Search

Rheological characterisation of mortar is complicated by phenomena of slip, the formation of shear bands and depletion. At relatively low solid fractions a typical Couette geometry and a medium-size mixer-type rheometer were used to determine flow curves. At higher solid fractions a large-size coaxial cylinder rheometer with multiple blade vane geometry was used up to the point where slippage occurred. The viscosity as a function of concentration responds to the Krieger-Dougherty law, when a mortar is considered as a suspension of sand in a matrix of binder slurry. The limits of this description corresponds to a critical solid fraction above which air is entrapped during the mixing procedure: air content measurements demonstrate this phenomenon. A clear relationship between mortar and slurries was established, based on the measured properties of both binder and sand particles, and on the Farris model for polydisperse suspensions. Intrinsic viscosity can be used as a tool to evaluate shape characteristics of the binder particles. A procedure for mixture optimisation of mortars using this model is demonstrated for the case of a trimodal mortar.

African trypanosomiasis is a widespread disease of human and veterinary importance caused by various Trypanosoma spp. with a globally devastating impact and a need for novel treatment options. We here provide a comprehensive preclinical evaluation of nucleoside analogues, 6-thioether-modified tubercidins, with curative activity against African trypanosomiasis. Promising hits were identified following in vitro screening against the most relevant trypanosome species. Selected hit compounds were extensively tested for in vitro metabolic stability, potency in in vivo mouse models for the various species, genotoxicity in an in vitro testing battery, and mode of action studies (i.e., genome-wide RNA interference library screening and metabolomics). Among the nucleoside analogues, analogue 3 was curative in mouse models with no indication of genotoxicity and a low ecotoxicological footprint. Mode-of-action studies revealed that P1-type nucleoside transporters and adenosine kinase are involved in the uptake and activation, respectively. Analogue 3 represents a potent, advanced lead fitting the preferred target product profile for a broad-spectrum trypanocide regardless of the causative species.

The development of inhaled drugs for respiratory diseases is frequently impacted by lung pathology in non-clinical safety studies. To enable design of novel candidate drugs with the right safety profile, predictive in vitro lung toxicity assays are required that can be applied during drug discovery for early hazard identification and mitigation. Here, we describe a novel high-content imaging-based screening assay that allows for quantification of the tight junction protein occludin in A549 cells, as a model for lung epithelial barrier integrity. We assessed a set of compounds with a known lung safety profile, defined by clinical safety or non-clinical in vivo toxicology data, and were able to correctly identify 9 of 10 compounds with a respiratory safety risk and 9 of 9 compounds without a respiratory safety risk (90% sensitivity, 100% specificity). The assay was sensitive at relevant compound concentrations to influence medicinal chemistry optimization programs and, with an accessible cell model in a 96-well plate format, short protocol and application of automated imaging analysis algorithms, this assay can be readily integrated in routine discovery safety screening to identify and mitigate respiratory toxicity early during drug discovery. Interestingly, when we applied physiologically-based pharmacokinetic (PBPK) modelling to predict epithelial lining fluid exposures of the respiratory tract after inhalation, we found a robust correlation between in vitro occludin assay data and lung pathology in vivo, suggesting the assay can inform translational risk assessment for inhaled small molecules., (© 2024. The Author(s).)

Accurate detection of viable Leishmania parasites is critical for evaluating visceral leishmaniasis (VL) treatment response at an early timepoint. We compared the decay of kinetoplast DNA (kDNA) and spliced-leader RNA (SL-RNA) in vitro, in vivo, and in a VL patient cohort. An optimized combination of blood preservation and nucleic acid extraction improved efficiency for both targets. SL-RNA degraded more rapidly during treatment than kDNA, and correlated better with microscopic examination. SL-RNA quantitative polymerase chain reaction emerges as a superior method for dynamic monitoring of viable Leishmania parasites. It enables individualized treatment monitoring for improved prognoses and has potential as an early surrogate endpoint in clinical trials., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Addressing the challenges of quiescence and post-treatment relapse is of utmost importance in the microbiology field. This study shows that Leishmania infantum and L. donovani parasites rapidly enter into quiescence after an estimated 2-3 divisions in both human and mouse bone marrow stem cells. Interestingly, this behavior is not observed in macrophages, which are the primary host cells of the Leishmania parasite. Transcriptional comparison of the quiescent and non-quiescent metabolic states confirmed the overall decrease of gene expression as a hallmark of quiescence. Quiescent amastigotes display a reduced size and signs of a rapid evolutionary adaptation response with genetic alterations. Our study provides further evidence that this quiescent state significantly enhances resistance to treatment. Moreover, transitioning through quiescence is highly compatible with sand fly transmission and increases the potential of parasites to infect cells. Collectively, this work identified stem cells in the bone marrow as a niche where Leishmania quiescence occurs, with important implications for antiparasitic treatment and acquisition of virulence traits., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Dirkx et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Keller's arthroplasty, arthrodesis and cheilectomy are well-known surgical interventions for hallux rigidus. This study aimed to evaluate the effects of these surgical interventions on gait, plantar pressure distribution and clinical outcome in patients treated for hallux rigidus 22 years after surgery., Methods: Spatio-temporal gait parameters and plantar pressure distribution, determined as pressure time integrals (PTIs) and peak pressures (PPs), were analyzed using a 7-foot tone analysis model. Patient-reported outcome was assessed using the Manchester-Oxford Foot Questionnaire (MOXFQ). Of the 73 patients (89 feet) from the original study, 27 patients (33 feet) and 13 healthy controls (26 feet) were available for evaluation 22 years after hallux rigidus surgery., Results: Spatio-temporal gait parameters were comparable between all groups and were in line with healthy controls ( P  > 0.05). No differences ( P  > 0.05) in PTIs and PPs were found in the seven plantar zones between groups and as compared to healthy controls. MOXFQ scores in all domains (walking/standing, range 21.4-24.1; pain, range 16.5-22.2 and social interaction, range 23.8-35.4) were not clinically and statistically different ( P  > 0.05) between the three different surgical interventions., Conclusion: These results suggest no long-term functional and biomechanical differences after these surgical interventions for hallux rigidus correction. The interventions seem to be appropriate treatment options for a selective group of patients with symptomatic hallux rigidus., Competing Interests: Thijs Smeets is the owner of Smeets Loopcomfort and was involved as researcher. Thijs Smeets and his company do not have financial or other competing interests with the performed research. The other authors declare that they have no competing interests., (©2023 de Bot et al.)

