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Abstract Background One of the main side effects of radiation therapy to the head and neck region is altered taste sensation. This causes significant morbidity and has profound effects on the quality of life (QoL) of patients. While radiation-associated toxicities like xerostomia and dysphagia are part of large investigations, data on taste impairment is sparse. Small cohort sizes in the majority of studies and a variety of analysis methods limit our current understanding of the underlying processes. None of the studies published to date used a taste-specific QoL questionnaire with differentiation of the different taste qualities (e.g. sour, bitter). Furthermore, data regarding the correlation of taste impairment with radiation-associated change in saliva composition is currently not available. The aim of the TASTE study is to fill this gap. Based on the acquired data, a normal tissue complication probability (NTCP) model for late radiation-associated taste impairment will be developed. Methods In this prospective, observational multicenter study 150 head and neck cancer patients undergoing radiation therapy will be recruited and undergo repetitive (semi-) objective and subjective assessment of their taste, smell and salivary function (questionnaires, taste and smell assessment, saliva analysis). Primary endpoint will be patient-reported taste impairment 12 months post radiation therapy using a standardized questionnaire. Secondary endpoints will include taste impairment measured using taste strips at 12 months and 2 years post radiation therapy. Differences between subgroups (radiation side, chemotherapy, etc.) and changes over time will be assessed while adjusting for confounding factors (e.g. age, sex, smoking history). Discussion This study sets out to further our understanding of taste impairment in patients undergoing radiation therapy to the head and neck region with the goal to prevent this common side effect in future patients. The results of the study may be used to evaluate taste-preserving radiotherapy for patients with head and neck cancer, which may significantly reduce the long-term burden in this patient cohort.

Background and purpose: The retrograde femoral approach is an established technique for implantation of nails for leg lengthening and correction and in cases of distal femoral fractures. The purpose of this study was to determine the 10-year outcome of this technique by analyzing the clinical long-term effects and radiological status of the knee after leg lengthening via a retrograde femoral approach. Patients and methods: This retrospective single-center study included 13 patients (median age at surgery 17 [range 15–20] years) who underwent unilateral, retrograde, femoral lengthening with a motorized nail. Outcome measurements were graded variables of the SF-36, ISKD score, and Lysholm score. MRI of both knees was performed in all patients. MRI was evaluated for the presence of degenerative changes and compared with the healthy contralateral knee. Cartilage condition was graded according to the International Cartilage Repair Society (ICRS) scoring system. Results: All patients were pain-free and had a full range of motion 10 (range 10.0–12.2) years after surgery. All postoperative knees showed fibrosis of Hoffa’s fat pad and moderate to severe cartilage defects (ICRS Grade 2–4) of the trochlear groove (nail entry site). 6 out of 13 operated knees exhibited retropatellar cartilage defects. Conclusion: Our study showed that patients were pain-free, but cartilage defects at the entry point and arthrofibrosis at Hoffa’s fat pad were observed without causing clinical impairment.

Abstract Background Spastic cerebral palsy is the most common cause of motor disability in children. It often leads to foot drop or equinus, interfering with walking. Ankle-foot orthoses (AFOs) are commonly used in these cases. However, AFOs can be too restrictive for mildly impaired patients. Functional electrical stimulation (FES) of the ankle-dorsiflexors is an alternative treatment as it could function as a dynamic functional orthosis. Despite previous research, high level evidence on the effects of FES on activities and participation in daily life is missing. The primary aim of this study is to evaluate whether FES improves the activity and participation level in daily life according to patients, and the secondary aim is to provide evidence of the effect of FES at the level of body functions and activities. Furthermore, we aim to collect relevant information for decisions on its clinical implementation. Methods A randomized crossover trial will be performed on 25 children with unilateral spastic cerebral palsy. Patients aged between 4 and 18 years, with Gross Motor Functioning Classification System level I or II and unilateral foot drop of central origin, currently treated with AFO or adapted shoes, will be included. All participants will undergo twelve weeks of conventional treatment (AFO/adapted shoes) and 12 weeks of FES treatment, separated by a six-week washout-phase. FES treatment consists of wearing the WalkAide® device, with surface electrodes stimulating the peroneal nerve during swing phase of gait. For the primary objective, the Goal Attainment Scale is used to test whether FES improves activities and participation in daily life. The secondary objective is to prove whether FES is effective at the level of body functions and structures, and activities, including ankle kinematics and kinetics measured during 3D-gait analysis and questionnaire-based frequency of falling. The tertiary objective is to collect relevant information for clinical implementation, including acceptability using the device log file and side effect registration, cost-effectiveness based on quality adjusted life years (QALYs) and clinical characteristics for patient selection. Discussion We anticipate that the results of this study will allow evidence-based use of FES during walking in children with unilateral spastic cerebral palsy. Trial registration ClinicalTrials.gov : NCT03440632 .

There is a paucity of information about potential molecular brakes on the activation of fibroblasts that drive tissue fibrosis. The transcription factor Krüppel-like factor 4 (KLF4) is best known as a determinant of cell stemness and a tumor suppressor. We found that its expression was diminished in fibroblasts from fibrotic lung. Gain- and loss-of-function studies showed that KLF4 inhibited fibroblast proliferation, collagen synthesis, and differentiation to myofibroblasts, while restoring their sensitivity to apoptosis. Conditional deletion of KLF4 from fibroblasts potentiated the peak degree of pulmonary fibrosis and abrogated the subsequent spontaneous resolution in a model of transient fibrosis. A small molecule inducer of KLF4 was able to restore its expression in fibrotic fibroblasts and elicit resolution in an experimental model characterized by more clinically relevant persistent pulmonary fibrosis. These data identify KLF4 as a pivotal brake on fibroblast activation whose induction represents a therapeutic approach in fibrosis of the lung and perhaps other organs.

Abstract Objective The ongoing pandemic of coronavirus disease (2019 coronavirus disease [COVID‐19]), caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2) virus, is highly contagious with high morbidity and mortality. The role of the nasal and paranasal sinus cavities is increasingly recognized for COVID‐19 symptomatology and transmission. We therefore conducted a systematic review, synthesizing existing scientific evidence about sinonasal pathophysiology in COVID‐19. Study Design Systematic review. Methods Systematic searches were performed of all indexed studies in PubMed/Medline and Cochrane databases through 28 March 2020 and studies searchable on preprints.com (including ArXiv and Scilit repositories) through 30 March 2020. Data extraction focused on sinonasal pathophysiology in COVID‐19. Results A total of 19 studies were identified. The sinonasal cavity may be a major site of infection by SARS‐CoV‐2, where susceptibility genes required for infection are expressed at high levels and may be modulated by environmental and host factors. Viral shedding appears to be highest from the nose, therefore reflecting a major source for transmission. This has been highlighted by multiple reports of health care‐associated infection (HAI) during rhinologic procedures, which are now consequently considered to be high risk for SARS‐CoV‐2 transmission to health care workers. While sinonasal symptomatology, such as rhinorrhea or congestion, appears to be a rarer symptom of COVID‐19, anosmia without nasal obstruction is reported as highly specific predictor of COVID‐19+ patients. Conclusion Sinonasal pathophysiology is increasingly important in our understanding of COVID‐19. The sinonasal tract may be an important site of infection while sinonasal viral shedding may be an important transmission mechanism—including HAI. Anosmia without nasal obstruction may be a highly specific indicator of COVID‐19. Level of Evidence 2a.

