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ABSTRACT: Background: The introduction of robotic assistance in surgical practice has led to advancements such as the MUSA-2 robotic system that was designed for microsurgical procedures. Advantages of this system include tremor filtration and motion scaling. Initial studies showed promising results in skill acquisition for robot-assisted microsurgery. This study evaluated the learning curve for microsurgical anastomosis with and without robotic assistance among surgeons of varying experience levels. Methods: Fifteen surgeons were divided into 3 groups (novice, intermediate, and expert) based on their microsurgical experience. They performed 10 anastomoses by hand and 10 with robotic assistance on synthetic polyvinyl alcohol vessels (diameter of 2 mm) in a laboratory setting. Participants were timed and mistakes such as backwall and leakage were assessed and recorded. Demographic information was collected. Results: Statistical differences were found in manual anastomosis times between the intermediate and novice groups compared to the experts (p < 0.01). However, no statistical difference was found in the mean time between groups for the robot-assisted anastomoses. Novice doctors had the steepest learning curve for hand-sewn anastomosis. Experts had the fastest completion time at the end of the 10th robotic session, finishing at 14 min, compared to 33 min at the 2nd session. All groups reduced their mean time in half through their 10 robotic sessions. Conclusion: This study indicated similarities in the learning curves for robot-assisted anastomosis among surgeons with varied experience levels. Experts excelled technically in manual anastomoses, but robot-assistance enabled novice and intermediate surgeons to perform comparably to the experts. Robotic assistance may aid more novice learners in performing microsurgical anastomosis safely at earlier points in their education.

Abstract Pediatric obstructive sleep apnea poses a significant health risk, with potential long‐term consequences on cardiovascular health. This study explores the dichotomous nature of neonatal cardiac response to chronic intermittent hypoxia (CIH) between males and females, aiming to fill a critical knowledge gap in the understanding of sex‐specific cardiovascular consequences of sleep apnea in early life. Neonates were exposed to CIH until p28 and underwent comprehensive in vivo physiological assessments, including whole‐body plethysmography, treadmill stress‐tests, and echocardiography. Results indicated that male CIH rats weighed 13.7% less than age‐matched control males (p = 0.0365), while females exhibited a mild yet significant increased respiratory drive during sleep (93.94 ± 0.84 vs. 95.31 ± 0.81;p = 0.02). Transcriptomic analysis of left ventricular tissue revealed a substantial sex‐based difference in the cardiac response to CIH, with males demonstrating a more pronounced alteration in gene expression compared to females (5986 vs. 3174 genes). The dysregulated miRNAs in males target metabolic genes, potentially predisposing the heart to altered metabolism and substrate utilization. Furthermore, CIH in males was associated with thinner left ventricular walls and dysregulation of genes involved in the cardiac action potential, possibly predisposing males to CIH‐related arrhythmia. These findings emphasize the importance of considering sex‐specific responses in understanding the cardiovascular implications of pediatric sleep apnea.

Abstract Linking clinical multi-omics with mechanistic studies may improve the understanding of rare cancers. We leverage two precision oncology programs to investigate rhabdomyosarcoma with FUS/EWSR1-TFCP2 fusions, an orphan malignancy without effective therapies. All tumors exhibit outlier ALK expression, partly accompanied by intragenic deletions and aberrant splicing resulting in ALK variants that are oncogenic and sensitive to ALK inhibitors. Additionally, recurrent CKDN2A/MTAP co-deletions provide a rationale for PRMT5-targeted therapies. Functional studies show that FUS-TFCP2 blocks myogenic differentiation, induces transcription of ALK and truncated TERT, and inhibits DNA repair. Unlike other fusion-driven sarcomas, TFCP2-rearranged tumors exhibit genomic instability and signs of defective homologous recombination. DNA methylation profiling demonstrates a close relationship with undifferentiated sarcomas. In two patients, sarcoma was preceded by benign lesions carrying FUS-TFCP2, indicating stepwise sarcomagenesis. This study illustrates the potential of linking precision oncology with preclinical research to gain insight into the classification, pathogenesis, and therapeutic vulnerabilities of rare cancers.

Abstract Background District Heating (DH) is a technology that provides heating and domestic hot water to buildings and is an important technology for supporting the European energy transition. As such the heating systems increasingly resort to renewable heat sources and waste heat, it is even more important that they operate in the most efficient way possible. DH companies have access to the primary network of which they can impact system performance. To maximize the efficiency of the system, however, it is important that the system at the building level, known as the secondary system, is also efficient; otherwise, overall system efficiency is reduced. To increase system efficiency, return temperatures from the secondary system into the primary system have been targeted through motivational tariffs. There is limited information on how to establish a motivational tariff that motivates the customer to improve both the primary and secondary systems, which is a gap that this paper aims to fill. Results In this paper, the impacts of retrofit actions in secondary systems are assessed through simulations. The identified relevant refurbishment measures to lower the return temperature to the primary system are variable flow pumps, low-temperature radiators, parallel heat exchangers, and a pass-through DHW system. Apart from simulated refurbishments, we also identify that the secondary system sometimes generates excess heat, which is valuable to recover, especially during peak load periods for the primary system. Hence, motivational tariffs targeting secondary system efficiency should also encompass an incentive for the customer to make use of waste heat in the secondary circuit to lower peak demand for the DH system. Conclusions To date, the most commonly used parameters introduced to customers are linked to the flow of water through the customer’s asset and the bonus malus principle. The results from simulations show that DH companies can introduce additional parameters to support customers in guiding their secondary system to perform more efficiently. Increased overall system efficiency has a positive impact on both costs and emissions.

Abstract Prosaposin (PSAP) modulates glycosphingolipid metabolism and variants have been linked to Parkinson’s disease (PD). Here, we find altered PSAP levels in the plasma, CSF and post-mortem brain of PD patients. Altered plasma and CSF PSAP levels correlate with PD-related motor impairments. Dopaminergic PSAP-deficient (cPSAPDAT) mice display hypolocomotion and depression/anxiety-like symptoms with mildly impaired dopaminergic neurotransmission, while serotonergic PSAP-deficient (cPSAPSERT) mice behave normally. Spatial lipidomics revealed an accumulation of highly unsaturated and shortened lipids and reduction of sphingolipids throughout the brains of cPSAPDAT mice. The overexpression of α-synuclein via AAV lead to more severe dopaminergic degeneration and higher p-Ser129 α-synuclein levels in cPSAPDAT mice compared to WT mice. Overexpression of PSAP via AAV and encapsulated cell biodelivery protected against 6-OHDA and α-synuclein toxicity in wild-type rodents. Thus, these findings suggest PSAP may maintain dopaminergic lipid homeostasis, which is dysregulated in PD, and counteract experimental parkinsonism.

Abstract Metabolism of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) to the neurotoxin MPP+ in the brain causes permanent Parkinson’s disease-like symptoms by destroying dopaminergic neurons in the pars compacta of the substantia nigra in humans and non-human primates. However, the complete molecular pathology underlying MPTP-induced parkinsonism remains poorly understood. We used dual polarity matrix-assisted laser desorption/ionization mass spectrometry imaging to thoroughly image numerous glycerophospholipids and sphingolipids in coronal brain tissue sections of MPTP-lesioned and control non-human primate brains (Macaca mulatta). The results revealed specific distributions of several sulfatide lipid molecules based on chain-length, number of double bonds, and importantly, hydroxylation stage. More specifically, certain long-chain hydroxylated sulfatides with polyunsaturated chains in the molecular structure were depleted within motor-related brain regions in the MPTP-lesioned animals, e.g., external and internal segments of globus pallidus and substantia nigra pars reticulata. In contrast, certain long-chain non-hydroxylated sulfatides were found to be elevated within the same brain regions. These findings demonstrate region-specific dysregulation of sulfatide metabolism within the MPTP-lesioned macaque brain. The depletion of long-chain hydroxylated sulfatides in the MPTP-induced pathology indicates oxidative stress and oligodendrocyte/myelin damage within the pathologically relevant brain regions. Hence, the presented findings improve our current understanding of the molecular pathology of MPTP-induced parkinsonism within primate brains, and provide a basis for further research regarding the role of dysregulated sulfatide metabolism in PD.