Background: Visceral leishmaniasis (VL), a life-threatening neglected tropical disease, is targeted for elimination from Nepal by the year 2026. The national VL elimination program is still confronted with many challenges including the increasingly widespread distribution of the disease over the country, local resurgence and the questionable efficacy of the key vector control activities. In this study, we assessed the status and risk of Leishmania donovani transmission based on entomological indicators including seasonality, natural Leishmania infection rate and feeding behavior of vector sand flies, Phlebotomus argentipes, in three districts that had received disease control interventions in the past several years in the context of the disease elimination effort., Methods: We selected two epidemiologically contrasting settings in each survey district, one village with and one without reported VL cases in recent years. Adult sand flies were collected using CDC light traps and mouth aspirators in each village for 12 consecutive months from July 2017 to June 2018. Leishmania infection was assessed in gravid sand flies targeting the small-subunit ribosomal RNA gene of the parasite (SSU-rRNA) and further sequenced for species identification. A segment (~ 350 bp) of the vertebrate cytochrome b (cytb) gene was amplified from blood-fed P. argentipes from dwellings shared by both humans and cattle and sequenced to identify the preferred host., Results: Vector abundance varied among districts and village types and peaks were observed in June, July and September to November. The estimated Leishmania infection rate in vector sand flies was 2.2% (1.1%-3.7% at 95% credible interval) and 0.6% (0.2%-1.3% at 95% credible interval) in VL and non-VL villages respectively. The common source of blood meal was humans in both VL (52.7%) and non-VL (74.2%) villages followed by cattle., Conclusions: Our findings highlight the risk of ongoing L. donovani transmission not only in villages with VL cases but also in villages not reporting the presence of the disease over the past several years within the districts having disease elimination efforts, emphasize the remaining threats of VL re-emergence and inform the national program for critical evaluation of disease elimination strategies in Nepal., (© 2023. The Author(s).)

JAK-STAT cytokines are critical in regulating immunity. Persistent activation of JAK-STAT signaling pathways by cytokines drives chronic inflammatory diseases such as asthma. Herein, we report on the discovery of a highly JAK1-selective, ATP-competitive series of inhibitors having a 1000-fold selectivity over other JAK family members and the approach used to identify compounds suitable for inhaled administration. Ultimately, compound 16 was selected as the clinical candidate, and upon dry powder inhalation, we could demonstrate a high local concentration in the lung as well as low plasma concentrations, suggesting no systemic JAK1 target engagement. Compound 16 has progressed into clinical trials. Using 16, we found JAK1 inhibition to be more efficacious than JAK3 inhibition in IL-4-driven Th2 asthma.

Background: Immunocompromised patients have been at an increased risk of succumbing to coronavirus disease 2019 (COVID-19) since the beginning of the pandemic., Methods: Here, we analyzed mortality and case fatality data from dialysis and kidney transplant patients, and compared each with an age-matched subgroup of the general population., Results: We found that both patients on dialysis and kidney transplant patients remain at increased risk of succumbing to COVID-19 despite all available countermeasures., Conclusions: The analyses underline the need for additional protection for this vulnerable population., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Background: Janus Kinase (JAK) inhibition has recently demonstrated therapeutic efficacy in both restoring hair growth and resolving inflammation in Alopecia Areata (AA). These effects are dose dependent and mainly efficacious at ranges close to a questionable risk profile., Objectives: We explored the possibility to separate the beneficial and adverse effects of JAK inhibition by selectively inhibiting JAK1 and thereby avoiding side effects associated with JAK2 blockade., Methods: The C3H/HeJ mouse model of AA was used to demonstrate therapeutic efficacy in vivo with different regimens of a selection of JAK inhibitors in regards to systemic versus local drug exposure. Human peripheral blood lymphocytes were stimulated in vitro to demonstrate translation to the human situation., Results: We demonstrate that selective inhibition of JAK1 produces fast resolution of inflammation and complete restoration of hair growth in the C3H/HeJ mouse model of AA. Furthermore, we show that topical treatment does not restore hair growth and that treatment needs to be extended well beyond that of restored hair growth in order to reach treatment-free remission. For translatability to human disease, we show that cytokines involved in AA pathogenesis are similarly inhibited by selective JAK1 and pan-JAK inhibition in stimulated human peripheral lymphocytes and specifically in CD8 + T cells., Conclusion: This study demonstrates that systemic exposure is required for efficacy in AA and we propose that a selective JAK1 inhibitor will offer a treatment option with a superior safety profile to pan-JAK inhibitors for these patients., Competing Interests: All authors are or were at the time employed by AstraZeneca., (© 2023 AstraZeneca. Skin Health and Disease published by John Wiley & Sons Ltd on behalf of British Association of Dermatologists.)

We present an approach to fabricate biological matrix composites made entirely from cultured plant cells. We utilize the cell’s innate ability to synthesize nanofibrillar cell walls, which serve as the composite’s fundamental building blocks. Following a controlled compression/dehydration process, the cells arrange into lamellar structures with hierarchical features. We demonstrate that the native cell wall nanofibrils tether adjacent cells together through fibrillar interlocking and intermolecular hydrogen bonding. These interactions facilitate intercellular adhesion and eliminate the need for other binders. Our fabrication process utilizes the entire plant cell, grown in an in vitro culture; requires no harsh chemical treatments or waste-generating extraction or selection processes; and leads to bulk biocomposites that can be produced in situ and biodegrade in soil. The final mechanical properties are comparable to commodity plastics and can be further modulated by introducing filler particles.

Studies on the mechanics of plant cells usually focus on understanding the effects of turgor pressure and properties of the cell wall (CW). While the functional roles of the underlying cytoskeleton have been studied, the extent to which it contributes to the mechanical properties of cells is not elucidated. Here, we study the contributions of the CW, microtubules (MTs) and actin filaments (AFs), in the mechanical properties of Nicotiana tabacum cells. We use a multiscale biomechanical assay comprised of atomic force microscopy and micro-indentation in solutions that (i) remove MTs and AFs and (ii) alter osmotic pressures in the cells. To compare measurements obtained by the two mechanical tests, we develop two generative statistical models to describe the cell's behaviour using one or both datasets. Our results illustrate that MTs and AFs contribute significantly to cell stiffness and dissipated energy, while confirming the dominant role of turgor pressure., Competing Interests: No conflicts of interest declared., (© The Author(s) 2022.)

Mammalian adenoviruses (AdVs) comprise more than ~350 types including over 100 human (HAdVs) and just three mouse AdVs (MAdVs). While most HAdVs initiate infection by high affinity/avidity binding of their fiber knob (FK) protein to either coxsackievirus AdV receptor (CAR), CD46 or desmoglein (DSG)-2, MAdV-1 (M1) infection requires arginine-glycine-aspartate (RGD) binding integrins. To identify the receptors mediating MAdV infection we generated five novel reporter viruses for MAdV-1/-2/-3 (M1, M2, M3) transducing permissive murine (m) CMT-93 cells, but not B16 mouse melanoma cells expressing mCAR, human (h) CD46 or hDSG-2. Recombinant M1 or M3 FKs cross-blocked M1 and M3 but not M2 infections. Profiling of murine and human cells expressing RGD-binding integrins suggested that αvβ6 and αvβ8 heterodimers are associated with M1 and M3 infections. Ectopic expression of mβ6 in B16 cells strongly enhanced M1 and M3 binding, infection, and progeny production comparable with mαvβ6-positive CMT-93 cells, whereas mβ8 expressing cells were more permissive to M1 than M3. Anti-integrin antibodies potently blocked M1 and M3 binding and infection of CMT-93 cells and hαvβ8-positive M000216 cells. Soluble integrin αvβ6, and synthetic peptides containing the RGDLXXL sequence derived from FK-M1, FK-M3 and foot and mouth disease virus coat protein strongly interfered with M1/M3 infections, in agreement with high affinity interactions of FK-M1/FK-M3 with αvβ6/αvβ8, determined by surface plasmon resonance measurements. Molecular docking simulations of ternary complexes revealed a bent conformation of RGDLXXL-containing FK-M3 peptides on the subunit interface of αvβ6/β8, where the distal leucine residue dips into a hydrophobic pocket of β6/8, the arginine residue ionically engages αv aspartate215, and the aspartate residue coordinates a divalent cation in αvβ6/β8. Together, the RGDLXXL-bearing FKs are part of an essential mechanism for M1/M3 infection engaging murine and human αvβ6/8 integrins. These integrins are highly conserved in other mammals, and may favour cross-species virus transmission., Competing Interests: The authors have declared that no competing interests exist

