4 fA/Hz with a 1/f corner of about 30 µHz and an effective input bias current well below 100 aA are demonstrated. For small direct currents, measurement uncertainties below 10 aA are achievable even without current reversal or on/off switching. Above about 1 pA, the stability of the ULCA's resistor network limits the relative measurement uncertainty to about 10 parts per million. The new setup is used to characterize and optimize the noise in the wiring installed on a dilution refrigerator for current measurements on single-electron transport pumps. In a test configuration connected to the wiring in a pulse tube refrigerator, a total noise floor of 0.44 fA/Hz was achieved including the contributions of amplifier and cryogenic wiring.

Effects of type II collagen epitope carbamylation and citrullination in human leucocyte antigen (HLA)-DR4(+) monozygotic twins discordant for rheumatoid arthritis.

The aim of this study is to investigate the effect of the native, citrullinated or carbamylated type II human collagen T cell- and B cell-epitopes on the adaptive immune response in rheumatoid arthritis (RA). Peripheral blood T and B cells obtained from a human leucocyte D4-related (antigen DR4(-) HLA-DR4)(+) woman with early RA, her healthy monozygotic twin and an unrelated HLA-DR3(+) woman with early RA were analysed for activation (CD154/CD69), apoptosis (annexin/7-aminoactinomycin), cytokine production [interferon (IFN)γ/interleukin (IL)-17/IL-4/IL-10/IL-6] and functional phenotype (CD45Ra/CCR7) after stimulation with the collagen native T cell epitope (T261-273), the K264 carbamylated T cell epitope (carT261-273), the native B cell epitope (B359-369) or the R360 citrullinated B cell epitope (citB359-369), and the combinations of these. The T cell memory compartment was activated by T cell epitopes in both discordant DR4(+) twins, but not in the DR3(+) RA. The collagen-specific activation of CD4(+) T cells was induced with both the native and carbamylated T cell epitopes only in the RA twin. Both T cell epitopes also induced IL-17 production in the RA twin, but a greater IL-4 and IL-10 response in the healthy twin. The citrullinated B cell epitope, particularly when combined with the carbamylated T cell epitope, induced B cell activation and an increased IL-6/IL-10 ratio in the RA twin compared to a greater IL-10 production in the healthy twin. Our data suggest that circulating collagen-specific T and B cells are found in HLA-DR4(+) subjects, but only RA activated cells express co-stimulatory molecules and produce proinflammatory cytokines. Carbamylation and citrullination further modulate the activation and cytokine polarization of T and B cells.

A novel method for high-throughput detection and quantification of neutrophil extracellular traps reveals ROS-independent NET release with immune complexes.

A newly-described first-line immune defence mechanism of neutrophils is the release of neutrophil extracellular traps (NETs). Immune complexes (ICxs) induce low level NET release. As such, the in vitro quantification of NETs is challenging with current methodologies. In order to investigate the role of NET release in ICx-mediated autoimmune diseases, we developed a highly sensitive and automated method for quantification of NETs. After labelling human neutrophils with PKH26 and extracellular DNA with Sytox green, cells are fixed and automatically imaged with 3-dimensional confocal laser scanning microscopy (3D-CLSM). NET release is then quantified with digital image analysis whereby the NET amount (Sytox green area) is corrected for the number of imaged neutrophils (PKH26 area). A high sensitivity of the assay is achieved by a) significantly augmenting the area of the well imaged (11%) as compared to conventional assays (0.5%) and b) using a 3D imaging technique for optimal capture of NETs, which are topologically superimposed on neutrophils. In this assay, we confirmed low levels of NET release upon human ICx stimulation which were positive for citrullinated histones and neutrophil elastase. In contrast to PMA-induced NET release, ICx-induced NET release was unchanged when co-incubated with diphenyleneiodonium (DPI). We were able to quantify NET release upon stimulation with serum from RA and SLE patients, which was not observed with normal human serum. To our knowledge, this is the first semi-automated assay capable of sensitive detection and quantification of NET release at a low threshold by using 3D CLSM. The assay is applicable in a high-throughput manner and allows the in vitro analysis of NET release in ICx-mediated autoimmune diseases.

Does ex vivo CD34+ positive selection influence outcome after autologous hematopoietic stem cell transplantation in systemic sclerosis patients?