Unconventional secretion and subsequent uptake of molecular cargo via extracellular vesicles (EVs) is an important mechanism by which cells can exert paracrine effects. While this phenomenon has been widely characterized in the context of their ability to promote inflammation, less is known about the ability of EVs to transfer immunosuppressive cargo. Maintenance of normal physiology in the lung requires suppression of potentially damaging inflammatory responses to the myriad of insults to which it is continually exposed. Recently, our laboratory has reported the ability of alveolar macrophages (AMs) to secrete suppressors of cytokine signaling (SOCS) proteins within microvesicles (MVs) and exosomes (Exos). Uptake of these EVs by alveolar epithelial cells (AECs) resulted in inhibition of pro-inflammatory STAT activation in response to cytokines. Moreover, AM packaging of SOCS within EVs could be rapidly tuned in response to exogenous or AEC-derived substances. In this article we will highlight gaps in knowledge regarding microenvironmental modulation of cargo packaging and utilization as well as EV secretion and uptake. Advances in these areas are critical for improving understanding of intercellular communication in the immune system and for therapeutic application of artificial vesicles aimed at treatment of diseases characterized by dysregulated inflammation.

In the First World War (WW1), different types of injuries became both political and economic factors for the main belligerent countries. This work illustrates the special role facial injuries played during and after the war and the profound impact they had on the field of Plastic Surgery in Britain and Germany.This is a historical work based on primary and secondary sources. English and German material was used to compile an overview of the development of Plastic and Reconstructive Surgery as a specialty during WW1 and up to the Second World War (WW2).Plastic and Reconstructive Surgery had the task of reconstructing male beauty and personalities, restoring the most intense, disfigured reminders of a terrible war in homes and the public. Looking at the significant role the specialty played during the war, remarkably, most of the contributors struggled to find financial support to develop designated specialized departments in the inter-war period. The specialty's connection to the emotional subject of facial disfigurement did not work in its favor at a time when people wanted to forget the horrors of the war.The developments in Britain and Germany during the WW1 were less isolated than often described and the inter-war period saw much effort for international collaboration. The different developments of WW2 further illustrate how essential the efforts of a strong group of individuals and support from authorities were for the evolution of Plastic Surgery as a specialty., Competing Interests: L.W. has received partial travel and participation funding from Abbvie Switzerland for the Annual Conference of the Swiss Society of Dermatology 2023, by Galderma Switzerland for the Euro-PDT Conference 2022 and 2023. Furthermore, she has received an invitation and paid travel expenses by Kerecis™ Iceland to attend the Northern Lights 2023 and Arctic Midnight Sun 2024 workshops in Iceland.M.S. is a member of the European Rhinologic Society Executive Committee, Chair of the Educational Committee of the European Rhinologic Society, and a Y-CEORL Rhinology Subgroup Board Member. All unpaid.H.S. reports being a paid lecturer by Medtronic on steroid-eluding implants. He is also a board member of the international Federation of Facial Plastic Surgery Societies, Treasurer of the European Board Examination Certification in Facial Plastic and Reconstructive Surgery, and Trustee of the Rhinology Fund at Imperial College. Also, past President of the European Academy of Facial Plastic Surgery and current senior advisor., (Thieme. All rights reserved.)

Objectives: To investigate potential presence and resolution of longer-term pulmonary diffusion limitation and microvascular perfusion impairment in COVID-19 convalescents., Materials and Methods: This prospective, longitudinal study was carried out between May 2020 and April 2023. COVID-19 convalescents repeatedly and age/sex-matched healthy controls once underwent MRI including hyperpolarized 129 Xe MRI. Blood samples were obtained in COVID-19 convalescents for immunophenotyping. Ratios of 129 Xe in red blood cells (RBC), tissue/plasma (TP), and gas phase (GP) as well as lung surface-volume ratio were quantified and correlations with CD4 + /CD8 + T cell frequencies were assessed using Pearson's correlation coefficient. Signed-rank tests were used for longitudinal and U tests for group comparisons., Results: Thirty-five participants were recruited. Twenty-three COVID-19 convalescents (age 52.1 ± 19.4 years, 13 men) underwent baseline MRI 12.6 ± 4.2 weeks after symptom onset. Fourteen COVID-19 convalescents underwent follow-up MRI and 12 were included for longitudinal comparison (baseline MRI at 11.5 ± 2.7 weeks and follow-up 38.0 ± 5.5 weeks). Twelve matched controls were included for comparison. In COVID-19 convalescents, RBC-TP was increased at follow-up (p = 0.04). Baseline RBC-TP was lower in patients treated on intensive care unit (p = 0.03) and in patients with severe/critical disease (p = 0.006). RBC-TP correlated with CD4 + /CD8 + T cell frequencies (R = 0.61/ - 0.60) at baseline. RBC-TP was not significantly different compared to matched controls at follow-up (p = 0.25)., Conclusion: Impaired microvascular pulmonary perfusion and alveolar membrane function persisted 12 weeks after symptom onset and resolved within 38 weeks after COVID-19 symptom onset., Clinical Relevance Statement: 129 Xe MRI shows improvement of microvascular pulmonary perfusion and alveolar membrane function between 11.5 ± 2.7 weeks and 38.0 ± 5.5 weeks after symptom onset in patients after COVID-19, returning to normal in subjects without significant prior disease., Key Points: • The study aims to investigate long-term effects of COVID-19 on lung function, in particular gas uptake efficiency, and on the cardiovascular system. • In COVID-19 convalescents, the ratio of 129 Xe in red blood cells/tissue plasma increased longitudinally (p = 0.04), but was not different from matched controls at follow-up (p = 0.25). • Microvascular pulmonary perfusion and alveolar membrane function are impaired 11.5 weeks after symptom onset in patients after COVID-19, returning to normal in subjects without significant prior disease at 38.0 weeks., (© 2024. The Author(s).)