This study identified the isoindolone ring as a scaffold for novel agents against Trypanosoma brucei rhodesiense and explored the structure-activity relationships of various aromatic ring substitutions. The compounds were evaluated in an integrated in vitro screen. Eight compounds exhibited selective activity against T. b. rhodesiense (IC 50 <2.2 μm) with no detectable side activity against T. cruzi and Leishmania infantum. Compound 20 showed low nanomolar potency against T. b. rhodesiense (IC 50 =40 nm) and no toxicity against MRC-5 and PMM cell lines and may be regarded as a new lead template for agents against T. b. rhodesiense. The isoindolone-based compounds have the potential to progress into lead optimization in view of their highly selective in vitro potency, absence of cytotoxicity and acceptable metabolic stability. However, the solubility of the compounds represents a limiting factor that should be addressed to improve the physicochemical properties that are required to proceed further in the development of in vivo-active derivatives., (© 2021 The Authors. Published by Wiley-VCH GmbH.)

Background: Recently, movement-based videogames (exergames) have gained popularity in improving the rehabilitation process after surgery. During exergaming, participants are physically challenged as the game component stimulates adherence to the training program. There is no literature on the effect of exergame training interventions in patients who received arthroscopic ankle arthrodesis., Objective: This pilot study assessed the potency of an existing exergaming tool for the rehabilitation program of patients who received arthroscopic ankle arthrodesis., Methods: A cross-sectional pilot study was performed, in which patients who received arthroscopic ankle arthrodesis (n=8) were subjected to an exergaming protocol. Gait analysis was performed with a treadmill system. A healthy age-matched control group (n=10) was used as the control group., Results: The patient group was capable of performing exergaming exercises and they showed no floor or ceiling effect. Only in case of the overall stability, the patient group performed significantly less better than the control group (P=.03). Gait analysis showed equal step length with increased external rotation of the affected limb., Conclusions: Exergaming seems to be a valuable tool for measuring the ability of patients who received AAA to perform activities of daily living and it has the potential to individualize rehabilitation programs. When exergaming is systematically integrated with patient-reported outcome measures and activity tracking, it has the potential to improve the quality of care., (©Roel Hendrickx, Tim van der Avoird, Peter Pilot, Gino Kerkhoffs, Martijn Schotanus. Originally published in JMIR Rehabilitation and Assistive Technology (https://rehab.jmir.org), 05.05.2021.)

Human African trypanosomiasis is a neglected parasitic disease for which the current treatment options are quite limited. Trypanosomes are not able to synthesize purines de novo and thus solely depend on purine salvage from the host environment. This characteristic makes players of the purine salvage pathway putative drug targets. The activity of known nucleoside analogues such as tubercidin and cordycepin led to the development of a series of C7-substituted nucleoside analogues. Here, we use RNA interference (RNAi) libraries to gain insight into the mode-of-action of these novel nucleoside analogues. Whole-genome RNAi screening revealed the involvement of adenosine kinase and 4E interacting protein into the mode-of-action of certain antitrypanosomal nucleoside analogues. Using RNAi lines and gene-deficient parasites, 4E interacting protein was found to be essential for parasite growth and infectivity in the vertebrate host. The essential nature of this gene product and involvement in the activity of certain nucleoside analogues indicates that it represents a potential novel drug target.

The purpose of this study was to retrospectively evaluate the efficacy of a tranexamic acid (TXA) perioperative protocol for primary hip- and knee arthroplasty, in terms of allogenic blood transfusion rates. A retrospective cohort study was conducted and included all primary hip and knee arthroplasty procedures in the period of 2014-2019. Patients who underwent surgery due to trauma or revision were excluded. A total amount of 5205 patients were eligible for inclusion. Two equal and weight depending doses of TXA were given, preoperative as an oral dose and intravenously at wound closure. The primary outcome was blood transfusion rate. Further analysis on patient characteristics (e.g. age, gender), blood loss, perioperative haemoglobin (Hb) levels and complication/readmission rate was performed. A total of 49 (0.9%) patients received perioperative allogenic blood transfusions. Mean age, distribution of gender, body-mass index, American Society of Anaesthesiologists score, duration of surgery, type of arthroplasty, estimated blood loss, perioperative Hb levels and length of stay were statistically significant different between transfused and not-transfused patients. The incidence of thromboembolic adverse events (e.g. deep vein thrombosis/lung embolism) was 0.5%. Low blood transfusion rate was found after implementation of a standardized perioperative TXA protocol for primary hip and knee arthroplasty.

Individual plant cells are the building blocks for all plantae and artificially constructed plant biomaterials, like biocomposites. Secondary cell walls (SCWs) are a key component for mediating mechanical strength and stiffness in both living vascular plants and biocomposite materials. In this paper, we study the structure and biomechanics of cultured plant cells during the cellular developmental stages associated with SCW formation. We use a model culture system that induces transdifferentiation of Arabidopsis thaliana cells to xylem vessel elements, upon treatment with dexamethasone (DEX). We group the transdifferentiation process into three distinct stages, based on morphological observations of the cell walls. The first stage includes cells with only a primary cell wall (PCW), the second covers cells that have formed a SCW, and the third stage includes cells with a ruptured tonoplast and partially or fully degraded PCW. We adopt a multi-scale approach to study the mechanical properties of cells in these three stages. We perform large-scale indentations with a micro-compression system in three different osmotic conditions. Atomic force microscopy (AFM) nanoscale indentations in water allow us to isolate the cell wall response. We propose a spring-based model to deconvolve the competing stiffness contributions from turgor pressure, PCW, SCW and cytoplasm in the stiffness of differentiating cells. Prior to triggering differentiation, cells in hypotonic pressure conditions are significantly stiffer than cells in isotonic or hypertonic conditions, highlighting the dominant role of turgor pressure. Plasmolyzed cells with a SCW reach similar levels of stiffness as cells with maximum turgor pressure. The stiffness of the PCW in all of these conditions is lower than the stiffness of the fully-formed SCW. Our results provide the first experimental characterization of the mechanics of SCW formation at single cell level.