Abstract The honey bee microbiota is involved in several important functions, and alterations in the composition could have a severe effect on honey bee health. Among the bacteria identified in the honey bee microbiome are a group of non‐pathogenic honey bee‐specific lactic acid bacteria (hbs‐LAB) that have been shown to inhibit the growth of bacterial pathogens such as Paenibacillus larvae, the causative agent of American foulbrood (AFB). While P. larvae only causes disease in larvae and not in adult honey bees, there are reports of the pathogen causing changes in the microbiota composition of the adults. The aim of this study was to investigate how AFB in the colony affect the hbs‐LAB composition in adult honey bees. Adult bees were collected from colonies with and without AFB during three outbreaks of AFB in Sweden. The hbs‐LAB was analyzed using qPCR to detect and quantify the number of ten hbs‐LAB (five Lactobacilli, two Apilactobacilli, one Bombilactobacilli, and two Bifidobacterium). The hbs‐LAB composition was compared between AFB outbreaks and depending on the AFB status of the honeybee colony at the time of sampling. The data analyses revealed differences in the abundance of individual hbs‐LAB between outbreaks and an overall difference in bacterial community composition depending on AFB status. Also, a higher hbs‐LAB diversity was observed in samples that were P. larvae culture positive.

Inotuzumab Ozogamicin (InO) is a CD22-directed antibody conjugated with calicheamicin. The Phase 1B of the ITCC-059 trial tested InO combined with chemotherapy in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Relapsed /refractory CD22+ BCP-ALL pediatric patients were enrolled. The primary objective was to establish the Recommended Phase 2 Dose (RP2D). Secondary objectives included preliminary efficacy and tolerability. InO was combined with 1.5 mg/m2 of vincristine (days 3, 10, 17, 24), 20 mg/m2 of dexamethasone (two 5-day blocks, then amended), and intrathecal therapy. A rolling-6 design was used testing InO from 0.8 to 1.8 mg/m2/cycle. Between May-2020 and Apr-2022, 30 patients were treated, and 29 were evaluable for dose limiting toxicities (DLTs). At 1.1 mg/m2/cycle, two out of four patients had DLTs (liver toxicity). InO was de-escalated to 0.8 mg/m2/cycle (n=6) without DLTs while awaiting a protocol amendment to reduce dexamethasone dose to 10 mg/m2. Post amendment, InO was re-escalated to 1.1 mg/m2/cycle (n=6, 1 DLT), then to 1.4 mg/m2/cycle (n=3, no DLTs), and finally to 1.8 mg/m2/cycle (n=7, 1 DLT). Three additional patients were treated in an expansion cohort. The pooled response rate was 80% (24/30; 95%CI: 61.4% to 92.3%) and, among responders, 66.7% achieved minimal residual disease negativity. The RP2D of InO combined with vincristine, dexamethasone and IT therapy was declared at 1.8 mg/m2/cycle (1.5 mg/m2/cycle after remission) in a fractionated schedule. This combination showed an response rate similar to the single agent cohorts of this trial, with liver toxicity issues at the initial higher dexamethasone dose. #NTR5736

BackgroundAdvances in treatment of childhood malignancies have improved overall cure rates to 80%. Nevertheless, cancer is still the most common cause of childhood mortality in Sweden. The prognosis is particularly poor for relapse of high-risk malignancies. In the international INFORM registry, tumor tissue from patients with relapsed, refractory, or progressive pediatric cancer as well as from very-high risk primary tumors is biologically characterized using next-generation sequencing to identify possible therapeutic targets. We analyzed data from Swedish children included in the INFORM registry concerning patient characteristics, survival, sequencing results and whether targeted treatment was administered to the children based on the molecular findings.MethodsA registry-based descriptive analysis of 184 patients included in the INFORM registry in Sweden during 2016–2021.ResultsThe most common diagnoses were soft tissue and bone sarcomas followed by high grade gliomas [including diffuse intrinsic pontine glioma (DIPG)]. Complete molecular analysis was successful for 203/212 samples originating from 184 patients. In 88% of the samples, at least one actionable target was identified. Highly prioritized targets, according to a preset scale, were identified in 48 (24%) samples from 40 patients and 24 of these patients received matched targeted treatment but only six children within a clinical trial. No statistically significant benefit in terms of overall survival or progression free survival was observed between children treated with matched targeted treatment compared to all others.ConclusionThis international collaborative study demonstrate feasibility regarding sequencing of pediatric high-risk tumors providing molecular data regarding potential actionable targets to clinicians. For a few individuals the INFORM analysis was of utmost importance and should be regarded as a new standard of care with the potential to guide targeted therapy.

Abstract Investigating organ biology often requires methodologies to induce genetically distinct clones within a living tissue. However, the 3D nature of clones makes sample image analysis challenging and slow, limiting the amount of information that can be extracted manually. Here we develop PECAn, a pipeline for image processing and statistical data analysis of complex multi-genotype 3D images. PECAn includes data handling, machine-learning-enabled segmentation, multivariant statistical analysis, and graph generation. This enables researchers to perform rigorous analyses rapidly and at scale, without requiring programming skills. We demonstrate the power of this pipeline by applying it to the study of Minute cell competition. We find an unappreciated sexual dimorphism in Minute cell growth in competing wing discs and identify, by statistical regression analysis, tissue parameters that model and correlate with competitive death. Furthermore, using PECAn, we identify several genes with a role in cell competition by conducting an RNAi-based screen.

ObjectivesThis study aimed to investigate the etiology, clinical features, and outcomes of community-acquired pneumonia (CAP) in adults. Understanding the causative pathogens is essential for effective treatment and prevention.DesignBetween 2016–2018, 518 hospitalized adults with CAP and 241 controls without symptoms were prospectively enrolled. Urine samples were collected for pneumococcal urinary antigen tests and nasopharyngeal swabs for viral and bacterial analysis, combined with routine diagnostic care.ResultsAmong the included CAP patients, Streptococcus pneumoniae was the most common pathogen, detected in 28% of patients, followed by Haemophilus influenzae in 16%. Viruses were identified in 28%, and concurrent viruses and bacteria were detected in 15%. There was no difference in mortality, length of stay, or symptoms at hospitalization when comparing patients with bacterial, viral, or mixed etiologies. Among the control subjects without respiratory symptoms, S. pneumoniae, H. influenzae, or Moraxella catarrhalis were detected in 5–7%, and viruses in 7%.ConclusionStreptococcus pneumoniae emerged as the predominant cause of CAP, followed closely by viruses and H. influenzae. Intriguingly, symptoms and outcome were similar regardless of etiology. These findings highlight the complexity of this respiratory infection and emphasize the importance of comprehensive diagnostic and treatment strategies.Clinical Trial Registration: ClinicalTrials.gov, identifier [NCT03606135].

Abstract With modern treatment most children with acute lymphoblastic leukemia (ALL) survive without relapse. However, for children who relapse the prognosis is still poor, especially in children with T‐cell phenotype (T‐ALL) and remains the major cause of death. The exact mechanism of relapse is currently not known. While contribution of RNA processing alteration has been linked to other hematological malignancies, its contribution in pediatric T‐ALL may provide new insights. Almost all human genes express more than one alternative splice isoform. Thus, gene modulation producing a diverse repertoire of the transcriptome and proteome have become a significant molecular marker of cancer and a potential therapeutic vulnerability. To study this, we performed RNA‐sequencing analysis on patient‐derived samples followed by splice isoform‐specific PCR. We uncovered a distinct RNA splice isoform expression pattern characteristic for relapse samples compared to the leukemia samples from the time of diagnosis. We also identified deregulated splicing and apoptosis pathways specific for relapse T‐ALL. Moreover, patients with T‐ALL displayed pro‐survival splice isoform switching favoring pro‐survival isoforms compared to normal healthy stem cells. Cumulatively, pro‐survival isoform switching and DFFB isoform regulation of SOX2 and MYCN may play a role in T‐ALL proliferation and survival, thus serving as a potential therapeutic option.