Hypothesis: Freeze-thaw cycles (FTC) in soils can cause the aggregation of dissolved organic matter but controlling factors are little understood., Experiments: In freeze-thaw experiments with tannic acid (TA) as model substance, we studied the effect of TA concentration, pH, electrolytes (NaCl, CaCl 2 , AlCl 3 ), and number of FTC on particle formation. Tannic acid (0.005 to 10 g L -1 ) was exposed to 1-20 FTC at pH 3 and 6. The size and shape of particles was determined by confocal laser scanning microscopy. Particle stability was deduced from the equivalent circle diameter (ECD) obtained in dry state and the hydrodynamic diameter measured in thawing solutions., Findings: Tannic acid particles occurred as plates and veins, resembling the morphology of ice grain boundaries. Low pH and presence of electrolytes favored the formation of large particles. The freeze-concentration effect was most intense at low TA concentrations and increased with the number of FTC. While ECD of particles formed at low TA concentrations were smaller than at high concentrations, it was vice versa in the thawed state. At low TA concentrations, higher crystallization pressure of ice caused enhanced stability of large particles. We conclude that FTC can strongly alter the physical state of dissolved organic matter, with likely consequences for its bioavailability., 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 © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: To investigate whether 3D phase-resolved functional lung (PREFUL)-MRI parameters are suitable to measure response to elexacaftor/tezacaftor/ivacaftor (ETI) therapy and their association with clinical outcomes in cystic fibrosis (CF) patients., Methods: Twenty-three patients with CF (mean age: 21; age range: 14-46) underwent MRI examination at baseline and 8-16 weeks after initiation of ETI. Morphological and 3D PREFUL scans assessed pulmonary ventilation. Morphological images were evaluated using a semi-quantitative scoring system, and 3D PREFUL scans were evaluated by ventilation defect percentage (VDP) values derived from regional ventilation (RVent) and cross-correlation maps. Improved ventilation volume (IVV) normalized to body surface area (BSA) between baseline and post-treatment visit was computed. Forced expiratory volume in 1 second (FEV 1 ) and mid-expiratory flow at 25% of forced vital capacity (MEF25), as well as lung clearance index (LCI), were assessed. Treatment effects were analyzed using paired Wilcoxon signed-rank tests. Treatment changes and post-treatment agreement between 3D PREFUL and clinical parameters were evaluated by Spearman's correlation., Results: After ETI therapy, all 3D PREFUL ventilation markers (all p < 0.0056) improved significantly, except for the mean RVent parameter. The BSA normalized IVV RVent was significantly correlated to relative treatment changes of MEF25 and mucus plugging score (all |r| > 0.48, all p < 0.0219). In post-treatment analyses, 3D PREFUL VDP values significantly correlated with spirometry, LCI, MRI global, morphology, and perfusion scores (all |r| > 0.44, all p < 0.0348)., Conclusions: 3D PREFUL MRI is a very promising tool to monitor CFTR modulator-induced regional dynamic ventilation changes in CF patients., Clinical Relevance Statement: 3D PREFUL MRI is sensitive to monitor CFTR modulator-induced regional ventilation changes in CF patients. Improved ventilation volume correlates with the relative change of mucus plugging, suggesting that reduced endobronchial mucus is predominantly responsible for regional ventilation improvement., Key Points: • 3D PREFUL MRI-derived ventilation maps show significantly reduced ventilation defects in CF patients after ETI therapy. • Significant post-treatment correlations of 3D PREFUL ventilation measures especially with LCI, FEV 1 %pred, and global MRI score suggest that 3D PREFUL MRI is sensitive to measure improved regional ventilation of the lung parenchyma due to reduced inflammation induced by ETI therapy in CF patients. • 3D PREFUL MRI-derived improved ventilation volume (IVV) correlated with MRI mucus plugging score changes suggesting that reduced endobronchial mucus is predominantly responsible for regional ventilation improvement 8-16 weeks after ETI therapy., (© 2023. The Author(s).)

Background Chronic lung allograft dysfunction (CLAD), the physiologic correlate of chronic rejection, remains a major barrier to long-term survival following lung transplant. Biomarkers for early prediction of future transplant loss or death due to CLAD might open a window of opportunity for early diagnosis and treatment of CLAD. Purpose To evaluate the prognostic use of phase-resolved functional lung (PREFUL) MRI in predicting CLAD-related transplant loss or death. Materials and Methods In this prospective, longitudinal, single-center study, PREFUL MRI-derived ventilation and parenchymal lung perfusion parameters of bilateral lung transplant recipients without clinically suspected CLAD were assessed 6-12 months (baseline) and 2.5 years (follow-up) after transplant. MRI scans were acquired between August 2013 and December 2018. Regional flow volume loop (RFVL)-based ventilated volume (VV) and perfused volume were calculated using thresholds and spatially combined as ventilation-perfusion (V/Q) matching. Spirometry data were obtained on the same day. Exploratory models were calculated using receiver operating characteristic analysis, and subsequent survival analyses (Kaplan-Meier, hazard ratios [HRs]) of CLAD-related graft loss were performed to compare clinical and MRI parameters as clinical end points. Results At baseline MRI examination, 132 clinically stable patients of 141 patients (median age, 53 years [IQR, 43-59 years]; 78 men) were included (nine were excluded for deaths not associated with CLAD), 24 of which had CLAD-related graft loss (death or retransplant) within the observational period of 5.6 years. PREFUL MRI-derived RFVL VV was a predictor of poorer survival (cutoff, 92.3%; log-rank P = .02; HR for graft loss, 2.5 [95% CI: 1.1, 5.7]; P = .02), while perfused volume ( P = .12) and spirometry ( P = .33) were not predictive of differences in survival. In the evaluation of percentage change at follow-up MRI (92 stable patients vs 11 with CLAD-related graft loss), mean RFVL (cutoff, 97.1%; log-rank P < .001; HR, 7.7 [95% CI: 2.3, 25.3]), V/Q defect (cutoff, 498%; log-rank P = .003; HR, 6.6 [95% CI: 1.7, 25.0]), and forced expiratory volume in the first second of expiration (cutoff, 60.8%; log-rank P < .001; HR, 7.9 [95% CI: 2.3, 27.4]; P = .001) were predictive of poorer survival within 2.7 years (IQR, 2.2-3.5 years) after follow-up MRI. Conclusion Phase-resolved functional lung MRI ventilation-perfusion matching parameters were predictive of future chronic lung allograft dysfunction-related death or transplant loss in a large prospective cohort who had undergone lung transplant. © RSNA, 2023 Supplemental material is available for this article. See also the editorial by Fain and Schiebler in this issue.

The emergence of drug-resistant Mycobacterium tuberculosis strains highlights the need to discover anti-tuberculosis drugs with novel mechanisms of action. Here we discovered a mycobactericidal strategy based on the prodrug activation of selected chemical derivatives classified as nitronaphthofurans (nNFs) mediated by the coordinated action of the sigH and mrx2 genes. The transcription factor SigH is a key regulator of an extensive transcriptional network that responds to oxidative, nitrosative, and heat stresses in M. tuberculosis. The nNF action induced the SigH stress response which in turn induced the mrx2 overexpression. The nitroreductase Mrx2 was found to activate nNF prodrugs, killing replicating, non-replicating and intracellular forms of M. tuberculosis. Analysis of SigH DNA sequences obtained from spontaneous nNF-resistant M. tuberculosis mutants suggests disruption of SigH binding to the mrx2 promoter site and/or RNA polymerase core, likely promoting the observed loss of transcriptional control over Mrx2. Mutations found in mrx2 lead to structural defects in the thioredoxin fold of the Mrx2 protein, significantly impairing the activity of the Mrx2 enzyme against nNFs. Altogether, our work brings out the SigH/Mrx2 stress response pathway as a promising target for future drug discovery programs., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Anti-eosinophilic therapy with interleukin-5/interleukin-5-receptor antibodies represents an established treatment for patients with severe eosinophilic asthma (SEA) but did not show clinical efficacy in patients with COPD. The objective of the present study was to evaluate treatment response to anti-eosinophilic antibody therapy in patients with asthma and COPD., Methods: A retrospective comparison of pulmonary function testing, oral corticosteroid intake, quality of life and pulmonary symptom control in patients with SEA and COPD and 1:1 propensity score matched patients suffering from SEA alone was performed. All patients received treatment with either mepolizumab or benralizumab. Data were assessed prior to antibody treatment start and after 6 months of therapy., Results: Data from 84 patients (42 patients with SEA and COPD and 42 patients with SEA) were analysed. After 6 months of treatment, patients in both groups showed improved forced expiratory volume in 1 s (improvement by 11% (IQR 5-18) in the SEA and COPD group versus 15% (IQR -3-23); p=0.637) and decreased oral corticosteroid dosages (median reduction by 3 mg in the SEA and COPD group versus 5 mg; p=0.070), without significant differences between groups. Pulmonary symptom control and quality of life improved in both groups. A significant decrease in eosinophils could be measured in both groups with similar cell numbers prior to treatment initiation (600 cells·µL -1 in the SEA and COPD group versus 500 cells·µL -1 )., Conclusion: Anti-eosinophilic therapy with interleukin-5/interleukin-5-receptor antibodies shows clinical efficacy in patients with SEA and COPD comparable to treatment response in patients with SEA alone., Competing Interests: Conflict of interest: N. Drick reports no conflict of interest. Conflict of interest: J. Fuge reports personal fees/speaker honoraria from AstraZeneca, outside the submitted work. Conflict of interest: B. Seeliger reports no conflict of interest. Conflict of interest: M. Speth reports no conflict of interest. Conflict of interest: J. Vogel-Claussen received grants, advisory/lecture/clinical trial fees and nonfinancial support from BMBF (German Ministry of Research and Education), Siemens Healthineers, AstraZeneca, Bayer, Boehringer Ingelheim, GSK, MSD, Novartis, AstraZeneca and Coreline Soft all outside the submitted work Conflict of interest: T. Welte and/or his institution received grants, advisory/lecture/clinical trial fees and nonfinancial support from DFG (German Research Council), BMBF, BMG (German Ministry of Health), EU, WHO, AstraZeneca, Basilea, Biotest, Bayer, Boehringer, Berlin Chemie, GSK, Infectopharm, MSD, Novartis, Pfizer, Roche, Gilead, and Janssen, all outside the submitted work. Conflict of interest: H. Suhling reports personal fees/speaker honoraria from AstraZeneca, GSK, Novartis and Sanofi, outside the submitted work., (Copyright ©The authors 2022.)