Background: Molecular detection techniques using peripheral blood are preferred over invasive tissue aspiration for the diagnosis and post-treatment follow-up of visceral leishmaniasis (VL) patients. This study aims to identify suitable stabilizing reagents to prevent DNA and RNA degradation during storage and transport to specialized laboratories where molecular diagnosis is performed., Methodology: The stabilizing capacities of different commercially available reagents were compared using promastigote-spiked human blood and peripheral blood of Syrian golden hamsters subjected to experimental infection, treatment (miltefosine or aminopyrazole DNDi-1044) and immunosuppression. The impact of various storage temperature conditions was tested in combination with an established kinetoplast DNA (kDNA) qPCR and a recently developed spliced leader RNA (SL-RNA) assay for Leishmania detection., Principal Findings: Irrespective of the blood type and stabilizer used, threshold (cT) values obtained with the SL-RNA qPCR were systematically lower than those obtained with the kDNA assay, confirming the advantage of the SL-RNA assay over the widely used kDNA assay for low-level Leishmania detection. Peripheral blood parasite levels correlated relatively well with hepatic burdens. RNA protect cell reagent provided the most optimal simultaneous DNA and RNA stabilization in both human and hamster blood. However, this stabilizer requires an erythrocyte lysis step, which can be challenging under field conditions. DNA/RNA shield provides a good alternative for downstream kDNA and SL-RNA assays, especially if sample storage capacity at 4 °C can be guaranteed., Conclusions/significance: The recommended stabilizing reagents are compatible with RNA- and DNA-based Leishmania detection in peripheral blood in the VL hamster model and spiked human blood. Since molecular detection techniques using peripheral blood are less invasive than microscopic assessment of tissue aspirates, the findings of this study may be applied to human VL clinical studies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: In eco-epidemiological studies, Leishmania detection in vectors and reservoirs is frequently accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast DNA (kDNA). A pan-Leishmania SYBR green quantitative PCR (qPCR) assay which detects the conserved spliced-leader RNA (SL RNA) sequence was developed recently. This study assessed the SL RNA assay performance combined with a crude extraction method for the detection of Leishmania in field-collected and laboratory-reared sand flies and in tissue samples from hyraxes as reservoir hosts., Methods: Field-collected and laboratory-infected sand fly and hyrax extracts were subjected to three different qPCR approaches to assess the suitability of the SL RNA target for Leishmania detection. Nucleic acids of experimentally infected sand flies were isolated with a crude extraction buffer with ethanol precipitation and a commercial kit and tested for downstream DNA and RNA detection. Promastigotes were isolated from culture and sand fly midguts to assess whether there was difference in SL RNA and kDNA copy numbers. Naive sand flies were spiked with a serial dilution of promastigotes to make a standard curve., Results: The qPCR targeting SL RNA performed well on infected sand fly samples, despite preservation and extraction under presumed unfavorable conditions for downstream RNA detection. Nucleic acid extraction by a crude extraction buffer combined with a precipitation step was highly compatible with downstream SL RNA and kDNA detection. Copy numbers of kDNA were found to be identical in culture-derived parasites and promastigotes isolated from sand fly midguts. SL RNA levels were slightly lower in sand fly promastigotes (ΔCq 1.7). The theoretical limit of detection and quantification of the SL RNA qPCR respectively reached down to 10 -3 and 10 parasite equivalents. SL RNA detection in stored hyrax samples was less efficient with some false-negative assay results, most likely due to the long-term tissue storage in absence of RNA stabilizing reagents., Conclusions: This study shows that a crude extraction method in combination with the SL RNA qPCR assay is suitable for the detection and quantification of Leishmania in sand flies. The assay is inexpensive, sensitive and pan-Leishmania specific, and accordingly an excellent assay for high-throughput screening in entomological research.

Purpose: Patient-specific instruments (PSI) are already widespread used in total knee arthroplasty (TKA). Either computed tomography (CT) scans or magnetic resonance imaging (MRI) scans are used pre-operatively to create jigs to guide resection during surgery. This study is a sequel of previous work that showed significantly more radiological outliers for posterior slope when CT-based guides were used. The aim of this study was to assess differences in revision rate and clinical outcome between the two groups at 2-year follow-up., Methods: At the 2-year follow-up, 124 patients were analysed in this prospective, randomised single-blind study. A survival analysis with revision of the TKA as endpoint was performed. Patients fulfilled four patient-reported outcome measurements (PROMs). Scores on the questionnaires were compared between both groups at the different follow-up visits., Results: At final follow-up, there was no significant difference in the survival rates of the CT- and MRI-based PSI surgery. Postoperatively, the PROMs significantly improved within each group compared with the pre-operative values. There were no significant differences for the PROMs between both groups at the 2-years follow-up., Conclusions: Although previous results showed more outliers regarding posterior slope for CT-based PSIs, no difference in revision rate or the outcome of PROMs was found at 2-year follow-up. Further research to determine what the influence is of radiological outliers on implant survival and clinical outcomes is necessary., Level of Evidence: I.

Background: Glucocorticoids are highly effective and widely used anti-inflammatory drugs, but their use is limited by serious side-effects, including glucocorticoid-induced hyperglycaemia and diabetes. AZD9567 is a non-steroidal, selective glucocorticoid receptor modulator that aims to reduce side-effects. We aimed to assess the safety, tolerability, and pharmacokinetics of AZD9567 in healthy volunteers., Methods: Two phase 1 clinical studies were done. First, a randomised, placebo-controlled, single-blind, single-ascending dose study was done in healthy men who received single oral doses of AZD9567 2 mg, 10 mg, 20 mg, 40 mg, 80 mg, 100 mg, 125 mg, or 155 mg, or prednisolone 60 mg (n=8 per dose group, randomly assigned [6:2] to receive active drug or placebo). Second, a randomised, active-controlled, single-blind, multiple-ascending dose study was done, in which men and women received oral AZD9567 or prednisolone once daily for 5 days. One cohort of volunteers with prediabetes received AZD9567 10 mg (n=7) or prednisolone 20 mg (n=2). All other cohorts comprised healthy volunteers, receiving AZD9567 20 mg, 40 mg, 80 mg, or 125 mg (n=7 per dose group), or prednisolone 5 mg (n=13), 20 mg (n=16), or 40 mg (n=13). Participants and study centre staff were masked to treatment assignment for each cohort, although data were unmasked for safety review between cohorts. The primary outcome of the single-ascending dose study was the safety, tolerability, and pharmacokinetics of single ascending doses of AZD9567; for the multiple-ascending dose study it was the safety and tolerability of AZD9567 following multiple ascending doses. As a secondary outcome, effects on glycaemic control were ascertained with oral glucose tolerance tests (OGTTs) done at baseline and on day 1 of the single-ascending dose study, and at baseline and on day 4 of the multiple-ascending dose study. These trials are registered at ClinicalTrials.gov, NCT02512575 and NCT02760316., Findings: In the single-ascending dose study, between Nov 18, 2015, and Sept 26, 2016, 72 healthy white men were enrolled, and all completed the study. In the multiple-ascending dose study, between May 2, 2016, and Sept 13, 2017, 77 predominantly white male volunteers (including nine individuals with prediabetes and eight women) were enrolled and 75 completed the study. All doses of AZD9567 and prednisolone were well tolerated, with no serious adverse events or events suggesting adrenal insufficiency. In the single-ascending dose study, nine adverse events of mild intensity were reported (five with AZD9567 and four with placebo); no adverse event was reported by more than one person. In the multiple-ascending dose study, 44 adverse events of mild or moderate intensity were reported (18 with AZD9567 and 26 with prednisolone). The most common were headache and micturition. Apparent clearance, volume of distribution, and half-life of AZD9567 were consistent across doses and for single versus repeated dosing. In the multiple-ascending dose study, OGTTs showed no significant difference with AZD9567 doses up to 80 mg compared with prednisolone 5 mg in glucose area under the curve from 0 h to 4 h post-OGTT (AUC 0-4h ) from baseline to day 4; the increase in glucose AUC 0-4h from baseline to day 4 was significantly lower with all AZD9567 doses versus prednisolone 20 mg (AZD9567 20 mg p<0·0001, 40 mg p=0·0001, 80 mg p=0·0001, and 125 mg p=0·0237)., Interpretation: AZD9567 appears to be safe and well tolerated in healthy, predominantly white male volunteers and shows promising initial evidence for improved post-prandial glucose control. Studies of longer duration, with a greater proportion of women and other ethnic groups, and in patients requiring anti-inflammatory treatment are needed to characterise the clinical efficacy and safety profile of AZD9567., Funding: AstraZeneca., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Synthetic glucocorticoids (GC) are essential for the treatment of a broad range of inflammatory diseases. However, their use is limited by target related adverse effects on, e.g., glucose homeostasis and bone metabolism. Starting from a nonsteroidal GR ligand (4) that is a full agonist in reporter gene assays, we exploited key functional triggers within the receptor, generating a range of structurally diverse partial agonists. Of these, only a narrow subset exhibited full anti-inflammatory efficacy and a significantly reduced impact on adverse effect markers in human cell assays compared to prednisolone. This led to the discovery of AZD9567 (15) with excellent in vivo efficacy when dosed orally in a rat model of joint inflammation. Compound 15 is currently being evaluated in clinical trials comparing the efficacy and side effect markers with those of prednisolone.

