Gene Expression Profiling of Peri-Implant Healing of PLGA-Li+ Implants Suggests an Activated Wnt Signaling Pathway In Vivo

Bone development and regeneration is associated with the Wnt signaling pathway that, according to literature, can be modulated by lithium ions (Li+). The aim of this study was to evaluate the gene expression profile during peri-implant healing of poly(lactic-co-glycolic acid) (PLGA) implants with incorporated Li+, while PLGA without Li+ was used as control, and a special attention was then paid to the Wnt signaling pathway. The implants were inserted in rat tibia for 7 or 28 days and the gene expression profile was investigated using a genome-wide microarray analysis. The results were verified by qPCR and immunohistochemistry. Histomorphometry was used to evaluate the possible effect of Li+ on bone regeneration. The microarray analysis revealed a large number of significantly differentially regulated genes over time within the two implant groups. The Wnt signaling pathway was significantly affected by Li+, with approximately 34% of all Wnt-related markers regulated over time, compared to 22% for non-Li+ containing (control; Ctrl) implants. Functional cluster analysis indicated skeletal system morphogenesis, cartilage development and condensation as related to Li+. The downstream Wnt target gene, FOSL1, and the extracellular protein-encoding gene, ASPN, were significantly upregulated by Li+ compared with Ctrl. The presence of beta-catenin, FOSL1 and ASPN positive cells was confirmed around implants of both groups. Interestingly, a significantly reduced bone area was observed over time around both implant groups. The presence of periostin and calcitonin receptor-positive cells was observed at both time points. This study is to the best of the authors' knowledge the first report evaluating the effect of a local release of Li+ from PLGA at the fracture site. The present study shows that during the current time frame and with the present dose of Li+ in PLGA implants, Li+ is not an enhancer of early bone growth, although it affects the Wnt signaling pathway

Immunomodulatory effects exerted by extracellular vesicles from <i>Staphylococcus epidermidis</i> and <i>Staphylococcus aureus</i> isolated from bone-anchored prostheses

Staphylococcus aureus and Staphylococcus epidermidis are the bacteria that most frequently cause osteomyelitis. This study aimed to determine whether staphylococci isolated from osteomyelitis associated with septic loosening of orthopedic prostheses release extracellular vesicles (EVs) and, if so, to determine tentative immunomodulatory effects on the human monocytic cell line THP-1. EVs were isolated from bacterial cultures using filtration and ultracentrifugation and characterized by scanning electron microscopy, nanoparticle tracking analysis and Western Blot. The cytotoxic effect of EVs was analyzed by NucleoCounter and lactate dehydrogenase (LDH) analyses. Confocal laser scanning microscopy was employed to visualize the uptake of EVs by THP-1 cells. Activation of the transcription factor nuclear factor-κB (NF-κB) was determined in THP1-Blue™ NF-κB cells, and the gene expression and secretion of cytokines were determined by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. All investigated strains, irrespective of their biofilm formation ability, were able to secrete EVs in vitro. The S. aureus strains produced significantly more EVs than the S. epidermidis strains. Both S. aureus-derived EVs and S. epidermidis-derived EVs were internalized by THP-1 cells, upregulated Toll-like receptor 3 (TLR3) gene expression, activated NF-κB, and promoted the gene expression and secretion of interleukin (IL)-8, monocyte chemoattractant protein (MCP)-1, matrix metallopeptidase (MMP)-9 and IL-10. Whereas EVs from both staphylococcal species upregulated the proapoptotic DNA damage-inducible transcript 4 (DDIT4) gene and downregulated the antiapoptotic B-cell lymphoma 2 (Bcl-2) gene, cytolysis was preferentially induced in S. aureus EV-stimulated cells, possibly related to the expression of cytolytic proteins predominantly in S. aureus EVs. In conclusion, staphylococcal EVs possess potent cytolytic and immunomodulatory properties

Targeting Acetylcholinesterase: Identification of Chemical Leads by High Throughput Screening, Structure Determination and Molecular Modeling