Abstract L-DOPA administration is the primary treatment for Parkinson’s disease (PD) but long-term administration is usually accompanied by hyperkinetic side-effects called L-DOPA-induced dyskinesia (LID). Signaling neuropeptides of the basal ganglia are affected in LID and changes in the expression of neuropeptide precursors have been described, but the final products formed from these precursors have not been well defined and regionally mapped. We therefore used mass spectrometry imaging to visualize and quantify neuropeptides in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine exposed parkinsonian and LID Macaca mulatta brain samples. We found that dyskinesia severity correlated with the levels of some abnormally processed peptides — notably, des-tyrosine dynorphins, substance P (1-7), and substance P (1-9) — in multiple brain regions. Levels of the active neuropeptides; dynorphin B, dynorphin A (1-8), α-neoendorphin, substance P (1-11), and neurokinin A, in the globus pallidus and substantia nigra correlated with putaminal levels of L-DOPA. Our results demonstrate that the abundance of selected active neuropeptides is associated with L-DOPA concentrations in the putamen, emphasizing their sensitivity to L-DOPA. Additionally, levels of truncated neuropeptides (which generally exhibit reduced or altered receptor affinity) correlate with dyskinesia severity, particularly for peptides associated with the direct pathway (i.e., dynorphins and tachykinins). The increases in tone of the tachykinin, enkephalin, and dynorphin neuropeptides in LID result in abnormal processing of neuropeptides with different biological activity and may constitute a functional compensatory mechanism for balancing the increased L-DOPA levels across the whole basal ganglia.

Abstract Plants employ different chemicals to protect themselves from herbivory. These defenses may be constitutive or triggered by stress. The chemicals can be toxic, act as repellents, phagosuppressants and/or phago-deterrents. The two-spotted spider mite (Tetranychus urticae) is a generalist arthropod herbivorous pest and its feeding causes extensive damage both to crops and wild plants. Cyclotides are cyclic peptides involved in host-plant defenses. A single Viola sp. can produce more than a hundred cyclotides with different biological activities and roles. The organ and tissue specific cyclotide patterns change over the seasons and/or with environment, but the role of biotic/abiotic stress in shaping them remains unclear. Here, we demonstrate the involvement of cyclotides in mutual interactions between violets and mites. We used immunohistochemistry and mass spectrometry imaging to show the ingested cyclotides in T. urticae and assess the Viola odorata response to mite feeding. Moreover, to assess how mites are affected by feeding on violets, acceptance and reproductive performance was compared between Viola uliginosa, V. odorata and Phaseolus vulgaris. We demonstrate that cyclotides had been taken in by mites feeding on the violets. The ingested peptides were found in contact with epithelial cells of the mite digestive system, in the fecal matter, feces, ovary and eggs. Mites preferred common bean plants (P. vulgaris) to any of the violet species; the latter affected their reproductive performance. The production of particular cyclotides in V. odorata (denoted by molecular weights: 2979, 3001, 3017, 3068, 3084, 3123) was activated by mite feeding and their levels were significantly elevated compared to the control after 5 and 21 days of infestation. Specific cyclotides may affect mites by being indigestible or through direct interaction with cells in the mite digestive tract and reproductive organs. A group of particular peptides in V. odorata appears to be involved in defense response against herbivores.

BackgroundPost-covid syndrome is an emerging condition involving a wide range of symptoms, including high rates of poor mental health. The diagnostic relevance and clinical severity of these symptoms are largely unknown, and evidence for treatment of post-covid mental health symptoms is lacking. This protocol describes a pilot randomized clinical trial, primarily aiming to assess feasibility, participant adherence and satisfaction in a novel phycho-therapeutic intervention on post-covid anxiety and depression symptoms ≥1 year after critically ill COVID-19. Whether the intervention may generate improvements in post-covid depression, anxiety, post-traumatic stress and health-related quality of life (HRQoL) will be addressed in a following larger trial.MethodsA multicenter, investigator-initiated randomized controlled trial (Clinical Trial Identifier number NCT05119608) including Intensive Care Unit (ICU)-treated COVID-19 survivors, who display symptoms of anxiety and/or depression at follow-up 12 months after hospitalization (Hospital Anxiety and Depression Scale ≥8 for depression or anxiety). Eligible individuals are referred to a psychiatrist for structured diagnostic assessment and inclusion in the trial. Participants will be randomized to either a 10-week cognitive behavioral therapy intervention with added acceptance and commitment therapy (CBT-ACT) or standard care (primary care referral). Primary study outcome measure is feasibility and patient adherence, defined as the proportion of participants who consent to randomization and remain in the study including follow-up. Secondary outcome measures include reduced symptoms in the HADS depression/anxiety subscales, post-traumatic symptoms, HRQoL and user satisfaction at 3 months after the intervention.DiscussionThis protocol describes a pilot trial to assess feasibility and preliminary effects of a structured psycho-therapeutic intervention to ameliorate mental health in a population severely affected by COVID-19, where evidence for structured psycho-therapy is lacking.

Paenibacillus larvae is the etiological agent of American Foulbrood (AFB), a highly contagious and worldwide spread bacterial disease that affects honeybee brood. In this study, all complete P. larvae genomes available on the NCBI database were analyzed in order to detect presence of prophages using the PHASTER software. A total of 55 intact prophages were identified in 11 P. larvae genomes (5.0 ± 2.3 per genome) and were further investigated for the presence of genes encoding relevant traits related to P. larvae. A closer look at the prophage genomes revealed the presence of several putative genes such as metabolic and antimicrobial resistance genes, toxins or bacteriocins, potentially influencing host performance. Some of the coding DNA sequences (CDS) were present in all ERIC-genotypes, while others were only found in a specific genotype. While CDS encoding toxins and antitoxins such as HicB and MazE were found in prophages of all bacterial genotypes, others, from the same category, were provided by prophages particularly to ERIC I (enhancin-like toxin), ERIC II (antitoxin SocA) and ERIC V strains (subunit of Panton-Valentine leukocidin system (PVL) LukF-PV). This is the first in-depth analysis of P. larvae prophages. It provides better knowledge on their impact in the evolution of virulence and fitness of P. larvae, by discovering new features assigned by the viruses.

Abstract Cyclotides are cyclic peptides produced by plants. Due to their insecticidal properties, they are thought to be involved in host defense. Violets produce complex mixtures of cyclotides, that are characteristic for each species and variable in different environments. Herein, we utilized mass spectrometry (LC–MS, MALDI-MS), transcriptomics and biological assays to investigate the diversity, differences in cyclotide expression based on species and different environment, and antimicrobial activity of cyclotides found in violets from the Canary Islands. A wide range of different habitats can be found on these islands, from subtropical forests to dry volcano peaks at high altitudes. The islands are inhabited by the endemic Viola palmensis, V. cheiranthifolia, V. anagae and the common V. odorata. The number of cyclotides produced by a given species varied in plants from different environments. The highest diversity was noted in V. anagae which resides in subtropical forest and the lowest in V. cheiranthifolia from the Teide volcano. Transcriptome sequencing and LC–MS were used to identify 23 cyclotide sequences from V. anagae. Cyclotide extracts exhibited antifungal activities with the lowest minimal inhibitory concentrations noted for V. anagae (15.62 μg/ml against Fusarium culmorum). The analysis of the relative abundance of 30 selected cyclotides revealed patterns characteristic to both species and populations, which can be the result of genetic variability or environmental conditions in different habitats. The current study exemplifies how plants tailor their host defense peptides for various habitats, and the usefulness of cyclotides as markers for chemosystematics.