This EBMT Autoimmune Disease Working Party study aimed to evaluate the influence of CD34+ positive graft selection (CD34+) on the outcome of systemic sclerosis (SSc) patients after autologous hematopoietic stem cell transplantation (AHSCT). Clinical and laboratory data from 138 SSc patients at diagnosis, before and after AHSCT were retrospectively analyzed. CD34+ selection was performed in 47.1% (n=65) patients. By multivariate analysis adjusting for all factors differing between the two groups (without or with CD34+), there was no statistically significant difference in terms of overall survival (hazard ratio (HR): 0.98, 95% confidence interval (CI) 0.40-2.39, P=0.96), PFS (HR: 1.55, 95% CI 0.83-2.88, P=0.17) and incidence of relapse or progression (HR: 1.70, 95% CI 0.85-3.38, P=0.13). We demonstrate that CD34+ does not add benefit to the outcome of SSc patient treated with AHSCT. These findings should be further confirmed by prospective randomized trials.

Ethyl-Zinc(II)-Cation Equivalents: Synthesis and Hydroamination Catalysis.

Ion-like ethylzinc(II) compounds with weakly coordinating aluminates [Al(OR(F))4](-) and [(R(F)O)3Al-F-Al(OR(F))3](-) (R(F)=C(CF3)3) were synthesized in a one-pot reaction and fully characterized by single-crystal X-ray diffraction, NMR and vibrational spectroscopy, and by quantum chemical calculations. The catalytic activity of ion-like Et-Zn[Al(OR(F))4] in intermolecular hydroamination and in the unusual double hydroamination of anilines and alkynes was investigated. Favorable performance was also found in comparison to the Et2Zn/[PhNMe2H](+)[B(C6F5)4](-) system generated in situ at lower catalyst loadings of 2.5 mol %.

Ultrastable low-noise current amplifier: a novel device for measuring small electric currents with high accuracy.

An ultrastable low-noise current amplifier (ULCA) is presented. The ULCA is a non-cryogenic instrument based on specially designed operational amplifiers and resistor networks. It involves two stages, the first providing a 1000-fold current gain and the second performing a current-to-voltage conversion via an internal 1 MΩ reference resistor or, optionally, an external standard resistor. The ULCA's transfer coefficient is highly stable versus time, temperature, and current amplitude within the full dynamic range of ±5 nA. The low noise level of 2.4 fA/√Hz helps to keep averaging times short at small input currents. A cryogenic current comparator is used to calibrate both input current gain and output transresistance, providing traceability to the quantum Hall effect. Within one week after calibration, the uncertainty contribution from short-term fluctuations and drift of the transresistance is about 0.1 parts per million (ppm). The long-term drift is typically 5 ppm/yr. A high-accuracy variant is available that shows improved stability of the input gain at the expense of a higher noise level of 7.5 fA/√Hz. The ULCA also allows the traceable generation of small electric currents or the calibration of high-ohmic resistors.

How undifferentiated arthritis evolves into chronic arthritis.

Undifferentiated arthritis (UA) is a frequently occurring clinical presentation with a variable outcome. While some forms of UA will spontaneously remit, other forms will progress to chronic arthritis; an outcome that would preferably be prevented. Which immunological factors are normally at the basis of resolution of inflammation, and what, on the other hand, causes inflammation to persist? This review provides an overview of the immunological mechanisms involved in these two scenarios, including specific examples of how these mechanisms apply, or can be influenced in rheumatic diseases. Furthermore, what do we know about risk factors for chronic arthritis, such as the development of autoantibodies? The recent years have provided many insights concerning risk factors for autoantibody-positive versus autoantibody-negative rheumatoid arthritis, which are discussed along with a possible pathophysiological model incorporating autoantibodies into the larger process of disease development. Finally, the evolution of the autoantibody response over time is described.

[Biomarkers and personalized medicine]. / Biomarker und personalisierte Medizin.

Biomarkers form the basis for patient stratification and the development of individualized treatment strategies. As the understanding of the pathophysiological processes underlying rheumatic diseases increases, novel biomarkers will become available and established markers can be used more efficiently. Autoantibodies in rheumatoid arthritis (RA), for example, define a subgroup of patients with a specific risk profile and response to therapy. For this reason they have been added to the classification criteria for RA and are part of current treatment guidelines. In addition, novel markers are being evaluated and validated. For the concept of personalized medicine this indicates that the use of future therapeutic substances will be more frequently coupled to the detection of specific biomarkers. While this will decrease the number of patients who become eligible for certain treatments, it will increase efficacy and safety for patients and potentially the cost-effectiveness.

Toward a data-driven evaluation of the 2010 American College of Rheumatology/European League Against Rheumatism criteria for rheumatoid arthritis: is it sensible to look at levels of rheumatoid factor?