Background: The ventilatory management of COVID-ARDS is controversial, especially with regard to the different subtypes and associated PEEP titration. A higher PEEP may be beneficial only in patients with potential for lung recruitment. The assessment of lung recruitment may be guided by lung imaging, such as electric impedance tomography or recruitment computed tomography, but is complex and not established in routine clinical practice. Therefore, bedside identification of recruitable ARDS phenotypes can aid in PEEP titration in clinical settings., Methods: In this retrospective consecutive cohort study in 40 patients with moderate-to-severe COVID-ARDS, we assessed lung recruitment using the recruitment-to-inflation ratio (R/I) in moderate-to-severe COVID-ARDS. Evidence of recruitment (R/I ≥ 0.5) was compared between clinical and computed tomography data., Results: Of the included patients, 28 (70%) were classified as recruiters by the R/I. Lung recruitment was associated with higher compliance and was not associated with a consolidated lung pattern assessed using CT. Even in the tertile of patients with the highest compliance (37-70 ml/mbar), eight (73%) patients were classified as recruitable. Patients classified as recruitable presented a lower reticular lung pattern (2% vs. 6%, p = 0.032)., Conclusions: Prediction of lung recruitment is difficult based on routine clinical data but may be improved by assessment of radiographic lung patterns. A bedside assessment of recruitment is necessary to guide clinical care. Even a high compliance may not rule out the potential for lung recruitment., (© 2022. The Author(s).)

With the increasing emergence of drug-resistant Mycobacterium tuberculosis strains, new and effective antibiotics against tuberculosis (TB) are urgently needed. However, the high frequency of poorly water-soluble compounds among hits in high-throughput drug screening campaigns is a major obstacle in drug discovery. Moreover, in vivo testing using conventional animal TB models, such as mice, is time consuming and costly, and represents a major bottleneck in lead compound discovery and development. Here, we report the use of the zebrafish embryo TB model for evaluating the in vivo toxicity and efficacy of five poorly water-soluble nitronaphthofuran derivatives, which were recently identified as possessing anti-TB activity in vitro. To aid solubilization, compounds were formulated in biocompatible polymeric micelles (PMs). Three of the five PM-formulated nitronaphthofuran derivatives showed low toxicity in vivo, significantly reduced bacterial burden and improved survival in infected zebrafish embryos. We propose the zebrafish embryo TB-model as a quick and sensitive tool for evaluating the in vivo toxicity and efficacy of new anti-TB compounds during early stages of drug development. Thus, this model is well suited for pinpointing promising compounds for further development., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Background: The developing zebrafish is an emerging tool in nanomedicine, allowing non-invasive live imaging of the whole animal at higher resolution than is possible in the more commonly used mouse models. In addition, several transgenic fish lines are available endowed with selected cell types expressing fluorescent proteins; this allows nanoparticles to be visualized together with host cells., Methods: Here, we introduce the zebrafish neural tube as a robust injection site for cancer cells, excellently suited for high resolution imaging. We use light and electron microscopy to evaluate cancer growth and to follow the fate of intravenously injected nanoparticles., Findings: Fluorescently labelled mouse melanoma B16 cells, when injected into this structure proliferated rapidly and stimulated angiogenesis of new vessels. In addition, macrophages, but not neutrophils, selectively accumulated in the tumour region. When injected intravenously, nanoparticles made of Cy5-labelled poly(ethylene glycol)-block-poly(2-(diisopropyl amino) ethyl methacrylate) (PEG-PDPA) selectively accumulated in the neural tube cancer region and were seen in individual cancer cells and tumour associated macrophages. Moreover, when doxorubicin was released from PEG-PDPA, in a pH dependant manner, these nanoparticles could strongly reduce toxicity and improve the treatment outcome compared to the free drug in zebrafish xenotransplanted with mouse melanoma B16 or human derived melanoma cells., Interpretation: The zebrafish has the potential of becoming an important intermediate step, before the mouse model, for testing nanomedicines against patient-derived cancer cells., Funding: We received funding from the Norwegian research council and the Norwegian cancer society., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Triggering or enhancing antitumor activity of tumor-associated macrophages is an attractive strategy for cancer treatment. We have previously shown that the cytokine interferon-γ (IFN-γ), a type II IFN, could synergize with toll-like receptor (TLR) agonists for induction of antitumor M1 macrophages. However, the toxicity of IFN-γ limits its clinical use. Here, we investigated whether the less toxic type I IFNs, IFN-α, and IFN-β, could potentially replace IFN-γ for induction of antitumor M1 macrophages. We measured in vitro the ability of type I and II IFNs to synergize with TLR agonists for transcription of inducible nitric oxide synthase (iNOS) mRNA and secretion of nitric oxide (NO) by mouse bone marrow-derived macrophages (BMDMs). An in vitro growth inhibition assay was used to measure both cytotoxic and cytostatic activity of activated macrophages against Lewis lung carcinoma (LLC) cancer cells. We found that both type I and II IFNs could synergize with TLR agonists in inducing macrophage-mediated inhibition of cancer cell growth, which was dependent on NO. The ability of high dose lipopolysaccharide (LPS) to induce tumoricidal activity in macrophages in the absence of IFN-γ was shown to depend on induction of autocrine type I IFNs. Antitumor M1 macrophages could also be generated in the absence of IFN-γ by a combination of two TLR ligands when using the TLR3 agonist poly(I:C) which induces autocrine type I IFNs. Finally, we show that encapsulation of poly(I:C) into nanoparticles improved its potency to induce M1 macrophages up to 100-fold. This study reveals the potential of type I IFNs for activation of antitumor macrophages and indicates new avenues for cancer immunotherapy based on type I IFN signaling, including combination of TLR agonists.