Acetylcholinesterase (AChE) is an essential enzyme that terminates cholinergic transmission by rapid hydrolysis of the neurotransmitter acetylcholine. Compounds inhibiting this enzyme can be used (inter alia) to treat cholinergic deficiencies (e.g. in Alzheimer's disease), but may also act as dangerous toxins (e.g. nerve agents such as sarin). Treatment of nerve agent poisoning involves use of antidotes, small molecules capable of reactivating AChE. We have screened a collection of organic molecules to assess their ability to inhibit the enzymatic activity of AChE, aiming to find lead compounds for further optimization leading to drugs with increased efficacy and/or decreased side effects. 124 inhibitors were discovered, with considerable chemical diversity regarding size, polarity, flexibility and charge distribution. An extensive structure determination campaign resulted in a set of crystal structures of protein-ligand complexes. Overall, the ligands have substantial interactions with the peripheral anionic site of AChE, and the majority form additional interactions with the catalytic site (CAS). Reproduction of the bioactive conformation of six of the ligands using molecular docking simulations required modification of the default parameter settings of the docking software. The results show that docking-assisted structure-based design of AChE inhibitors is challenging and requires crystallographic support to obtain reliable results, at least with currently available software. The complex formed between C5685 and Mus musculus AChE (C5685•mAChE) is a representative structure for the general binding mode of the determined structures. The CAS binding part of C5685 could not be structurally determined due to a disordered electron density map and the developed docking protocol was used to predict the binding modes of this part of the molecule. We believe that chemical modifications of our discovered inhibitors, biochemical and biophysical characterization, crystallography and computational chemistry provide a route to novel AChE inhibitors and reactivators

Incidence and time trends of second primary malignancies after non-Hodgkin lymphoma:a Swedish population-based study

Considering treatment changes and an improved prognosis of non-Hodgkin lymphoma (NHL) over time, knowledge regarding long-term health outcomes, including late effects of treatment, has become increasingly important. We report on time trends of second primary malignancies (SPMs) in Swedish NHL patients, encompassing the years before as well as after the introduction of anti-CD20 antibody therapy. We identified NHL patients in the Swedish Cancer Register 1993 to 2014 and matched comparators from the Swedish Total Population Register. The matched cohort was followed through 2017. By linking to the Swedish Lymphoma Register, subcohort analyses by NHL subtype were performed. Flexible parametric survival models were used to estimate hazard ratios (HRs) with 95% confidence intervals (CIs) of SPM among patients and comparators. Among 32 100 NHL patients, 3619 solid tumors and 217 myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) cases were observed, corresponding to a 40% higher rate of solid tumors (HR(solid tumors) = 1.4; 95% CI, 1.4-1.5) and a 5-fold higher rate of MDS/AML (HR(MDS/AML )= 5.2; 95% CI, 4.4-6.2) than for comparators. Overall, the observed excess risks for solid tumors or MDS/AML remained stable over the study period, except for follicular lymphoma, where the excess rate of MDS/AML attenuated with time (P for trend = .012). We conclude that NHL survivors have an increased risk of both solid tumors and hematologic malignancies, in particular MDS/AML. Stable excess risks over time indicate that contemporary treatment standards are not associated with modified SPM risk. Encouragingly, decreasing rates of MDS/AML were noted among patients with follicular lymphoma, possibly due to the increasing use of nonchemotherapy-based treatments

Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages

<p>Abstract</p> <p>Background</p> <p>Exosomes are 30-100 nm membrane vesicles of endocytic origin produced by numerous cells. They can mediate diverse biological functions, including antigen presentation. Exosomes have recently been shown to contain functional RNA, which can be delivered to other cells. Exosomes may thus mediate biological functions either by surface-to-surface interactions with cells, or by the delivery of functional RNA to cells. Our aim was therefore to determine the presence of RNA in exosomes from human saliva, plasma and breast milk and whether these exosomes can be taken up by macrophages.</p> <p>Method</p> <p>Exosomes were purified from human saliva, plasma and breast milk using ultracentrifugation and filtration steps. Exosomes were detected by electron microscopy and examined by flow cytometry. Flow cytometry was performed by capturing the exosomes on anti-MHC class II coated beads, and further stain with anti-CD9, anti-CD63 or anti-CD81. Breast milk exosomes were further analysed for the presence of Hsc70, CD81 and calnexin by Western blot. Total RNA was detected with a Bioanalyzer and mRNA was identified by the synthesis of cDNA using an oligo (dT) primer and analysed with a Bioanalyzer. The uptake of PKH67-labelled saliva and breast milk exosomes by macrophages was examined by measuring fluorescence using flow cytometry and fluorescence microscopy.</p> <p>Results</p> <p>RNA was detected in exosomes from all three body fluids. A portion of the detected RNA in plasma exosomes was characterised as mRNA. Our result extends the characterisation of exosomes in healthy humans and confirms the presence of RNA in human saliva and plasma exosomes and reports for the first time the presence of RNA in breast milk exosomes. Our results also show that the saliva and breast milk exosomes can be taken up by human macrophages.</p> <p>Conclusions</p> <p>Exosomes in saliva, plasma and breast milk all contain RNA, confirming previous findings that exosomes from several sources contain RNA. Furthermore, exosomes are readily taken up by macrophages, supporting the notion that exosomal RNA can be shuttled between cells.</p