Febrile neutropenia is a common complication during chemotherapy in paediatric cancer care. In this setting, clinical features and current diagnostic tests do not reliably distinguish between bacterial and viral infections. Children with cancer (n = 63) presenting with fever and neutropenia were recruited for extensive microbiological and blood RNA sampling. RNA sequencing was successful in 43 cases of febrile neutropenia. These were classified as having probable bacterial infection (n = 17), probable viral infection (n = 13) and fever of unknown origin (n = 13) based on microbiological defined infections and CRP cut-off levels. RNA expression data with focus on the 2-transcript signature (FAM89A and IFI44L), earlier shown to identify bacterial infections with high specificity and sensitivity, was implemented as a disease risk score. The median disease risk score was higher in the probable bacterial infection group, −0.695 (max 2.795; min −5.478) compared to the probable viral infection group −3.327 (max 0.218; min −7.861), which in ROC analysis corresponded to a sensitivity of 0.88 and specificity of 0.54 with an AUC of 0.80. To further characterise the immune signature, analysis of significantly expressed genes and pathways was performed and upregulation of genes associated to antibacterial responses was present in the group classified as probable bacterial infection. Our results suggest that the 2-transcript signature may have a potential use as a diagnostic tool to identify bacterial infections in immunosuppressed children with febrile neutropenia.

Various subsets of bone marrow mesenchymal stromal cells (BM MSCs), including fibroblasts, endothelial, fat and reticular cells, are implicated in the regulation of the hematopoietic microenvironment and the survival of long-lived antibody-secreting cells (ASCs). Nowadays it is widely acknowledged that vaccine-induced protective antibody levels are diminished in adults and children that are treated for hematological cancers. A reason behind this could be damage to the BM MSC niche leading to a diminished pool of ASCs. To this end, we asked whether cell cytotoxic treatment alters the capacity of human BM MSCs to support the survival of ASCs. To investigate how chemotherapy affects soluble factors related to the ASC niche, we profiled a large number of cytokines and chemokines from in vitro-expanded MSCs from healthy donors or children who were undergoing therapy for acute lymphoblastic leukemia (ALL), following exposure to a widely used anthracycline called doxorubicin (Doxo). In addition, we asked if the observed changes in the measured soluble factors after Doxo exposure impacted the ability of the BM niche to support humoral immunity by co-culturing Doxo-exposed BM MSCs with in vitro-differentiated ASCs from healthy blood donors, and selective neutralization of cytokines. Our in vitro results imply that Doxo-induced alterations in BM MSC-derived interleukin 6 (IL-6), CXCL12 and growth and differentiation factor 15 (GDF-15) are not sufficient to disintegrate the support of IgG-producing ASCs by the BM MSC niche, and that serological memory loss may arise during later stages of ALL therapy.

Paenibacillus larvae is a spore-forming bacterium causing American foulbrood (AFB) in honey bee larvae. The remains of a diseased larva contains billions of extremely resilient P. larvae spores viable for decades. Burning clinically symptomatic colonies is widely considered the only workable strategy to prevent further spread of the disease, and the management practices used for decontamination requires high concentrations of chemicals or special equipment. The aim of this study was to test and compare the biocidal effect of two commercially available disinfectants, “Disinfection for beekeeping” and Virkon S on P. larvae. The two products were applied to P. larvae spores in suspension as well as inoculated on two common beehive materials, wood and Styrofoam. “Disinfection for beekeeping” had a 100 % biocidal effect on P. larvae spores in suspension compared to 87.0–88.6% for Virkon S which, however, had a significantly better effect on P. larvae on Styrofoam. The two disinfectants had similar effect on infected wood material.

Abstract Background The Pediatric Quality of Life Inventory (PedsQL) is a modular instrument, designed to integrate generic and disease specific measures, and includes both self- and proxy-reports. The aim of the study was to assess the reliability and limited validity of the Swedish version of the disease specific Pediatric Quality of Life Inventory 3.0 Cancer Module Scales (PedsQL 3.0), in a sample of Swedish children diagnosed with cancer. Method A total of 94 families at The Queen Silvia Children’s Hospital, Sahlgrenska University participated in the study. The Pediatric Quality of Life Inventory 4.0 Generic Core Scales (PedsQL 4.0) and the PedsQL 3.0 were administered to 63 children (aged 5–18 years) with cancer and to 94 parents of children with cancer aged 2–18 years. Results The internal consistency of the PedsQL 3.0, reached or exceeded Cronbach’s alpha values of 0.70 for both -self- and proxy-reports. The PedsQL 4.0 and PedsQL 3.0 were highly correlated (r = 0.94 for proxy-reports and r = 0.91 for self-reports), indicating convergent validity. Conclusion PedsQL 3.0 Cancer Module Scales can be used as a valuable tool for measuring cancer-specific HRQOL in child populations, both in research and in clinical practice.

Proportionality reasoning is an established form of legal argumentation under international human rights law, employed by the European Court of Human Rights and the United Nations (UN) human rights treaty bodies alike. However, relatively little has been written about its precise role and content in relation to equality norms. Proportionality scholars tend to draw on other examples to demonstrate how proportionality reasoning works in practice, and legal scholarship on equality and non-discrimination has not fully explored whether or how proportionality argumentation can assist us in distinguishing lawful state practices from unlawful ones. This article picks up these loose ends and develops a model of proportionality assessment tailored to the non-discrimination context. The model breaks down proportionality argumentation into a step-by-step process and sets out clear criteria to be fulfilled at each step. It illustrates the distinctive features of balancing as a part of discrimination analysis and provides useful guidance to national authorities tasked with such balancing. It is anchored in existing non-discrimination jurisprudence but structured so as to facilitate more predictable outcomes than existing justification tests.

Individuals aged 65 years and above are at increased risk of complications and death from influenza compared with any other age group. Enhanced vaccines, as the MF59®-adjuvanted quadrivalent influenza vaccine (aQIV) and the high-dose quadrivalent influenza vaccine (HD-QIV), provide increased protection for older adults in comparison to the traditional standard-dose quadrivalent influenza vaccines (SD-QIV). This study aimed to assess the cost-effectiveness of aQIV compared to SD-QIV and HD-QIV in Denmark, Norway, and Sweden for adults aged ≥65 years. A static decision tree model was used to evaluate costs and outcomes of different vaccination strategies from healthcare payer and societal perspectives. This model projects that compared to SD-QIV, vaccination with aQIV could prevent a combined total of 18,772 symptomatic influenza infections, 925 hospitalizations, and 161 deaths in one influenza season across the three countries. From a healthcare payer perspective, the incremental costs per quality adjusted life year (QALY) gained with aQIV versus SD-QIV were EUR 10,170/QALY in Denmark, EUR 12,515/QALY in Norway, and EUR 9894/QALY in Sweden. The aQIV was cost saving compared with HD-QIV. This study found that introducing aQIV to the entire population aged ≥65 years may contribute to reducing the disease and economic burden associated with influenza in these countries.