Tumor-associated macrophages may either promote or suppress tumor growth depending on their activation status. Interferon-γ (IFN-γ) has been identified as a key factor for inducing tumoricidal M1 phenotype in macrophages. However, it remains unclear whether IFN-γ is sufficient or if additional stimuli are required. Here, we tested IFN-γ and a panel of toll-like receptor (TLR) agonists for the ability to activate murine macrophages toward a tumoricidal M1 phenotype. The following TLR ligands were used: TLR1/TLR2 agonist Pam3CSK4, TLR2/TLR6 agonist lipotechoic acid, TLR3 agonist poly(I:C), TLR4 agonist lipopolysaccharide (LPS), TLR5 agonist flagellin, TLR7 agonist CL264, and TLR9 agonist CpG. We used an in vitro growth inhibition assay to measure both cytotoxic and cytostatic activity of mouse macrophages against Lewis lung carcinoma (LLC) and MOPC315 plasmacytoma tumor cells. Production of nitric oxide (NO) and cytokines by activated macrophages was quantified. We found that IFN-γ alone was not able to render macrophages tumoricidal. Similarly, macrophage activation with single TLR agonists was inefficient. In sharp contrast, IFN-γ was shown to synergize with TLR agonists for induction of macrophage tumoricidal activity and production of both NO and pro-inflammatory cytokines (TNF-α, IL-12p40, and IL-12p70). Furthermore, IFN-γ was shown to suppress macrophage IL-10 secretion induced by TLR agonists. NO production was necessary for macrophage tumoricidal activity. We conclude that two signals from the microenvironment are required for optimal induction of antitumor M1 macrophage phenotype. Combination treatment with IFN-γ and TLR agonists may offer new avenues for macrophage-based cancer immunotherapy.

Encapsulating antibiotics such as rifampicin in biodegradable nanoparticles provides several advantages compared to free drug administration, including reduced dosing due to localized targeting and sustained release. Consequently, these characteristics reduce systemic drug toxicity. However, new nanoformulations need to be tested in complex biological systems to fully characterize their potential for improved drug therapy. Tuberculosis, caused by infection with the bacterium Mycobacterium tuberculosis, requires lengthy and expensive treatment, and incomplete therapy contributes to an increasing incidence of drug resistance. Recent evidence suggests that standard therapy may be improved by combining antibiotics with bacterial efflux pump inhibitors, such as thioridazine. However, this drug is difficult to use clinically due to its toxicity. Here, we encapsulated thioridazine in poly(lactic-co-glycolic) acid nanoparticles and tested them alone and in combination with rifampicin nanoparticles, or free rifampicin in macrophages and in a zebrafish model of tuberculosis. Whereas free thioridazine was highly toxic in both cells and zebrafish embryos, after encapsulation in nanoparticles no toxicity was detected. When combined with rifampicin nanoparticles, the nanoparticles loaded with thioridazine gave a modest increase in killing of both Mycobacterium bovis BCG and M. tuberculosis in macrophages. In the zebrafish, the thioridazine nanoparticles showed a significant therapeutic effect in combination with rifampicin by enhancing embryo survival and reducing mycobacterial infection. Our results show that the zebrafish embryo is a highly sensitive indicator of drug toxicity and that thioridazine nanoparticle therapy can improve the antibacterial effect of rifampicin in vivo.

There is a constant need to increase the efficiency of vaccines in the aquaculture industry. Although several nano-based vaccine formulations have been reported, to the best of our knowledge so far only one of them have been implemented in the industry. Here we report on chitosan-poly(I:C) nanoparticles (NPs) that could be used as a non-specific adjuvant in antiviral vaccines in aquaculture. We have characterized the physical parameters of the NPs, studied the in vivo and in vitro bio-distribution of fluorescent NPs and verified NP uptake by zebrafish leucocytes. We used the zebrafish model to test the protective efficiency of the recombinant glycoprotein G (rgpG) of VHSV compared to inactivated whole virus (iV) against VHSV using NPs as an adjuvant in both formulations. In parallel we tested free poly(I:C) and rgpG (pICrgpG), and free chitosan and rgpG (CSrgpG) vaccine formulations. While the iV group (with NP adjuvant) provided the highest overall survival, all vaccine formulations with poly(I:C) provided a significant protection against VHSV; possibly through an early induction of an anti-viral state. Our results suggest that chitosan-poly(I:C) NPs are a promising adjuvant candidate for future vaccine formulations., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This study reports a single-centre experience of the Medos Deltastream diagonal-pump (DP3) for extracorporeal cardiac, pulmonary, or combined support in a single-center pediatric cohort. Twenty-seven consecutive patients with 28 runs of the DP3 between January 2013 and June 2014 were included for analysis. Median patient age, weight, and duration of support were 278 days (range: 0 days-14.2 years), 7.2 kg (range: 2.5-39 kg), and 8 days (range: 2-69 days). Midline sternotomy (n = 20, 71.4%) or cervical approaches (n = 8, 28.6%) were used for cannulation. The DP3 was employed for either veno-arterial extracorporeal life support (ECLS, n = 16), veno-venous extracorporeal membrane oxygenation (ECMO, n = 5), or ventricular assist devices (right ventricular assist device [RVAD], n = 1; left ventricular assist device [LVAD], n = 1; and univentricular assist device [UNIVAD], n = 5). Three patients initially supported with ECLS were switched to UNIVAD and one patient with UNIVAD was changed to ECLS. Required flow for neonates (n = 8) ranged between 0.2 and 0.75 L/min. Irreversible pump damage occurred in one patient during deairing after air block. Successful weaning, 30 day and hospital survival were 89.3% (n = 25), 85.7% (n = 24), and 71.4% (n = 20). All patients on UNIVAD, who did not require further extracorporeal respiratory assist, survived. In conclusion, the DP3 can be used for individual patient demands and adapted to their most suitable method of support. Meticulous flow adjustments render this pump highly effective for extracorporeal support particularly in pediatric patients.

Objectives: Assessment of the clinical severity of Fabry disease (FD), an X-linked, rare, progressive disorder based on a genetic defect in alpha-galactosidase is challenging, especially regarding cardiac involvement. The aim of the study was to evaluate the diagnostic value of cardiac troponin I (cTnI) in discriminating FD patients with cardiac involvement in a large FD patient cohort., Methods: cTnI levels were measured with a contemporary sensitive assay in plasma samples taken routinely from FD patients. The assay was calibrated to measure cTnI levels ≥0.01 ng/ml. Elevated cTnI values (cut-off ≥0.04 ng/ml) were correlated with clinical data., Results: cTnI was assessed in 62 FD patients (median age: 47 years, males: 36%). Elevated cTnI levels were detected in 23 (37%) patients. Patients with a cTnI elevation were older (median 55 years versus 36 years, p<0.001). Elevated cTnI levels were associated with the presence of a LVH (16/23 versus 1/39; OR 65.81, CI: 6.747-641.859; p<0.001). In almost all patients with a left ventricular hypertrophy (LVH) elevated cTnI levels were detected (16/17, 94%). Absolute cTnI levels in patients with LVH were higher than in those without (median 0.23 ng/ml versus 0.02 ng/ml; p<0.001). A cTnI level <0.04ng/ml had a high negative predictive value regarding the presence of a LVH (38/39, 97%). In a control group of non-FD patients (n = 17) with LVH (due to hypertension) none showed cTnI levels ≥0.01 ng/ml., Conclusions: Elevated cTnI levels are common in FD patients, reflecting cardiac involvement. FD patients might benefit from a continuous cTnI monitoring.

Therapeutic nanoparticles (NPs) have great potential to deliver drugs against human diseases. Encapsulation of drugs in NPs protects them from being metabolized, while they are delivered specifically to a target site, thereby reducing toxicity and other side-effects. However, non-specific tissue accumulation of NPs, for example in macrophages, especially in the spleen and liver is a general problem with many NPs being developed for cancer therapy. To address the problem of non-specific tissue accumulation of NPs we describe the development of the zebrafish embryo as a transparent vertebrate system for characterization of NPs against cancer. We show that injection of human cancer cells results in tumor-like structures, and that subsequently injected fluorescent NPs, either made of polystyrene or liposomes can be imaged in real-time. NP biodistribution and general in vivo properties can be easily monitored in embryos having selective fluorescent labeling of specific tissues. We demonstrate in vitro, by using optical tweezer micromanipulation, microscopy and flow cytometry that polyethylene glycol (PEG) coating of NPs decreases the level of adhesion of NPs to macrophages, and also to cancer cells. In vivo in zebrafish embryos, PEG coating resulted in longer NP circulation times, decreased macrophage uptake, and reduced adhesion to the endothelium. Importantly, liposomes were observed to accumulate passively and selectively in tumor-like structures comprised of human cancer cells. These results show that zebrafish embryo is a powerful system for microscopy-based screening of NPs on the route to preclinical testing.