Translational pharmacokinetic (PK) models are needed to describe and predict drug concentration-time profiles in lung tissue at the site of action to enable animal-to-man translation and prediction of efficacy in humans for inhaled medicines. Current pulmonary PK models are generally descriptive rather than predictive, drug/compound specific, and fail to show successful cross-species translation. The objective of this work was to develop a robust compartmental modeling approach that captures key features of lung and systemic PK after pulmonary administration of a set of 12 soluble drugs containing single basic, dibasic, or cationic functional groups. The model is shown to allow translation between animal species and predicts drug concentrations in human lungs that correlate with the forced expiratory volume for different classes of bronchodilators. Thus, the pulmonary modeling approach has potential to be a key component in the prediction of human PK, efficacy, and safety for future inhaled medicines., (© 2017 The Authors CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.)

A class of potent, nonsteroidal, selective indazole ether-based glucocorticoid receptor modulators (SGRMs) was developed for the inhaled treatment of respiratory diseases. Starting from an orally available compound with demonstrated anti-inflammatory activity in rat, a soft-drug strategy was implemented to ensure rapid elimination of drug candidates to minimize systemic GR activation. The first clinical candidate 1b (AZD5423) displayed a potent inhibition of lung edema in a rat model of allergic airway inflammation following dry powder inhalation combined with a moderate systemic GR-effect, assessed as thymic involution. Further optimization of inhaled drug properties provided a second, equally potent, candidate, 15m (AZD7594), that demonstrated an improved therapeutic ratio over the benchmark inhaled corticosteroid 3 (fluticasone propionate) and prolonged the inhibition of lung edema, indicating potential for once-daily treatment.

Background: In this case report, we describe a left-arm both-bone forearm fracture in a 15-year-old girl who fell off a swing. Conservative treatment with an above-elbow cast failed, resulting in a malunion with functional impairment. The pro- and supination were 90/0/10, respectively. The patient complained of difficulties performing daily activities. For this pediatric case, a corrective osteotomy was proposed using a CT-guided technique aiming for maximum anatomical and functional outcome. It was the first time this technique was used in our hospital., Methods: A corrective osteotomy of the patient's left arm was performed using 3D printed templates to guide the osteotomy orientation. These templates were produced using specialized software in which CT images of her malunited left forearm were overlaid with the mirrored images of her healthy right forearm., Results: The postoperative CT-scan showed a near-anatomical reduction with close to 1° correction in all three planes, as compared to the preoperative planning. Three months after surgery, the patient had regained full function of her left forearm., Conclusion: Although this was the first time this technique was used in our hospital, it resulted in excellent anatomical and functional outcomes making it a safe, reliable and precise treatment option that may be useful for even more complex corrections., Level of Evidence: Level V., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Background: Adenoviruses are common pathogens infecting animals and humans. They are classified based on serology, or genome sequence information. These methods have limitations due to lengthy procedures or lack of infectivity data. Adenoviruses are easy to produce and amenable to genetic and biochemical modifications, which makes them a powerful tool for biological studies, and clinical gene-delivery and vaccine applications. Antibodies directed against adenoviral proteins are important diagnostic tools for virus identification in vivo and in vitro, and are used to elucidate infection mechanisms, often in combination with genomic sequencing and type specific information from hyper-variable regions of structural proteins., Results: Here we describe a novel and readily useable method for cloning, expressing and purifying small fragments of hyper-variable regions 1-6 of the adenoviral hexon protein. We used these polypeptides as antigens for generating polyclonal rabbit antibodies against human adenovirus 3 (HAdV-B3), mouse adenovirus 1 (MAdV-1) and MAdV-2 hexon. In Western immunoblots with lysates from cells infected from thirteen human and three mouse viruses, these antibodies bound to homologous full-length hexon protein and revealed variable levels of cross-reactivity to heterologous hexons. Results from immuno-fluorescence and electron microscopy studies indicated that HAdV-B3 and MAdV-2 hexon antibodies recognized native forms of hexon., Conclusions: The procedure described here can in principle be applied to any adenovirus for which genome sequence information is available. It provides a basis for generating novel type-specific tools in diagnostics and research, and extends beyond the commonly used anti-viral antibodies raised against purified viruses or subviral components.