Background: The clinical-stage drug candidate EBL-1003 (apramycin) represents a distinct new subclass of aminoglycoside antibiotics for the treatment of drug-resistant infections. It has demonstrated best-in-class coverage of resistant isolates, and preclinical efficacy in lung infection models. However, preclinical evidence for its utility in other disease indications has yet to be provided. Here we studied the therapeutic potential of EBL-1003 in the treatment of complicated urinary tract infection and acute pyelonephritis (cUTI/AP). Methods: A combination of data-base mining, antimicrobial susceptibility testing, time-kill experiments, and four murine infection models was used in a comprehensive assessment of the microbiological coverage and efficacy of EBL-1003 against Gram-negative uropathogens. The pharmacokinetics and renal toxicology of EBL-1003 in rats was studied to assess the therapeutic window of EBL-1003 in the treatment of cUTI/AP. Findings: EBL-1003 demonstrated broad-spectrum activity and rapid multi-log CFU reduction against a phenotypic variety of bacterial uropathogens including aminoglycoside-resistant clinical isolates. The basicity of amines in the apramycin molecule suggested a higher increase in positive charge at urinary pH when compared to gentamicin or amikacin, resulting in sustained drug uptake and bactericidal activity, and consequently in potent efficacy in mouse infection models. Renal pharmacokinetics, biomarkers for toxicity, and kidney histopathology in adult rats all indicated a significantly lower nephrotoxicity of EBL-1003 than of gentamicin. Interpretation: This study provides preclinical proof-of-concept for the efficacy of EBL-1003 in cUTI/AP. Similar efficacy but lower nephrotoxicity of EBL-1003 in comparison to gentamicin may thus translate into a higher safety margin and a wider therapeutic window in the treatment of cUTI/API. Funding: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Abstract Background Campylobacter colonise the gastrointestinal tract of warm-blooded animals and are major enteropathogens in humans. C. coli is less common than C. jejuni and accounts for about 10% of the total number of Campylobacter infections although the two species seem to share many virulence determinants. Campylobacter bacteraemia is rare, estimated to occur in less than 1% of the infections, and the exact mechanisms regulating the progression of the infection from the gastrointestinal tract to the blood stream are unclear. Here, we looked at the contribution of C. coli to Campylobacter infections and further compared various virulence traits in C. coli clade 1 blood and stool isolates. Results We assessed the numbers of C. jejuni and C. coli among typed isolates in the PubMLST database and found that C. coli accounted for 25.9% of blood isolates, but only 8.9% of the stool isolates. Phylogenetic analysis of 128 C. coli clade 1 whole genome sequences deposited to NCBI revealed no specific clustering of the human blood, stool or animal isolates. Of the six C. coli isolates chosen for phenotypic analyses, stool isolates adhered significantly better to human HT-29 colon cancer cells than the blood isolates, while there was no difference in induced IL-8 levels between the isolates. Furthermore, the stool isolates had two- to fourfold higher RNA expression levels of the flpA, ciaB, iamA and cdt virulence genes than the blood isolates. Finally, we looked at the gene structure of the cdtA, B and C toxin genes and found numerous nucleotide additions and deletions disrupting the open reading frames. In contrast to 58% isolates of animal origin, only 38% and 32% of human blood and stool isolates, respectively, had all three cdt genes intact, a prerequisite to produce functional toxins. Conclusions This study reveals interesting differences between C. coli clade 1 isolates of human and animal origin on one hand, and also between human blood and stool isolates, on the other. The results suggest that C. coli might downregulate and/or inactivate various virulence determinants as the isolates pass from the animal host to the human gastrointestinal tract and enter the human blood stream.

Cyclotides are macrocycle peptides produced by plants from several families, including Violaceae. These compounds have the potential for applications in medicine, bioengineering and crop protection thanks to their multiple biological activities. In most cases, cyclotides are extracted from plant material. Plant cell culture provides a viable and sustainable form of plant biomass production Cyclotides are host defense peptides. The aim of the current study was to test whether different plant stress hormones and biological elicitors have effects on cyclotide production in Viola uliginosa suspension cultures. Different concentrations of jasmonic acid (JA), salicylic acid (SA), abscisic acid (ABA) and neutralized pathogens were tested. The cyclotide production was assessed using MALDI-MS. Five major peptides produced by V. uliginosa cultures were chosen for analysis, of which one was sequenced de novo. The treatments had little influence on the suspension’s growth, with the exception of 100 μM SA, which enhanced the biomass increase, and 100 μM ABA, which was toxic. Significant increases in the production of three cyclotides (viul M, cyO13 and cyO3) were observed in suspensions primed with JA (50 μM, 100 μM, 200 μM) after 14 days of culturing. Biotic elicitors had no observable effect on cyclotide production. The current study indicates that some cyclotides in V. uliginosa are triggered in response to JA. The stress plant hormones can be used to enhance plant cell culture-based production systems.

The immune system plays a major role in recognizing and eliminating malignant cells, and this has been exploited in the development of immunotherapies aimed at either activating or reactivating the anti-tumor activity of a patient's immune system. A wide range of therapeutic approaches involving T lymphocytes, such as programmed cell death protein ligand-1 (PDL-1) inhibitors, cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) blockers, and CD19-targeted T-cell therapy through chimeric antigen receptor (CAR)-T cells or CD19/CD3 bi-specific T-cell engagers, have been introduced to the field of oncology, leading to significant improvements in overall survival of adult cancer patients. During the past few years, the availability and approval of T-cell based immunotherapies have become a reality also for the treatment of childhood cancers. However, the distribution, ratio of regulatory to effector cells and the quality of T-cell responses early in life are distinct from those during adolescence and adulthood, raising the possibility that these differences impact the efficacy of immunotherapy. Herein we provide a brief overview of the properties of conventional T cell subsets during early life. Focusing on the most common cancer type during childhood, acute lymphoblastic leukemia (ALL), we describe how current conventional therapies used against ALL influence the T-cell compartment of small children. We describe early life T-cell responses in relation to immunotherapies engaging T-cell anticancer reactivity and present our opinion that it is not only immaturity of the adaptive immune system, but also the impact of an immunosuppressive environment that may prove disadvantageous in the setting of immunotherapies targeting pediatric cancer cells.

Neuropeptides are important signalling molecules in the brain and alterations in their expression levels have been linked to neurological disorders such as Parkinson's disease. It is challenging to map neuropeptide changes across and within brain regions because of their low in vivo concentrations and complex post-translational processing. Consequently, the role of neuropeptides in Parkinson's disease is not well understood. Thus, we have developed and evaluated a method to image multiple neuropeptides simultaneously in both rat and primate brain tissue sections by matrix-assisted laser desorption/ionisation mass spectrometry imaging at high lateral resolution. Using a unilateral 6-hydroxydopamine rat model of Parkinson's disease, we imaged changes in enkephalins, dynorphins, tachykinins and neurotensin associated with the dopaminergic denervation and L-DOPA treatment in multiple brain regions. L-DOPA administration significantly affected neuropeptides in the globus pallidus, while neuropeptides in the caudate-putamen were mostly affected by dopamine depletion. Using high lateral resolution imaging, we observed an increase of neurotensin in the dorsal sub-region of the globus pallidus after dopamine depletion. This study highlights the capacity of mass spectrometry imaging to elucidate the dynamics of neuropeptide signalling during Parkinson's disease and its treatment.

Abstract Objectives Loss of vaccine‐induced antibodies (Abs) after chemotherapy against paediatric acute lymphoblastic leukaemia (ALL) is common and often necessitates re‐immunisation after cessation of treatment. Even so, some ALL survivors fail to mount or to maintain protective Abs. Germinal centres (GCs) are clusters of proliferating B cells in follicles of secondary lymphoid tissues (SLTs) formed during adaptive immune responses and the origins of long‐lived memory B and plasma cells that are the source of Abs. Furthermore, productive GC reactions depend on T follicular helper (TFH) cells. To understand why chemotherapy induces deficits in Ab responses, we examined how SLTs were affected by chemotherapy. Methods Rhesus macaques were infused with either three cycles of the anthracycline doxorubicin or saline, followed by immunisation with a de novo and booster antigen. Spleen and lymph nodes were removed, and memory B, bulk T and TFH cells were examined. Results Despite adequate GC morphology, a diminished memory and IgG+ B‐cell population along with diminished total and booster vaccine‐specific IgG‐producing memory B cells were noted in the spleens of macaques with past doxorubicin exposure compared to the saline‐treated controls (P

Treatment of respiratory disease with a drug delivered via inhalation is generally held as being beneficial as it provides direct access to the lung target site with a minimum systemic exposure. There is however only limited information of the regional localization of drug retention following inhalation. The aim of this study was to investigate the regional and histological localization of salmeterol retention in the lungs after inhalation and to compare it to systemic administration. Lung distribution of salmeterol delivered to rats via nebulization or intravenous (IV) injection was analyzed with high-resolution mass spectrometry imaging (MSI). Salmeterol was widely distributed in the entire section at 5 min after inhalation, by 15 min it was preferentially retained in bronchial tissue. Via a novel dual-isotope study, where salmeterol was delivered via inhalation and d3-salmeterol via IV to the same rat, could the effective gain in drug concentration associated with inhaled delivery relative to IV, expressed as a site-specific lung targeting factor, was 5-, 31-, and 45-fold for the alveolar region, bronchial sub-epithelium and epithelium, respectively. We anticipate that this MSI-based framework for quantifying regional and histological lung targeting by inhalation will accelerate discovery and development of local and more precise treatments of respiratory disease.