There is an urgent need for more efficient viral vaccines in finfish aquaculture worldwide. Here, we report the use of poly(I:C) stabilized with chitosan as an adjuvant for development of better finfish vaccines. The adjuvant was co-injected with inactivated viral hemorrhagic septicemia virus (VHSV) (CSpIC+iV vaccine) in adult zebrafish and its efficiency in protection against VHSV infection was compared to a live, attenuated VHS virus vaccine (aV). Both free and stabilized poly(I:C) were strong inducers of an antiviral state, measured by transcriptional activation of the genes of viral sensors: toll-like receptors, interferons, and interferon-stimulated genes, such as MXa within 48 h after injection. Both the CSpIC+iV and the aV formulations provided a significant protection against VHSV-induced mortality. However, when plasma from survivors was tested for neutralizing antibodies in an in vitro protection assay, we could not demonstrate any protective effect. On the contrary, plasma from aV vaccinated fish enhanced cytopathic effects, indicating that antibody-dependent entry may play a role in this system. Our results show that poly(I:C) is a promising candidate as an adjuvant for fish vaccination against viral pathogens, and that the zebrafish is a promising model for aquaculture-relevant vaccination studies.

The objective of the present study was to evaluate the effects of different pectins on strawberry anthocyanins in viscous model solutions at pH 3.0. For this purpose, low esterified amidated, low and high methyl esterified citrus and apple pectins, and a sugar beet pectin (SBP), respectively, were added to strawberry extracts. The latter were predominantly composed of pelargonidin-glycosides, containing either reduced (E-1) or original amounts of non-anthocyanin phenolics (E-2). Model systems were stored for 18 weeks at 20±0.5 °C protected from light, and anthocyanins were quantitated in regular intervals by HPLC-DAD analyses. Half-life (t1/2) and destruction (D) values were calculated based on first-order kinetics. Generally, significant differences in pigment retention could be ascribed to differing pectin sources, while variation in the degree of esterification and amidation, respectively, had negligible effects. Compared to systems without added pectin, apple pectins and SPB enhanced anthocyanin stability moderately, while stabilising effects of citrus pectins were poor or even imperceptible. Generally, the amount of non-anthocyanin phenolics and the addition of citrate did not markedly affect anthocyanin stability. However, pectins had no influence on total phenolic contents (Folin-Ciocalteu assay) and antioxidant capacities (FRAP and TEAC assay) of strawberry phenolics over time. For pelargonidin-3-glucoside and -rutinoside largely consistent stabilities were found in all model systems. In contrast, pelargonidin-3-malonylglucoside was less stable in the blank, and stabilisation by pectins was always negligible. The findings of the present study are contrary to results reported previously for the stabilisation of cyanidin- and delphinidin-glycosides in similar model systems prepared with black currant extracts, indicating a high impact of the number of hydroxyl groups in the anthocyanin B-ring., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The effects of different pectins on the stabilisation of black currant anthocyanins in viscous model solutions at pH 3.0 were investigated. For this purpose, low esterified amidated (AM), low (LM) and high (HM) methoxylated citrus and apple pectins and a sugar beet pectin were added to a purified anthocyanin extract (ACN-E) and to an extract containing anthocyanins and non-anthocyanin phenolics (PP-E). Model systems were stored at 20±0.5°C in the dark. Anthocyanin contents were monitored by HPLC analysis over a period of 18 weeks, and half-life and destruction values were calculated. In all pectic model solutions anthocyanin stability was significantly improved compared to stability of the extracts without added pectins (blank). Best stabilisation was obtained with AM pectin, followed by LM and HM pectins. In model systems containing citrus pectins, anthocyanin stabilisation was better compared to that of apple pectins having similar degrees of esterification and amidation, respectively. This was primarily due to the strong interaction of delphinidin glycosides with the citrus pectins, whereas stabilisation of cyanidin derivatives was less important. Sugar beet pectin improved anthocyanin stability only to a limited extent. In the presence of non-anthocyanin phenolics (PP-E) the impact of the pectin source was even more pronounced than the effect of the pectin type. Addition of citrate to pectic systems accelerated anthocyanin decay. Stabilising effects of pectins were hardly noticeable when evaluating total phenolic content (TPC, Folin-Ciocalteu) and antioxidant capacity during storage. Highest TPC, TEAC- and FRAP values were observed in systems containing citrus pectin, which was in contrast to sugar beet pectin, where values fell below those of the blank after storage., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The calpain inhibitor A-705253 and the Na(+)/H(+)-exchange inhibitor Cariporide were studied in isolated perfused rabbit hearts subjected to 60 min occlusion of the ramus interventricularis of the left coronary artery (below the origin of the first diagonal branch), followed by 120 min of reperfusion. The inhibitors were added to the perfusion fluid solely or in combination at the beginning of reperfusion. Hemodynamic monitoring and biochemical analysis of perfusion fluid from the coronary outflow were performed. Myocardial infarct size and area at risk (transiently not perfused myocardium) were determined from left ventricular slices after a special staining procedure with Evans blue and 2,3,5-triphenyltetrazolium chloride. The infarcted area (dead myocardium) was 72.7+/-4.0% of the area at risk in untreated controls, but was significantly smaller in the presence of the inhibitors. The largest effect was seen with 10(-6) m A-705253, which reduced the infarcted area to 49.2+/-4.1% of the area at risk, corresponding to a reduction of 33.6%. Cariporide at 10(-6) m reduced the infarct size to the same extent. The combination of both inhibitors, however, did not further improve cardioprotection. No statistical difference was observed between the experimental groups in coronary perfusion, left ventricular pressure, heart rate, and in the release of lactate dehydrogenase and creatin kinase from heart muscle.

Purpose: Transient cardiac ventricular dysfunction or sudden cardiac deaths have been reported for male athletes participating in marathon racing. Less is known about the myocardial response in females. We examined natriuretic peptides and cardiac troponins in female athletes after a marathon., Methods: At the 31st real,- Berlin Marathon plasma levels of NT-pro-BNP, BNP, cTnI and cTnT were measured in 15 women (age 35+/-6 years; finishing times between 3:22 h and 5:21 h) at four different time points (before, immediately after, day one and day three)., Results: An increase in [NT-pro-BNP] was observed immediately after the marathon (median [NT-pro-BNP] before: 39.6 pg ml(-1), after: 138.6 pg ml(-1), p=0.003) with a further increase on day one. [BNP] did not increase immediately after the marathon but increased on day one (median [BNP] before: 15 pg ml(-1), day one: 27.35 pg ml(-1), p=0.006). On day three, [NT-pro-BNP] and [BNP] returned to initial values. [cTnI] was under the detection limit prior to the marathon in all runners. [cTnT] was under the detection limit before the marathon except in one runner who presented a concentration of 0.03 ng ml(-1). Cardiac troponins (median [cTnl] after: 0.098 ng ml(-1), p=0.028; median [cTnT] after: 0.032 ng ml(-1), p=0.012) increased immediately after the marathon and returned to initial values on day one [cTnT] and three [cTnI]., Discussion: Parameters representing cardiac stress increased in females after a marathon. Different kinetics of natriuretic peptides BNP and NT-pro-BNP post-marathon could be due to their different half-lives and dependence on renal function. The increase of cTnI and cTnT may result from minor myocardial lesions.