A structure-based design approach led to the identification of a novel class of indazole ether based, non-steroidal glucocorticoid receptor (GR) modulators. Several examples were identified that displayed cell potency in the picomolar range, inhibiting LPS-induced TNF-α release by primary peripheral blood mononuclear cells (PBMCs). Additionally, an improved steroid hormone receptor binding selectivity profile, compared to classical steroidal GR agonists, was demonstrated. The indazole ether core tolerated a broad range of substituents allowing for modulation of the physiochemical parameters. A small sub-set of indazole ethers, with pharmacokinetic properties suitable for oral administration, was investigated in a rat antigen-induced joint inflammation model and demonstrated excellent anti-inflammatory efficacy., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Many genes are candidates for involvement in epileptic encephalopathy (EE) because one or a few possibly pathogenic variants have been found in patients, but insufficient genetic or functional evidence exists for a definite annotation., Methods: To increase the number of validated EE genes, we sequenced 26 known and 351 candidate genes for EE in 360 patients. Variants in 25 genes known to be involved in EE or related phenotypes were followed up in 41 patients. We prioritized the candidate genes, and followed up 31 variants in this prioritized subset of candidate genes., Results: Twenty-nine genotypes in known genes for EE (19) or related diseases (10), dominant as well as recessive or X-linked, were classified as likely pathogenic variants. Among those, likely pathogenic de novo variants were found in EE genes that act dominantly, including the recently identified genes EEF1A2, KCNB1 and the X-linked gene IQSEC2. A de novo frameshift variant in candidate gene HNRNPU was the only de novo variant found among the followed-up candidate genes, and the patient's phenotype was similar to a few recent publications., Conclusion: Mutations in genes described in OMIM as, for example, intellectual disability gene can lead to phenotypes that get classified as EE in the clinic. We confirmed existing literature reports that de novo loss-of-function HNRNPUmutations lead to severe developmental delay and febrile seizures in the first year of life.

Febrile seizures (FS) are the most common seizure syndrome and are potentially a prelude to more severe epilepsy. Although zinc (Zn(2+)) metabolism has previously been implicated in FS, whether or not variation in proteins essential for Zn(2+) homeostasis contributes to susceptibility is unknown. Synaptic Zn(2+) is co-released with glutamate and modulates neuronal excitability. SLC30A3 encodes the zinc transporter 3 (ZNT3), which is primarily responsible for moving Zn(2+) into synaptic vesicles. Here we sequenced SLC30A3 and discovered a rare variant (c.892C > T; p.R298C) enriched in FS populations but absent in population-matched controls. Functional analysis revealed a significant loss-of-function of the mutated protein resulting from a trafficking deficit. Furthermore, mice null for ZnT3 were more sensitive than wild-type to hyperthermia-induced seizures that model FS. Together our data suggest that reduced synaptic Zn(2+) increases the risk of FS and more broadly support the idea that impaired synaptic Zn(2+) homeostasis can contribute to neuronal hyperexcitability.

Human adenoviruses are the most widely used vectors in gene medicine, with applications ranging from oncolytic therapies to vaccinations, but adenovirus vectors are not without side effects. In addition, natural adenoviruses pose severe risks for immunocompromised people, yet infections are usually mild and self-limiting in immunocompetent individuals. Here we describe how adenoviruses are recognized by the host innate defense system during entry and replication in immune and nonimmune cells. Innate defense protects the host and represents a major barrier to using adenoviruses as therapeutic interventions in humans. Innate response against adenoviruses involves intrinsic factors present at constant levels, and innate factors mounted by the host cell upon viral challenge. These factors exert antiviral effects by directly binding to viruses or viral components, or shield the virus, for example, soluble factors, such as blood clotting components, the complement system, preexisting immunoglobulins, or defensins. In addition, Toll-like receptors and lectins in the plasma membrane and endosomes are intrinsic factors against adenoviruses. Important innate factors restricting adenovirus in the cytosol are tripartite motif-containing proteins, nucleotide-binding oligomerization domain-like inflammatory receptors, and DNA sensors triggering interferon, such as DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 and cyclic guanosine monophosphate-adenosine monophosphate synthase. Adenovirus tunes the function of antiviral autophagy, and counters innate defense by virtue of its early proteins E1A, E1B, E3, and E4 and two virus-associated noncoding RNAs VA-I and VA-II. We conclude by discussing strategies to engineer adenovirus vectors with attenuated innate responses and enhanced delivery features.

Background: Mutations in the proline-rich transmembrane protein 2 (PRRT2) gene have been identified in patients with benign (familial) infantile convulsions (B(F)IC), infantile convulsions with choreoathetosis (ICCA) and paroxysmal dyskinesias (PDs). However it remains unknown whether PRRT2 mutations are causal in other epilepsy syndromes. After we discovered a PRRT2 mutation in a large family with ICCA containing one individual with febrile seizures (FS) and one individual with West syndrome, we analysed PRRT2 in a heterogeneous cohort of patients with different types of infantile epilepsy., Methods: We screened a cohort of 460 patients with B(F)IC or ICCA, fever related seizures or infantile epileptic encephalopathies. All patients were tested for point mutations using direct sequencing., Results: We identified heterozygous mutations in 16 individuals: 10 familial and 6 sporadic cases. All patients were diagnosed with B(F)IC, ICCA or PD. We were not able to detect mutations in any of the other epilepsy syndromes. Several mutation carriers had learning disabilities and/or impaired fine motor skills later in life., Conclusions: PRRT2 mutations do not seem to be involved in the aetiology of FS or infantile epileptic encephalopathies. Therefore B(F)IC, ICCA and PD remain the core phenotypes associated with PRRT2 mutations. The presence of learning disabilities or neuropsychiatric problems in several mutation carriers calls for additional clinical studies addressing this developmental aspect in more detail.

Objective: To identify novel epilepsy genes using a panel approach and describe the functional consequences of mutations., Methods: Using a panel approach, we screened 357 patients comprising a vast spectrum of epileptic disorders for defects in genes known to contribute to epilepsy and/or intellectual disability (ID). After detection of mutations in a novel epilepsy gene, we investigated functional effects in Xenopus laevis oocytes and screened a follow-up cohort., Results: We revealed de novo mutations in GRIN2B encoding the NR2B subunit of the N-methyl-D-aspartate (NMDA) receptor in 2 individuals with West syndrome and severe developmental delay as well as 1 individual with ID and focal epilepsy. The patient with ID and focal epilepsy had a missense mutation in the extracellular glutamate-binding domain (p.Arg540His), whereas both West syndrome patients carried missense mutations within the NR2B ion channel-forming re-entrant loop (p.Asn615Ile, p.Val618Gly). Subsequent screening of 47 patients with unexplained infantile spasms did not reveal additional de novo mutations, but detected a carrier of a novel inherited GRIN2B splice site variant in close proximity (c.2011-5&#95;2011-4delTC). Mutations p.Asn615Ile and p.Val618Gly cause a significantly reduced Mg(2+) block and higher Ca(2+) permeability, leading to a dramatically increased Ca(2+) influx, whereas p.Arg540His caused less severe disturbance of channel function, corresponding to the milder patient phenotype., Interpretation: We identified GRIN2B gain-of-function mutations as a cause of West syndrome with severe developmental delay as well as of ID with childhood onset focal epilepsy. Severely disturbed channel function corresponded to severe clinical phenotypes, underlining the important role of facilitated NMDA receptor signaling in epileptogenesis., (© 2014 The Authors. Annals of Neurology published by Wiley Periodicals, Inc. on behalf of Child Neurology Society/American Neurological Association.)