Campylobacter jejuni fecal isolates of eight international travelers, 5 of which had traveled to Ecuador and 3 to Bangladesh, were characterized, and the possible relationship between bacterial traits and clinical symptoms was further analyzed. All eight isolates belonged to the same Multi-Locus Sequence Type clonal complex (ST353CC). The three isolates from Bangladesh were all of the same sequence type (ST-9438), and when compared to isolates of various other sequence types, they had a larger quantity of unique genetic content, higher expression levels of some putative virulence genes involved in adhesion and invasion (flpA, ciaB and iamA), and showed higher adhesion levels to human HT-29 colon cancer cells in an in vitro infection model. However, in contrast to the seemingly higher pathogenic potential of these bacterial isolates, travelers infected with the ST-9438 isolates had no or only very mild symptoms, whereas the other individuals, whose bacterial isolates seemed to have less pathogenic potential, generally reported severe symptoms. When studying the 16S rRNA gene-based fecal microbiota in samples collected prior to travel, there was an individual variation in the relative abundance of the three major bacterial phyla Actinobacteria, Bacteroidetes and Firmicutes, but there were no associations between composition and diversity of microbiota and development of severe symptoms from the infection. It remains to be confirmed by larger studies whether an individual’s characteristics such as gut microbiota, might be related to the severity of symptoms in Campylobacter infections.

Access to modern energy services is a precondition to improving livelihoods and building resilience against climate change. Still, electricity reaches only about half of the population in Sub-Saharan Africa (SSA), while about 40% live under the poverty line. Heavily reliant on the agriculture sector and increasingly affected by prolonged droughts, small-scale irrigation could be instrumental for development and climate change adaptation in SSA countries. A bottom-up understanding of the demand for irrigation and associated energy services is essential for designing viable energy supply options in an effective manner. Using Uganda as a case study, the study introduces a GIS-based methodology for the estimation of groundwater irrigation requirements through which energy demand is derived. Results are generated for two scenarios: (a) a reference scenario and (b) a drought scenario. The most critical need is observed in the northern and southern regions of the country. The total annual irrigation demand is estimated to be ca. 90 thousand m3, with the highest demand observed in the months of December through February, with an average irrigation demand of 445 mm per month. The highest energy demand is observed in the northern part of the study area in January, reaching 48 kWh/ha. The average energy demand increases by 67% in the drought scenario. The study contributes to current gaps in the existing literature by providing a replicable methodological framework and data aimed at facilitating energy system planning through the consideration of location-specific characteristics at the nexus of energy–water–agriculture.

Abstract HBV vaccine has 95% efficacy in children to prevent HBV infection and related cancer. We conducted a prospective study in HIV-1 infected children receiving ART (n = 49) and controls (n = 63) to assess humoral and cellular responses to HBV vaccine provided with three doses under an accelerated schedule of 4 weeks apart. At 1 month post-vaccination all children, except 4 HIV-1 infected, displayed protective antibody (ab) titers to HBV vaccine; ab titers were lower in infected children (P

Abstract Knowledge about the region-specific absorption profiles from the gastrointestinal tract of orally administered drugs is a critical factor guiding dosage form selection in drug development. We have used a novel approach to study three well-characterized permeability and absorption marker drugs in the intestine. Propranolol and metoprolol (highly permeable compounds) and atenolol (low-moderate permeability compound) were orally co-administered to rats. The site of drug absorption was revealed by high spatial resolution matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and complemented by quantitative measurement of drug concentration in tissue homogenates. MALDI-MSI identified endogenous molecular markers that illustrated the villi structures and confirmed the different absorption sites assigned to histological landmarks for the three drugs. Propranolol and metoprolol showed a rapid absorption and shorter transit distance in contrast to atenolol, which was absorbed more slowly from more distal sites. This study provides novel insights into site specific absorption for each of the compounds along the crypt-villus axis, as well as confirming a proximal-distal absorption gradient along the intestine. The combined analytical approach allowed the quantification and spatial resolution of drug distribution in the intestine and provided experimental evidence for the suggested absorption behaviour of low and highly permeable compounds.

Abstract Salmonella Typhimurium causes a self-limiting gastroenteritis that may lead to systemic disease. Bacteria invade the small intestine, crossing the intestinal epithelium from where they are transported to the mesenteric lymph nodes (MLNs) within migrating immune cells. MLNs are an important site at which the innate and adaptive immune responses converge but their architecture and function is severely disrupted during S. Typhimurium infection. To further understand host-pathogen interactions at this site, we used mass spectrometry imaging (MSI) to analyse MLN tissue from a murine model of S. Typhimurium infection. A molecule, identified as palmitoylcarnitine (PalC), was of particular interest due to its high abundance at loci of S. Typhimurium infection and MLN disruption. High levels of PalC localised to sites within the MLNs where B and T cells were absent and where the perimeter of CD169+ sub capsular sinus macrophages was disrupted. MLN cells cultured ex vivo and treated with PalC had reduced CD4+CD25+ T cells and an increased number of B220+CD19+ B cells. The reduction in CD4+CD25+ T cells was likely due to apoptosis driven by increased caspase-3/7 activity. These data indicate that PalC significantly alters the host response in the MLNs, acting as a decisive factor in infection outcome.

Sequence variants in genes involved in the immune system have previously been linked to neutropenia as well as infections in cancer patients. Sequence variants in genes coding for TLR4, MBL, and IL-1Ra were investigated in relation to clinical utility of identifying severe episodes of febrile neutropenia (FN) in a cohort of children undergoing treatment for acute lymphoblastic leukemia. The study included 122 children, where data on FN and microbiological findings were retrospectively collected from medical records. Sequence variants in genes coding for MBL, TLR4, and IL-1Ra were identified by pyrosequencing, TaqMan SNP genotyping assay, and gel electrophoresis. A total of 380 episodes of FN were identified and in 139 episodes, there was a microbiological defined infection. Age and treatment intensity were all associated with the risk of developing FN. No sequence variant was associated to increased numbers of FN episodes. Two sequence variants in the TLR4 gene increased the risk of viral infection, whilst sequence variants in the IL-1Ra gene were associated to a decreased risk of bacterial blood-stream infection (BSI). The investigated sequence variants did not associate with increased risk for FN or to severe infections, as to why the clinical utility as a risk-stratification tool is low. Most episodes of FN were classified as fever with unknown origin, emphasizing the need for improved microbial detection methods.