We have recently shown that a polyphenol-rich insoluble dietary fibre preparation from carob pulp (Ceratonia siliqua L; carob fibre) decreased postprandial acylated ghrelin, TAG and NEFA during an acute liquid meal challenge test. However, delayed effects of carob fibre consumption are unknown. Therefore, a randomized controlled crossover study in nineteen healthy volunteers consuming foods with or without 50 g carob fibre was conducted. On the subsequent day (day 2), glucose, TAG, total and acylated ghrelin as well as insulin, NEFA and leptin were assessed at baseline and at timed intervals for 300 min after ingestion of standardized bread. Consumption of carob fibre-enriched foods did not affect fasting concentrations of glucose, TAG, total ghrelin, NEFA, insulin and leptin. Fasting acylated ghrelin was increased on the day subsequent to carob fibre consumption compared with control (P = 0.046). After consumption of the standard bread on day 2, glucose response (P = 0.029) was increased, and TAG (P = 0.033) and NEFA (P < 0.001) responses were decreased compared with control. Postprandial responses of total and acylated ghrelin, insulin and leptin on day 2 were unaffected by carob fibre consumption the previous day. In conclusion, an increase in total and acylated plasma ghrelin accompanied by enhanced lipid metabolism after carob fibre consumption suggests higher lipid utilization and suppressed lipolysis on the day subsequent to carob fibre consumption. However, elevated glucose levels after carob fibre consumption need to be addressed in future studies.

Objective: Arabinoxylan (AX) consumption is associated with metabolic improvement during diabetes and with modulation of ghrelin, an orexigenic gut hormone. The effect of AX consumption on ghrelin secretion in disturbed metabolic states is unknown. Therefore, we investigated the postprandial responses to AX consumption of serum glucose, insulin and triglycerides and plasma total and acylated ghrelin in subjects with impaired glucose tolerance (IGT)., Design: Randomized, single-blind, controlled, crossover intervention trial., Subjects: Seven female and four male adults with IGT, aged 55.5 years, and body mass index (BMI) 30.1 kg/m(2)., Intervention: Subjects received either placebo or 15 g AX supplement for 6 weeks with a 6-week washout period in-between., Main Outcome Measurements: Postprandial responses of serum glucose, insulin and triglycerides, and plasma total and acylated ghrelin after a liquid meal challenge test (LMCT) measured at the beginning and at the end of the dietary intervention at -20, -5, 0, 15, 30, 45, 60, 90, 120, 150, 180, 210 and 240 min., Results: After LMCT, AX consumption resulted in lower postprandial responses in serum glucose, insulin and triglycerides (P<0.05). Compared to placebo, total plasma ghrelin was also reduced by 42+/-8 pg/ml (P<0.001) after AX consumption with no difference in plasma acylated ghrelin., Conclusion: AX consumption improved postprandial metabolic responses after an LMCT in subjects with IGT and reduced total ghrelin response. However, acylated ghrelin responses were unchanged, suggesting that the acylated ghrelin-mediated orexigenic regulation is not improved as only total plasma ghrelin decreased.

Wnt/beta-catenin signaling regulates many processes during vertebrate development. To study transcriptional targets of canonical Wnt signaling, we used the conditional Cre/loxP system in mouse to ectopically activate beta-catenin during central nervous system development. We show that the activation of Wnt/beta-catenin signaling in the embryonic mouse telencephalon results in the up-regulation of Sp5 gene, which encodes a member of the Sp1 transcription factor family. A proximal promoter of Sp5 gene is highly evolutionarily conserved and contains five TCF/LEF binding sites that mediate direct regulation of Sp5 expression by canonical Wnt signaling. We provide evidence that Sp5 works as a transcriptional repressor and has three independent repressor domains, called R1, R2, and R3, respectively. Furthermore, we show that the repression activity of R1 domain is mediated through direct interaction with a transcriptional corepressor mSin3a. Finally, our data strongly suggest that Sp5 has the same DNA binding specificity as Sp1 and represses Sp1 target genes such as p21. We conclude that Sp5 transcription factor mediates the downstream responses to Wnt/beta-catenin signaling by directly repressing Sp1 target genes.

The consumption of arabinoxylan, a soluble fibre fraction, has been shown to improve glycemic control in type 2 diabetic subjects. Soluble dietary fibre may modulate gastrointestinal or adipose tissue hormones regulating food intake. The present study investigated the effects of arabinoxylan consumption on serum glucose, insulin, lipids, leptin, adiponectin and resistin in subjects with impaired glucose tolerance. In a randomized, single-blind, controlled, crossover intervention trial, 11 adults consumed white bread rolls as either placebo or supplemented with 15 g arabinoxylan for 6 weeks with a 6-week washout period. Fasting serum glucose, insulin, triglycerides, unesterified fatty acids, apolipoprotein A1 and B, adiponectin, resistin and leptin were assessed before and after intervention. Fasting serum glucose, serum triglycerides and apolipoprotein A-1 were significantly lower during arabinoxylan consumption compared to placebo (p=0.029, p=0.047; p=0.029, respectively). No effects of arabinoxylan were observed for insulin, adiponectin, leptin and resistin as well as for apolipoprotein B, and unesterified fatty acids. In conclusion, the consumption of AX in subjects with impaired glucose tolerance improved fasting serum glucose, and triglycerides. However, this beneficial effect was not accompanied by changes in fasting adipokine concentrations.

Objective: Human resistin has been stated to influence preadipocyte cell numbers and to stimulate adipocyte triglyceride lipolysis in vivo and in vitro. However, its role in human obesity remains unclear., Design: Cross-sectional study for comparisons of lean and obese subjects, and subsequent longitudinal 4-month weight loss intervention study in obese subjects., Subjects: Healthy subjects, lean (n=20, BMI<25) and overweight (n=43, BMI>or=25)., Measurements: Serum resistin, body weight, body fat, waist-to-hip ratio, as well as markers of insulin resistance and lipid metabolism at baseline and after 4 months of intervention., Results: Serum resistin was positively correlated to HOMA-IR (partial r=0.288; P=0.055), serum fructosamines (partial r=0.280; P=0.062), serum NEFA (partial r=0.276; P=0.066) and negatively to age (partial r=-0.349; P=0.019) and serum apolipoprotein A-1 (partial r=-0.363; P=0.014). During the intervention, serum resistin increased significantly (P<0.001). The increase was inversely related to changes in waist-to-hip ratio (P=0.025) and positively to serum apolipoprotein B (P=0.011). In males only, the increase in resistin during weight loss was predicted by total serum cholesterol at baseline (r=0.703, P=0.007). No relation was observed between changes in resistin and changes in HOMA-IR., Conclusion: The present study indicates an association between serum resistin and markers of abdominal fat distribution as well as the regulation of lipid metabolism. However, human resistin is unlikely to play an independent role in the regulation of glucose metabolism.