Objectives: To determine the frequency of KCNQ2 mutations in patients with neonatal epileptic encephalopathy (NEE), and to expand the phenotypic spectrum of KCNQ2 epileptic encephalopathy., Methods: Eighty-four patients with unexplained NEE were screened for KCNQ2 mutations using classic Sanger sequencing. Clinical data of 6 additional patients with KCNQ2 mutations detected by gene panel were collected. Detailed phenotyping was performed with particular attention to seizure frequency, cognitive outcome, and video-EEG., Results: In the cohort, we identified 9 different heterozygous de novo KCNQ2 missense mutations in 11 of 84 patients (13%). Two of 6 missense mutations detected by gene panel were recurrent and present in patients of the cohort. Seizures at onset typically consisted of tonic posturing often associated with focal clonic jerking, and were accompanied by apnea with desaturation. One patient diagnosed by gene panel had seizure onset at the age of 5 months. Based on seizure frequency at onset and cognitive outcome, we delineated 3 clinical subgroups, expanding the spectrum of KCNQ2 encephalopathy to patients with moderate intellectual disability and/or infrequent seizures at onset. Recurrent mutations lead to relatively homogenous phenotypes. One patient responded favorably to retigabine; 5 patients had a good response to carbamazepine. In 6 patients, seizures with bradycardia were recorded. One patient died of probable sudden unexpected death in epilepsy., Conclusion: KCNQ2 mutations cause approximately 13% of unexplained NEE. Patients present with a wide spectrum of severity and, although rare, infantile epilepsy onset is possible.

The role of mutations in the melanocortin-3 receptor (MC3R) gene, which is implicated in the regulation of energy homeostasis, is still under debate. Animal studies have clearly proven that, together with the melanocortin-4 receptor (MC4R), the MC3R is a critical receptor for melanocortin peptides within the leptin-melanocortin signaling cascade. However, as several mutations have been found in lean individuals and not all mutations seem to cause receptor dysfunction, results from mutation screens in obese humans remain controversial. In the present study, we screened for rare variants in the MC3R gene of obese children and lean controls to assess the prevalence of MC3R mutations in the Belgian population. We screened 249 severely overweight and obese children and adolescents and 239 lean adults for mutations in the coding region of MC3R. Mutation screening was performed by high resolution melting curve analysis and direct sequencing. We identified four non-synonymous coding variations in the obese population, all of which had been reported previously. In addition, we also found four novel rare MC3R variants in the lean control population, suggesting that not all MC3R mutations are disease-causing. Overall, the total prevalence of rare MC3R variants was 1 % in Belgian obese children and adolescents compared to 1.02 % in lean controls. Ultimately, cosegregation studies combined with comprehensive functional analysis is required to determine the potential pathogenic role of rare MC3R variants in causing human obesity.

Recently the clinical importance of human organic cation transporters 1 (hOCT1/SLC22A1) and 2 (hOCT2/SLC22A2) in drug disposition, for example, clearance, toxicity, and drug-drug interactions, have been highlighted [Annu. Rev. Pharmacol. Toxicol. 2012, 52, 249-273; Nat. Rev. Drug Discovery 2010, 9 (3), 215-236]. Consequently, there is an extensive need for experimental assessment of structure-transport relationships as well as tools to predict drug uptake by these transporters in ADMET (absorption, distribution, metabolism, excretion, toxicity) investigations. In the present study, we developed a robust assay for screening unlabeled compound uptake by hOCT1 and hOCT2 using transfected HEK293 cells. For the first time, an extensive data set comprising uptake of 354 compounds is presented. As expected, there was a large overlap in substrate specificity between the two organic cation transporters. However, several compounds selectively taken up by either hOCT1 or hOCT2 were identified. In particular, a chemical series of phenylthiophenecarboxamide ureas was identified as selective hOCT1 substrates. Moreover, the drivers for transport differed: molecular volume was the most important determinant of hOCT1 substrates, whereas H-bonding parameters like polar surface area (PSA) dominated for hOCT2.

Mutations in STXBP1 have been identified in a subset of patients with early onset epileptic encephalopathy (EE), but the full phenotypic spectrum remains to be delineated. Therefore, we screened a cohort of 160 patients with an unexplained EE, including patients with early myoclonic encephalopathy (EME), Ohtahara syndrome, West syndrome, nonsyndromic EE with onset in the first year, and Lennox-Gastaut syndrome (LGS). We found six de novo mutations in six patients presenting as Ohtahara syndrome (2/6, 33%), West syndrome (1/65, 2%), and nonsyndromic early onset EE (3/64, 5%). No mutations were found in LGS or EME. Only two of four mutation carriers with neonatal seizures had Ohtahara syndrome. Epileptic spasms were present in five of six patients. One patient with normal magnetic resonance imaging (MRI) but focal seizures underwent epilepsy surgery and seizure frequency dropped drastically. Neuropathology showed a focal cortical dysplasia type 1a. There is a need for additional neuropathologic studies to explore whether STXBP1 mutations can lead to structural brain abnormalities., (Wiley Periodicals, Inc. © 2013 International League Against Epilepsy.)

Objective: Reliable fusion and optimal correction of the alignment of the ankle joint using a 2-incision, 3-screw technique., Indications: Symptomatic osteoarthritis of the ankle joint after insufficient other treatment, severe deformity of the osteoarthritic ankle joint, or salvation procedure after failed arthroplasty., Contraindications: Active osteomyelitis, very poor soft tissues, or severe peripheral arterial occlusive disease., Surgical Technique: Osteotomy and excision medial malleolus. Osteotomy, dislocation, and denudation of the distal fibula. Osteotomy of distal tibia and talus in the desired position for optimal alignment. Temporary tibiotalar fixation with two Weber reposition clamps. Final tibiotalar fixation with a medial and lateral 4.5 mm full threaded cortical lag screw. Reduction of the distal fibula and fibulotibial fixation using a similar technique., Postoperative Management: A nonweight-bearing circular below knee cast for 6 weeks, followed by a walker or weight-bearing cast for another 6 weeks., Results: A total of 30 ankle fusions in 26 patients were analyzed. Mean age at fusion was 50 years (range 11-72.1 years). Mean follow-up time was 9.2 years (3.4-18.8 years). Radiological and clinical fusion in 28 (93%) of the primary arthrodeses. The mean American Orthopedic Foot and Ankle Society (AOFAS) Ankle-Hindfoot score was 66 ± 12. Of the 26 patients, 22 (85%) were satisfied. Deep wound infection occurred once (3%). No other serious complications were encountered. The described technique provides reliable long-term follow-up results for a wide range of indications for ankle fusion.

Herein we describe the scalable diastereoselective and enantioselective syntheses of eight enantiomers of hydroxy metabolites of saperconazole. The in vitro antifungal activity of the eight stereoisomers (compounds 1-8) was compared against a broad panel of Candida spp. (n=93), Aspergillus spp. (n=10), Cryptococcus spp. (n=19), and dermatophytes (n=27). The four 2S isomers 1-4 of the new agent were generally slightly more active than the four 2R isomers 5-8. All eight isomers were tested in a model of experimental A. fumigatus infection in guinea pigs by intravenous inoculation of the fungal conidia. Treatment doses were 1.25 mg kg(-1) and 2.5 mg kg(-1) per day. Infection severity was measured in terms of mean survival time (MST) after infection and mean tissue burdens in brain, liver, spleen, and kidney at postmortem examination. Among the eight isomers, the 2S diastereomers 1-4 showed a generally higher level of activity than the 2R diastereomers 5-8, revealing compounds 1 and 4 as the most potent overall in eradicating tissue burden and MST. Compared with reference compounds itraconazole and saperconazole, the hydroxy isomers 1-8 are less potent inhibitors of the growth of A. fumigatus in vitro and of ergosterol biosynthesis in both A. fumigatus and C. albicans.