The trace amine-associated receptor 1 (TAAR1) is expressed by dopaminergic neurons, but the precise influence of trace amines upon their functional activity remains to be fully characterized. Here, we examined the regulation of tyrosine hydroxylase (TH) by tyramine and beta-phenylethylamine (β-PEA) compared to 3-iodothyronamine (T1AM). Immunoblotting and amperometry were performed in dorsal striatal slices from wild-type (WT) and TAAR1 knockout (KO) mice. T1AM increased TH phosphorylation at both Ser19 and Ser40, actions that should promote functional activity of TH. Indeed, HPLC data revealed higher rates of L-dihydroxyphenylalanine (DOPA) accumulation in WT animals treated with T1AM after the administration of a DOPA decarboxylase inhibitor. These effects were abolished both in TAAR1 KO mice and by the TAAR1 antagonist, EPPTB. Further, they were specific inasmuch as Ser845 phosphorylation of the post-synaptic GluA1 AMPAR subunit was unaffected. The effects of T1AM on TH phosphorylation at both Ser19 (CamKII-targeted), and Ser40 (PKA-phosphorylated) were inhibited by KN-92 and H-89, inhibitors of CamKII and PKA respectively. Conversely, there was no effect of an EPAC analog, 8-CPT-2Me-cAMP, on TH phosphorylation. In line with these data, T1AM increased evoked striatal dopamine release in TAAR1 WT mice, an action blunted in TAAR1 KO mice and by EPPTB. Mass spectrometry imaging revealed no endogenous T1AM in the brain, but detected T1AM in several brain areas upon systemic administration in both WT and TAAR1 KO mice. In contrast to T1AM, tyramine decreased the phosphorylation of Ser40-TH, while increasing Ser845-GluA1 phosphorylation, actions that were not blocked in TAAR1 KO mice. Likewise, β-PEA reduced Ser40-TH and tended to promote Ser845-GluA1 phosphorylation. The D1 receptor antagonist SCH23390 blocked tyramine-induced Ser845-GluA1 phosphorylation, but had no effect on tyramine- or β-PEA-induced Ser40-TH phosphorylation. In conclusion, by intracellular cascades involving CaMKII and PKA, T1AM, but not tyramine and β-PEA, acts via TAAR1 to promote the phosphorylation and functional activity of TH in the dorsal striatum, supporting a modulatory influence on dopamine transmission.

During anti-retroviral therapy (ART) HIV-1 persists in cellular reservoirs, mostly represented by CD4+ memory T cells. Several approaches are currently being undertaken to develop a cure for HIV-1 infection through elimination (or reduction) of these reservoirs. Few studies have so far been conducted to assess the possibility of reducing the size of HIV-1 reservoirs through vaccination in virologically controlled HIV-1-infected children. We recently conducted a vaccination study with a combined hepatitis A virus (HAV) and hepatitis B virus (HBV) vaccine in 22 HIV-1-infected children. We assessed the size of the virus reservoir, measured as total HIV-1 DNA copies in blood cells, pre- and postvaccination. In addition, we investigated by immunostaining whether the frequencies of CD4+ and CD8+ T cells and parameters of immune activation and proliferation on these cells were modulated by vaccination. At 1 month from the last vaccination dose, we found that 20 out of 22 children mounted a serological response to HBV; a majority of children had antibodies against HAV at baseline. The number of HIV-1 DNA copies in blood at 1 month postvaccination was reduced in comparison to baseline although this reduction was not statistically significant. A significant reduction of HIV-1 DNA copies in blood following vaccination was found in 12 children. The frequencies of CD4+ (naïve, effector memory) and CD8+ (central memory) T-cell subpopulations changed following vaccinations and a reduction in the activation and proliferation pattern of these cells was also noticed. Multivariate linear regression analysis revealed that the frequency of CD8+ effector memory T cells prior to vaccination was strongly predictive of the reduction of HIV-1 DNA copies in blood following vaccination of the 22 HIV-1-infected children. The results of this study suggest a beneficial effect of vaccination to reduce the size of virus reservoir in HIV-1-infected children receiving ART. A reduced frequency of activated CD4+ cells and an increase in central memory CD8+ T cells were associated with this finding. Further studies should assess whether vaccination is a possible tool to reduce HIV-1 reservoirs.

BackgroundEpstein–Barr virus (EBV) and cytomegalovirus (CMV) are ubiquitous and persistent herpesviruses commonly acquired during childhood. Both viruses have a significant impact on the immune system, especially through mediating the establishment of cellular immunity, which keeps these viruses under control for life. Far less is known about how these viruses influence B-cell responses.ObjectivesTo evaluate the impact of latent EBV and CMV infection on rubella- and measles-specific antibody responses as well as on the B-cell compartment in a prospective birth cohort followed during the first 10 years of life.MethodsIgG titers against rubella and measles vaccines were measured in plasma obtained from the same donors at 2, 5, and 10 years of age. Peripheral B-cell subsets were evaluated ex vivo at 2 and 5 years of age. Factors related to optimal B-cell responses including IL-21 and CXCL13 levels in plasma were measured at all-time points.ResultsEBV carriage in the absence of CMV associated with an accelerated decline of rubella and measles-specific IgG levels (p = 0.003 and p = 0.019, respectively, linear mixed model analysis), while CMV carriage in the absence of EBV associated with delayed IgG decay over time for rubella (p = 0.034). At 5 years of age, EBV but not CMV latency associated with a lower percentage of plasmablasts, but higher IL-21 levels in the circulation.ConclusionOur findings suggest that EBV carriage in the absence of CMV influences the B-cell compartment and the dynamics of antibody responses over time during steady state in the otherwise healthy host.

Drug addiction is a chronic, debilitating disease that affects millions of people around the world causing a substantial societal burden. Despite decades of research efforts, treatment possibilities remain limited and relapse represents the most treatment-resistant element. Neurosteroid sigma-1 receptors have been meticulously studied in psychostimulant reinforced Pavlovian learning, while the sigma-2 receptor subtype has remained unexplored. Recent development of selective sigma-2 receptor ligands have now made it possible to investigate if the sigma-2 receptor system is a potential target to treat drug addiction. We examined the effect of the sigma-2 receptor agonist Siramesine (Lu 28-179) on cocaine-associated locomotion, Pavlovian learning, and reward neurocircuitry using electrophysiology recordings and in vivo microdialysis. We found that Siramesine significantly attenuated conditioned place preference acquisition and expression, as well as it completely blocked cocaine-primed reinstatement. Siramesine, in a similar manner as the selective sigma-1 receptor antagonist BD 1063, decreased acute locomotor responses to cocaine. Immunohistochemistry suggests co-expression of progesterone receptor membrane component 1/sigma-2 receptors and vesicular glutamate transporter 1 in presynaptic boutons of the nucleus accumbens (NAc). Whole-cell voltage clamp recordings of neurons in the NAc indicated that Siramesine decreases the presynaptic release probability of glutamate. Further, we demonstrated, via in vivo microdialysis, that Siramesine significantly decreased cocaine-evoked dopamine release in the striatum of freely moving mice. Collectively, these findings demonstrate that sigma-2 receptors regulate neurocircuitry responsible for positive reinforcement and thereby play a role in cocaine-reinforced Pavlovian behaviors.

Campylobacter jejuni is the most common cause of bacterial gastroenteritis. Major reservoirs are warm-blooded animals, poultry in particular, but Campylobacter can also be transmitted via water. In this paper, we have taken a closer look at the biology and potential virulence of C. jejuni water isolates. Seven C. jejuni isolates from incoming surface water at water plants in Sweden were characterized with whole genome sequencing and phenotypical testing. Multi locus sequence typing analysis revealed that these isolates belonged to groups known to include both common (ST48CC) and uncommon (ST1275CC, ST683, ST793 and ST8853) human pathogens. Further genomic characterization revealed that these isolates had potential for arsenic resistance (due to presence of arsB gene in all isolates), an anaerobic dimethyl sulfoxide oxidoreductase (in three isolates) and lacked the MarR-type transcriptional regulator gene rrpB (in all but one isolate) earlier shown to be involved in better survival under oxidative and aerobic stress. As putative virulence factors were concerned, there were differences between the water isolates in the presence of genes coding for cytolethal distending toxin (cdtABC), Type VI secretion system and sialylated LOS, as well as in biofilm formation. However, all isolates were motile and could adhere to and invade the human HT-29 colon cancer cell line in vitro and induce IL-8 secretion suggesting potential to infect humans. This is, to the best of our knowledge, the first study where C. jejuni water isolates have been characterized using whole genome sequencing and phenotypical assays. We found differences and shared traits among the isolates but also potential to infect humans.