Ghrelin is an orexigenic hormone that may affect substrate utilization in humans. Ghrelin is influenced by macronutrients, but the effects of insoluble dietary fiber and polyphenols are unknown. We investigated the effects of a polyphenol-rich insoluble dietary fiber preparation from carob pulp (carob fiber) on postprandial ghrelin responses and substrate utilization. Dose-dependent effects of the consumption of carob fiber were investigated in a randomized, single-blind, crossover study in 20 healthy subjects, aged 22-62 y. Plasma total and acylated ghrelin, triglycerides, and serum insulin and nonesterified fatty acids (NEFA) levels were repeatedly assessed before and after ingestion of an isocaloric standardized liquid meal with 0, 5, 10, or 20 g of carob fiber over a 300-min period. The respiratory quotient (RQ) was determined after consumption of 0 or 20 g of carob fiber. Carob fiber intake lowered acylated ghrelin to 49.1%, triglycerides to 97.2%, and NEFA to 67.2% compared with the control meal (P < 0.001). Total ghrelin and insulin concentrations were not affected by consumption of a carob fiber-enriched liquid meal. Postprandial energy expenditure was increased by 42.3% and RQ was reduced by 99.9% after a liquid meal with carob fiber compared with a control meal (P < 0.001). We showed that the consumption of a carob pulp preparation, an insoluble dietary fiber rich in polyphenols, decreases postprandial responses of acylated ghrelin, triglycerides, and NEFA and alters RQ, suggesting a change toward increased fatty acid oxidation. These results indicate that carob fiber might exert beneficial effects in energy intake and body weight.

Two novel calpain inhibitors (A-705239 and A-705253) were studied in isolated perfused rabbit hearts subjected to 60-min occlusion of the ramus interventricularis of the left coronary artery (below the origin of the first diagonal branch), followed by 120 min of reperfusion. The inhibitors were added to the perfusion fluid in various final concentrations from the beginning of the experiments before the coronary artery was blocked. Hemodynamic monitoring and biochemical analysis of perfusion fluid from the coronary outflow were carried out. Myocardial infarct size and the area at risk (transiently non-perfused myocardium) were determined from left ventricular slices after a special staining procedure with Evans blue and 2,3,5-triphenyltetrazolium chloride. The infarcted area (dead myocardium) was 77.9+/-2.3% of the area at risk in untreated controls ( n =12). The infarct size was significantly reduced in the presence of both calpain inhibitors. The best effect was achieved with 10 -8 M A-705253 ( n =8), which reduced ( p <0.001) the infarcted area to 49.3+/-3.9% of the area at risk, corresponding to an infarct reduction of 61.8%. No statistical difference was observed between the experimental groups in coronary perfusion, left ventricular pressure, and in the release of lactate dehydrogenase and creatine kinase from heart muscle.

The effects of the novel calpain inhibitor A-705239 were studied in isolated perfused rabbit hearts subjected to 45 min of global ischemia, followed by 60 min of reperfusion. During 15 min of perfusion the inhibitor accumulated in myocardial tissue up to 16 times the concentration in the perfusate. Almost complete recovery and survival of heart function (90%) was seen with an inhibitor concentration of 10(-8) M in the perfusion fluid when the compound was administered prior to ischemia. Left ventricular pressure amplitude and coronary flow showed significantly higher values during reperfusion in the presence of the inhibitor. A-705239 significantly reduced the release of creatine kinase, from 166+/-49 U/l in untreated hearts to 44+/-10 U/l, and diminished the release of lactate dehydrogenase from 118+/-20 U/l in untreated hearts to 63+/-4 U/l. Mitochondrial dysfunction following ischemia and reperfusion was markedly attenuated by the inhibitor. Thus, the state 3 respiration rate only decreased to 4.2 in contrast to 2.6 nmol O2/(min x mg s.w.) in untreated hearts, reflecting a reduced damage of oxidative phosphorylation. Furthermore, in the presence of the inhibitor the inner mitochondrial membranes became less permeable as indicated by a smaller leak respiration. The excellent properties of A-705239 should make this compound a valuable tool for further pharmacological studies.

Objectives: Heparin is thought to play a crucial role in the clinical monitoring of patients with acute coronary syndrome as well as after coronary bypass surgery in that it interferes with different commercial immunoassay test systems for cardiac troponin T (cTnT) and troponin I (cTnI). The mechanism, however, by which heparin apparently affects the cTnT and cTnI levels in plasma is not yet resolved., Design and Methods: We analyzed the effect of heparin by simultaneously collecting serum and heparin plasma samples from 32 patients after coronary bypass surgery. The cTnT and cTnI levels were determined using the Roche/Elecsys, the Dade-Behring/Opus and the Bayer/ACS:Centaur immunoassay systems in the absence or in the presence of either heparinase or protamine. Association between the cardiac troponins and the anticoagulant has been demonstrated by affinity chromatography using heparin as the ligand., Results: The data obtained indicate that heparin produces an apparent decrease in cTnT as well as of cTnI levels, analyzed either by the Elecsys, the Opus or the ACS:Centaur immunoassay systems. Individual patients show a wide variation in the discrepancies between serum and heparin plasma troponin especially in the cTnT immunoassay. Pretreatment of the heparin plasma samples either with heparinase or protamine cannot completely reverse the heparin-induced decrease in cTnT and cTnI levels and therefore addition of these reagents to the commercial test systems could not significantly improve the performance of the assay. When serum is supplemented with increasing concentrations of heparin, and cardiac troponin levels were reanalysed, significantly lower recoveries for the cTnT than for the cTnI immunoassays were detectable. Affinity chromatography with heparin Sepharose demonstrates that cTnT and cTnI interact differentially with the negatively charged ligand. Whereas cTnI shows minor affinity to the immobilized heparin and is eluted at near physiological conditions, cTnT is bound and can be quantitatively recovered only by solutions of high ionic strength., Conclusions: We conclude, therefore, that the apparent decrease in cTnT values by addition of heparin is a result of direct molecular interaction between the negatively charged glycosaminoglycan and clusters of basic residues within the sequence of the cardiac protein. In contrast, the effect of heparin on the cTnI immunoassay systems, is primarily indirect, most possibly induced by changes within the sample matrix itself., (Copyright 2002 The Canadian Society of Clinical Chemists)

A highly active and soluble glucose-6-phosphatase has been purified to near homogeneity from rat liver. Successful purification has been initiated by covalent labeling of the enzyme in native rat liver microsomes with pyridoxal 5'-phosphate and NaBH4, followed by solubilization of the microsomes with Triton X-100, chromatography on phenyl-Sepharose, hydroxyapatite, DEAE-Sephacel and a second chromatography step on hydroxyapatite. The final enzyme preparation obtained was approximately 700-fold purified over the activity of starting microsomes. As judged by SDS/PAGE the purified glucose-6-phosphatase is composed of a single protein with a molecular mass of 35 kDa. The present work demonstrates that the purified glucose-6-phosphatase must be arranged in the native microsomal membrane so that it is accessible to pyridoxal 5'-phosphate from the cytoplasmic side.

Controlled proteolytic digestion by trypsin or bacterial proteases limited to the cytosolic side of the native microsomal membrane is not efficient to inhibit glucose-6-phosphate hydrolysis. Modification of the microsomes with deoxycholate prior to protease treatment is prerequisite to allow accessibility of the integral protein and inhibition of enzyme activity. Glucose-6-phosphatase of native microsomes, however, is rapidly inactivated by micromolar concentrations of TPCK as well as TLCK. In deoxycholate-modified microsomes both reagents do not affect glucose-6-phosphate hydrolysis. These results indicate that in the native, intact microsomal membrane glucose-6-phosphatase is not accessible to proteolytic attack from the cytoplasmic surface. The putative inhibitory effect of some trypsin or bacterial protease preparations on glucose-6-phosphatase of native microsomes observed most possibly is a result of contaminating agents as TPCK or TLCK.