The functional classification system for wheelchair basketball is based on an analysis of the players' functional resources through field-testing and game observation. Under this system, players are assigned a score of 1 to 4.5. The aim of this study was to determine if the wheelchair basketball player classification system reflects the existing differences in performance of elite female players. During the World Championship for Wheelchair Basketball in Sydney 1998, eight teams were videotaped for three 40-min games for a total of 120 min per team. Fifty-nine female players (Class I [1 and 1.5 points] n=12; Class II [2 and 2.5 points] n=20; Class III [3 and 3.5 points] n=13; Class IV [4 and 4.5 points] n=14) were retained for a detailed performance analysis by means of the Comprehensive Basketball Grading System (CBGS). Two-way analysis of variance showed high point players to perform better compared with low point players for the majority of variables determining the quality of game performance. A lack of significant differences between two adjacent classes was explained on the basis of the methodological approach, the class-position relationship in this sample, and the performance level of women's wheelchair basketball. We conclude that the performance of elite female wheelchair basketball players is dependent on functional ability.

Objective: To determine the effect of dialysis fluid containing various glucose concentrations on the phagocytosis and killing of Staphylococcus aureus by rat peritoneal cells under conditions mimicking the in vivo situation., Design: Phagocytosis and killing were evaluated by quantitation of the killing capacity of macrophages after in vivo phagocytosis of the bacteria as well as by an in vitro flow cytometric assay of the phagocytosis and killing of adhered bacteria by peritoneal cells., Animals: Male Wistar rats., Main Outcome Measure: It was expected that the intraperitoneal administration of dialysis fluid would impair the capacity of peritoneal cells to eliminate bacteria., Results: The first test revealed no effects of glucose concentration or dwell time on the killing of phagocytosed bacteria by macrophages, median percentages ranging between 29% and 64%. In the second series of experiments no effect of glucose concentration on the phagocytosis and killing of adhered bacteria was found either; however, longer dwell times significantly enhanced both the phagocytosis (at a dwell time of 1 hour, under 20%; at dwell times of 4 or 18 hours, above 20%, p < 0.02) and the killing (at a dwell time of 1 hour, under 53%; at dwell times of 4 and 18 hours, above 70%, p < 0.01)., Conclusions: Glucose concentration has no effect on the phagocytosis and killing of Staphylococcus aureus, whereas the dwell time significantly enhances both of these functional capacities of peritoneal cells if the bacteria are adhered to surfaces.

To study the effect of glucose concentration and dwell time of dialysis fluid on peritoneal antibacterial defence, an experimental infection with Staphylococcus aureus was induced in rats. For this purpose rats were inoculated intraperitoneally with Staphylococcus aureus at different intervals after the administration of various dialysis fluids. Twenty-four hours later the numbers of bacteria and cells in the peritoneal cavity were determined. The number of bacteria was correlated positively with the glucose concentration. Furthermore, an inverse correlation between dwell time and the number of bacteria was observed. Neither finding could be attributed to a glucose-dependent growth of the bacteria or disruption of the killing capacity of peritoneal cells in vitro. A glucose-dependent increase in the volume of the peritoneal fluid could partially explain the differences found in vivo. It is concluded that the glucose in dialysis fluid impairs antibacterial defence in the peritoneal cavity and that longer dwell times enhance this defence.

Background: A major drawback of continuous ambulatory peritoneal dialysis (CAPD) is the occurrence of peritoneal infection. This might be explained by a non-optimal phagocytic capacity of peritoneal cells which can be improved by stimulating factors., Aim: To investigate the effect of addition of interferon-gamma (IFN) to dialysis fluid with various glucose concentrations or to saline (as control) on the peritoneal defence against Staphylococcus aureus in an experimental dialysis model in rats., Methods: Twenty-four hours after the administration of either dialysis fluid containing various glucose concentrations or saline with or without IFN, bacteria were injected intraperitoneally. At the time of the bacterial infection and 24 h later cellular and bacterial parameters were studied., Results: The addition of IFN to dialysis fluid or saline resulted in a significant (P < 0.01) increase in the number of peritoneal macrophages at the time of infection; this was accompanied by a significant increase in both the number of Ia-positive peritoneal macrophages (P < 0.01) and the production of nitrite by macrophages (P < 0.05) at the time. IFN in dialysis fluid as well as in saline significantly (P < 0.01) reduced the recovery of bacteria from the peritoneal cavity 24 h after infection. Only the absence of IFN glucose increased the recovery of bacteria from the peritoneal cavity at the same time., Conclusion: In this experimental model the addition of IFN to dialysis fluid lowered the recovery of staphylococci from the peritoneal cavity by means of activation of an increased number of macrophages.

The effect of the number of bacteria and the volume of the inoculum was studied in an experimental infection model to establish a peritoneal dialysis model in the rat. Staphylococcus aureus was used in all experiments, and Staphylococcus epidermidis only in the volume experiments. A bacterial number between 10(8) and 10(9) colony-forming units (cfu) resulted in a time-dependent decrease of bacteria collected from the peritoneal cavity. Higher concentrations resulted in the death of animals, while lower concentrations were rapidly cleared. There was a positive correlation between the volume in which 3 x 10(8) cfu were dissolved and the number of bacteria isolated from the peritoneal cavity 24 hours after infection. The results of this study led to an experimental dialysis model using 10 mL of dialysis fluid and 0.5 mL of a suspension containing 3 x 10(8) cfu of Staphylococcus aureus.

In the milky spots, precursors of cells belonging to the mononuclear phagocyte system (MPS), such as monoblasts, can be found on the basis of ultrastructural endogenous peroxidase cytochemistry. Therefore, in the present study, we investigated the milky spots using a panel of monoclonal antibodies, especially antibodies (ER-MP) that recognize macrophage precursor antigens. Early macrophage precursor antigens ER-MP12 and ER-MP58 were detected only on cells localized inside the milky spots. On the other hand, an antigen which disappears late in the course of macrophage differentiation, ER-MP20, was detected in high amounts on cells both inside and around the milky spots. This clearly indicates that macrophage precursors are centrally localized inside the milky spots, while more differentiated cells are found in peripheral areas. Moreover, long-term culture of milky spot tissue resulted in the forming of a monolayer of stromal cells which supported macrophage proliferation in vitro. In conclusion, both in situ and in vitro studies demonstrated that mouse milky spots have a microenvironment in which precursor cells of the MPS can home and proliferate, illustrating that milky spots play a role as a source of local macrophage generation, e.g. that of the free peritoneal macrophages.