A role for enhanced peptidergic transmission, either opioidergic or not, has been proposed for the generation of l-3,4-dihydroxyphenylalanine (l-DOPA)-induced dyskinesia (LID) on the basis of in situ hybridization studies showing that striatal peptidergic precursor expression consistently correlates with LID severity. Few studies, however, have focused on the actual peptides derived from these precursors. We used mass-spectrometry to study peptide profiles in the putamen and globus pallidus (internalis and externalis) collected from 1-methyl-4-phenyl-1,2,4,6-tetrahydropyridine treated macaque monkeys, acutely or chronically treated with l-DOPA. We identified that parkinsonian and dyskinetic states are associated with an abnormal production of proenkephalin-, prodynorphin- and protachykinin-1-derived peptides in both segments of the globus pallidus. Moreover, we report that peptidergic processing is dopamine-state dependent and highly structure-specific, possibly explaining the failure of previous clinical trials attempting to rectify abnormal peptidergic transmission.

Coronaviruses (CoVs) are found in a wide variety of wild and domestic animals, and constitute a risk for zoonotic and emerging infectious disease. In poultry, the genetic diversity, evolution, distribution and taxonomy of some coronaviruses have been well described, but little is known about the features of CoVs in wild birds. In this study we screened 764 samples from 22 avian species of the orders Anseriformes and Charadriiformes in Sweden collected in 2006/2007 for CoV, with an overall CoV prevalence of 18.7%, which is higher than many other wild bird surveys. The highest prevalence was found in the diving ducks--mainly Greater Scaup (Aythya marila; 51.5%)--and the dabbling duck Mallard (Anas platyrhynchos; 19.2%). Sequences from two of the Greater Scaup CoV fell into an infrequently detected lineage, shared only with a Tufted Duck (Aythya fuligula) CoV. Coronavirus sequences from Mallards in this study were highly similar to CoV sequences from the sample species and location in 2011, suggesting long-term maintenance in this population. A single Black-headed Gull represented the only positive sample from the order Charadriiformes. Globally, Anas species represent the largest fraction of avian CoV sequences, and there seems to be no host species, geographical or temporal structure. To better understand the eitiology, epidemiology and ecology of these viruses more systematic surveillance of wild birds and subsequent sequencing of detected CoV is imperative.

Campylobacters are major enteropathogens worldwide with a substantial financial burden. Matrix metalloproteinases (MMPs) are proteolytic metalloendopeptidases with ability to modify immune response and shown to be upregulated in patients with several tissue destructive diseases, including infections. We measured here serum concentrations of MMP-8 and MMP-9 together with their regulators myeloperoxidase (MPO), human neutrophil elastase (HNE), and tissue inhibitor of metalloproteinases (TIMP)-1 in 80 Campylobacter and 25 Salmonella patients as well as in 27 healthy controls. Paired serum samples were available for 73 and 23 patients, respectively. When the initial serum samples were compared to those from controls, both Campylobacter and Salmonella patients showed elevated concentrations of all biomarkers tested (p ≤ 0.037). In the follow-up samples, collected about 25 days afterwards, MMP-8 levels of Campylobacter patients had already turned to normal but all the other biomarkers still showed elevated, although from the initial levels significantly dropped, levels. For the follow-up samples of Salmonella patients, only MMP-9 and MPO levels were at a significantly higher level than in controls. It remains to be studied if the systematically enhanced neutrophil-derived proteolytic and oxidative stress, induced by Campylobacter infection as shown here and persisting for several weeks, is important for the development of late sequelae.

Drug toxicity observed in animal studies during drug development accounts for the discontinuation of many drug candidates, with the kidney being a major site of tissue damage. Extensive investigations are often required to reveal the mechanisms underlying such toxicological events and in the case of crystalline deposits the chemical composition can be problematic to determine. In the present study, we have used mass spectrometry imaging combined with a set of advanced analytical techniques to characterize such crystalline deposits in situ. Two potential microsomal prostaglandin E synthase 1 inhibitors, with similar chemical structure, were administered to rats over a seven day period. This resulted in kidney damage with marked tubular degeneration/regeneration and crystal deposits within the tissue that was detected by histopathology. Results from direct tissue section analysis by matrix-assisted laser desorption ionization mass spectrometry imaging were combined with data obtained following manual crystal dissection analyzed by liquid chromatography mass spectrometry and nuclear magnetic resonance spectroscopy. The chemical composition of the crystal deposits was successfully identified as a common metabolite, bisulphonamide, of the two drug candidates. In addition, an un-targeted analysis revealed molecular changes in the kidney that were specifically associated with the area of the tissue defined as pathologically damaged. In the presented study, we show the usefulness of combining mass spectrometry imaging with an array of powerful analytical tools to solve complex toxicological problems occurring during drug development.

Readouts that define the physiological distributions of drugs in tissues are an unmet challenge and at best imprecise, but are needed in order to understand both the pharmacokinetic and pharmacodynamic properties associated with efficacy. Here we demonstrate that it is feasible to follow the in vivo transport of unlabeled drugs within specific organ and tissue compartments on a platform that applies MALDI imaging mass spectrometry to tissue sections characterized with high definition histology. We have tracked and quantified the distribution of an inhaled reference compound, tiotropium, within the lungs of dosed rats, using systematic point by point MS and MS/MS sampling at 200 microm intervals. By comparing drug ion distribution patterns in adjacent tissue sections, we observed that within 15 min following exposure, tiotropium parent MS ions (mass-to-charge; m/z 392.1) and fragmented daughter MS/MS ions (m/z 170.1 and 152.1) were dispersed in a concentration gradient (80 fmol-5 pmol) away from the central airways into the lung parenchyma and pleura. These drug levels agreed well with amounts detected in lung compartments by chemical extraction. Moreover, the simultaneous global definition of molecular ion signatures localized within 2-D tissue space provides accurate assignment of ion identities within histological landmarks, providing context to dynamic biological processes occurring at sites of drug presence. Our results highlight an important emerging technology allowing specific high resolution identification of unlabeled drugs at sites of in vivo uptake and retention.

Class switch recombination and somatic hypermutation occur in mature B-cells in response to antigen stimulation. These processes are crucial for the generation of functional antibodies. During HIV-1 infection, loss of memory B-cells, together with an altered differentiation of naïve B-cells result in production of low quality antibodies, which may be due to impaired immunoglobulin affinity maturation. In the current study, we evaluated the effect of HIV-1 infection on class switch recombination and somatic hypermutation by studying the expression of activation-induced cytidine deaminase (AID) in peripheral B-cells from a cohort of chronically HIV-1 infected patients as compared to a group of healthy controls. In parallel, we also characterized the phenotype of B-cells and their ability to produce immunoglobulins in vitro. Cells from HIV-1 infected patients showed higher baseline levels of AID expression and increased IgA production measured ex-vivo and upon CD40 and TLR9 stimulation in vitro. Moreover, the percentage of CD27(-)IgA+ and CD27(-)IgG+ B-cells in blood was significantly increased in HIV-1 infected patients as compared to controls. Interestingly, our results showed a significantly increased number of somatic hypermutations in the VH genes in CD27(-) cells from patients. Taken together, these results show that during HIV-1 infection, CD27(-) B-cells can also produce class switched and somatically hypermutated antibodies. Our data add important information for the understanding of the mechanisms underlying the loss of specific antibody production observed during HIV-1 infection.