Aspirin intake and breast cancer survival – a nation-wide study using prospectively recorded data in Sweden

Background: Aspirin (ASA) use has been associated with improved breast cancer survival in several prospective studies. Methods: We conducted a nested case–control study of ASA use after a breast cancer diagnosis among women using Swedish National Registries. We assessed prospectively recorded ASA exposure during several different time windows following cancer diagnosis using conditional logistic regression with breast cancer death as the main outcome. Within each six-month period of follow-up, we categorized dispensed ASA doses into three groups: 0, less than 1, and 1 or more daily doses. Results: We included 27,426 women diagnosed with breast cancer between 2005 and 2009; 1,661 died of breast cancer when followed until Dec 31, 2010. There was no association between ASA use and breast cancer death when exposure was assessed either shortly after diagnosis, or 3–12 months before the end of follow-up. Only during the period 0–6 months before the end of follow-up was ASA use at least daily compared with non-use associated with a decreased risk of breast cancer death: HR (95% CI) = 0.69 (0.56-0.86). However, in the same time-frame, those using ASA less than daily had an increased risk of breast cancer death: HR (95% CI) = 1.43 (1.09-1.87). Conclusions: Contrary to other studies, we did not find that ASA use was associated with a lower risk of death from breast cancer, except when assessed short term with no delay to death/end of follow-up, which may reflect discontinuation of ASA during terminal illness

Risk of diabetes and the impact on preexisting diabetes in patients with lymphoma treated with steroid-containing immunochemotherapy

First-line treatments for lymphomas often include high doses of prednisolone, but the risks of new-onset diabetes mellitus (DM) or worsening of preexisting DM following treatment with cyclic high dose corticosteroids is unknown. This cohort study matched non-Hodgkin lymphoma (NHL) patients treated with steroid-containing immunochemotherapy (ie, R-CHOP[-like] and R-CVP) between 2002 and 2015 to individuals from the Danish population to investigate the risks of new-onset DM. For patients with preexisting DM, the risks of insulin dependency and anthracycline-associated cardiovascular diseases (CVDs) were assessed. In total, 5672 NHL patients and 28 360 matched comparators were included. Time-varying incidence rate ratios (IRRs) showed increased risk of DM in the first year after treatment compared with matched comparators, with the highest IRR being 2.7. The absolute risks were higher among patients in the first 2 years, but the difference was clinically insignificant. NHL patients with preexisting DM had increased risks of insulin prescriptions with 0.5-, 5-, and 10-year cumulative risk differences of insulin treatment of 15.3, 11.8, and 6.0 percentage units as compared with the DM comparators. In a landmark analysis at 1 year, DM patients with lymphoma had decreased risks of insulin dependency compared with comparators. Time-varying IRRs showed a higher CVD risk for NHL patients with DM as compared with comparators in the first year after treatment. NHL patients treated with steroid-containing immunochemotherapy regimens have a clinically insignificant increased risk of DM in the first year following treatment, and patients with preexisting DM have a temporary increased risk of insulin prescriptions and CVD

Longitudinal genome-wide DNA methylation changes in response to kidney failure replacement therapy

Chronic kidney disease (CKD) is an emerging public health priority associated with high mortality rates and demanding treatment regimens, including life-style changes, medications or even dialysis or renal transplantation. Unavoidably, the uremic milieu disturbs homeostatic processes such as DNA methylation and other vital gene regulatory mechanisms. Here, we aimed to investigate how dialysis or kidney transplantation modifies the epigenome-wide methylation signature over 12 months of treatment. We used the Infinium HumanMethylation450 BeadChip on whole blood samples from CKD-patients undergoing either dialysis (n = 11) or kidney transplantation (n = 12) and 24 age- and sex-matched population-based controls. At baseline, comparison between patients and controls identified several significant (PFDR &lt; 0.01) CpG methylation differences in genes with functions relevant to inflammation, cellular ageing and vascular calcification. Following 12 months, the global DNA methylation pattern of patients approached that seen in the control group. Notably, 413 CpG sites remained differentially methylated at follow-up in both treatment groups compared to controls. Together, these data indicate that the uremic milieu drives genome-wide methylation changes that are partially reversed with kidney failure replacement therapy. Differentially methylated CpG sites unaffected by treatment may be of particular interest as they could highlight candidate genes for kidney disease per se