OBJECTIVE: Recently, new classification criteria for rheumatoid arthritis (RA) have been devised by methodology that used first a quantitative approach (data from databases), then a qualitative approach (consensus; based on paper patients), and finally a common sense-based approach (evaluation of the former phases). Now the individual items that make up these criteria are being evaluated. This study was undertaken to analyze the item "autoantibodies," in particular rheumatoid factor (RF) level. METHODS: Three separate cohorts comprising a total of 972 patients with undifferentiated arthritis were studied for RA development (according to the 1987 American College of Rheumatology criteria) and arthritis persistence. The positive predictive value (PPV), negative predictive value (NPV), and likelihood ratios (LRs) were compared between different levels of RF and the presence of anti-citrullinated protein antibody (ACPA). A similar comparison was made in 686 RA patients for the rate of joint destruction and achievement of sustained disease-modifying antirheumatic drug-free remission during 7 years of followup. The variation in RF levels obtained by different measurement methods in the same RF-positive sera was explored. RESULTS: Compared to high RF levels, presence of ACPA had a better balance between positive LR and negative LR and between PPV and NPV for RA development. The additive value of ACPA assessment after testing for RF level was higher than vice versa. The association between high RF level and RA severity was not as strong as that between ACPA antibodies and RA severity. The RF level obtained by different methods in the same patients' sera varied considerably. CONCLUSION: Our findings indicate that determination of RF level is subject to large variation; high RF level has limited additive prognostic value compared to ACPA positivity. Thus, omitting RF level and using RF presence, ACPA presence, and ACPA level may improve the 2010 criteria for RA.

Anti-citrullinated protein antibodies have a low avidity compared with antibodies against recall antigens.

OBJECTIVES: Anti-citrullinated protein antibodies (ACPA) are highly specific for rheumatoid arthritis (RA) and have been implicated in disease pathogenesis. Recent ongoing evidence indicates that the ACPA response broadens before precipitation of full-blown RA, as indicated by a more extensive isotype usage and an increased citrullinated epitope recognition profile. Nonetheless, the evolution of the ACPA response is still poorly understood and might intrinsically differ from the protective responses against pathogens. METHODS: The avidity and the avidity maturation of ACPA in relation to the avidity of antibodies against recall antigens were analysed. RESULTS: The avidity of ACPA was significantly lower than the avidity of antibodies to the recall antigens tetanus toxoid and diptheria toxoid. Moreover, ACPA did not show avidity maturation during longitudinal follow-up and ACPA avidity was also relatively low in patients who displayed extensive isotype switching. CONCLUSIONS: These observations indicate that the natural evolution of ACPA differs from the development of antibodies against recall antigens. These data also indicate that ACPA avidity maturation and isotype switching are disconnected, whereby extensive isotype switching occurs in the setting of restricted avidity maturation. Intrinsic differences between the RA-specific autoantibody system and protective antibody responses against pathogens could be of relevance for designing novel B cell-targeted therapies for RA.

Effect of long term organic amendments on adsorption-desorption of thiram onto a luvisol soil derived from loess.

The objective of this work was to assess the influence of soil organic amendments on the sorption properties of the fungicide thiram. The organic amendments studied were organic household compost (COM), sewage sludge from municipal water treatment facilities (SLU) and farmyard manure (FYM), which were compared to mineral fertilizer application (MIN). Sorption-desorption experiments were performed using the batch method and the results indicated that the adsorption isotherms were non-linear and were found to conform to the Brunauer-Emmett-Teller (BET) model, suggesting multilayer adsorption and adsorbate-adsorbate interactions after the saturation of the surface layer. In general, distribution coefficient values, K(D), are dependent on, but not proportional to, the initial concentration of thiram. For a fixed thiram initial concentration, a significant correlation (r(2)>0.851; p<0.001) between K(D) values and the soil organic carbon content (OC) was observed. The highest value of K(D) was observed for the soil amended with compost, which is the one with the highest organic carbon content. K(D) values were divided by the soil organic carbon contents in order to obtain organic carbon partition coefficients K(OC). Comparing K(OC) means from 3 (initial concentrations) x 4 (soil organic matter compositions) x 3 (replicates) factorial ANOVA allow us to conclude that there is a significant but not proportional influence of the initial concentration of thiram on those values, but changes in the soil organic matter composition, associated to different soil amendments, have no significant influence on adsorption of thiram. To evaluate the reversibility of thiram adsorption, two consecutive desorption cycles were performed with CaCl(2) 0.01 mol L(-1). The desorption K(D) values were consistently higher (approximately twice) than those for adsorption at the same equilibrium concentrations for all soil samples supporting the existence of hysteresis in the adsorption-desorption behavior of thiram. Despite the fact that the adsorption K(D) values were proportionally increased with increasing total organic carbon content, this was not the case for the desorption K(D) values.

The improvement of middle ear ventilation by laser ablation of the epipharyngeal eustachian tube: a prospective study.

A long-lasting dysfunction of the eustachian tube seems to be the etiologic origin for development of chronic otitis media (COM) with mesotympanic perforation, otitis media with effusion (OME), and chronic atelectasis of the middle ear. Surgical interventions in the middle ear generally treat the sequelae of the tube dysfunction but not the dysfunction itself. This prospective clinical study investigated how far fiber-guided laser ablation of the posterior half of the epipharyngeal tubal ostium led to better middle ear ventilation in the otologic disease patterns mentioned below. There were 38 adult patients included in the analysis. The patients in one group had a perforated tympanic membrane [COM before primary tympanoplasty (n = 14) or revision tympanoplasty (n = 5)]; the patients in a second group had an intact eardrum [OME resistant to therapy (n = 3), with an atelectasis of the middle ear (n = 2) or problems of pressure equalization with fast changes in ambient pressure (diving, flying) (n = 14)]. Laser ablation of the posterior half of the epipharyngeal tubal ostium was performed, generally with local anesthesia, if tubal function testing was pathologic (Valsalva maneuver, passive tube opening, tympanogram). In patients with COM the procedure was performed 8 weeks before the middle ear surgery. All patients were checked 8 weeks postoperatively and in the course of the following year. The intervention seemed to have had a positive effect on tube function in 68.4% of patients operated on (P = 0.001). In 26 of the 38 patients that had undergone operation, an improvement the results of tubal function tests could be seen in the postoperative follow-up. In the COM group the Valsalva maneuver improved in 14 of 19 patients (73.6%) (P = 0.001), and the passive tube opening improved in nine of 18 patients (50%). In the group with an intact eardrum the Valsalva maneuver improved in 13 of 18 patients (72.2%) (P = 0.001). The resulting condition remained stable after 1 year. None of the patients showed any complications as a result of the therapy. Minimally invasive shaping of the distal eustachian tube under topical anesthesia can be recommended for patients with the above-mentioned diagnoses who have pathologic middle ear ventilation. Especially prior to tympanoplasties, and especially in otologic revision procedures, where middle ear ventilation is a prerequisite for successful otologic surgery, the function of the eustachian tube can be optimized in 70% of the patients, particularly if there are pathological findings (tubal tonsil, narrow orifice of the tubal ostium, adenoids). The placement of permanent ear tubes in adults with recurrent OME can also be avoided by the procedure described. The resultant conditions remained stable for the next year. Patients with tympanic ventilation problems due to rapid pressure changes (flying, diving) can also benefit from this procedure.

[Influence of different head-trunk positions to the upbeat-nystagmus]. / Einfluss der Kopf-Rumpf-Positionsänderung auf den Upbeat-Nystagmus.

The neck torsion test (NTT) can be used to evaluate the cervico-ocular reflex (COR). We think there are two main reasons why NRT has failed to gain general acceptance in clinical practice: the test does not fully exploit the somatosensory examination potential, and electronystagmography has been most commonly used but is inferior to videonystagmography for a very low-amplitude nystagmus. These considerations have led us to develop a modified neck torsion test (mNTT) with three-dimensional video-oculographic (3D-VOG) recording. Sensors on the video glasses enable three-dimensional and continuous registration of the head position. Horizontal, vertical and rotatory eye movements are determined in six different head-body positions (right/left head tilt, ante/retroflexion of the head, and right/left body rotation). The basis for assessing the mNTT is described in detail. We used the mNTT to examine prospectively compensated, vestibularly deficient (n = 52), and cervically deficient (n = 93) patients with vertigo against a control group (n = 22). Our results show that upbeat-nystagmus (UBN) significantly differentiated the groups. This occurs most frequently in the tonic analysis phase of head inclination and reclination. UBN is differential-diagnostically discussed on the basis of these examination findings, and its cervical origin is elucidated with the aid of model conceptions. We conclude that our modified NRT is an improved instrument for COR determination. A suspected cervical somatosensory reduction warrants testing particularly for UBN.