An accelerated, clinical-grade protocol to generate high yields of type 1-polarizing messenger RNA-loaded dendritic cells for cancer vaccination.

BACKGROUND: Many efforts have been devoted to improve the performance of dendritic cell (DC)-based cancer vaccines. Ideally, a DC vaccine should induce robust type 1-polarized T-cell responses and efficiently expand antigen (Ag)-specific cytotoxic T-cells, while being applicable regardless of patient human leukocyte antigen (HLA) type. Production time should be short, while maximally being good manufacturing practice (GMP)-compliant. We developed a method that caters to all of these demands and demonstrated the superiority of the resulting product compared with DCs generated using a well-established "classical" protocol. METHODS: Immunomagnetically purified monocytes were cultured in a closed system for 3 days in GMP-compliant serum-free medium and cytokines, and matured for 24 h using monophosphoryl lipid A (MPLA)+ interferon-gamma (IFN-γ). Mature DCs were electroporated with messenger RNA (mRNA) encoding full-length antigen and cryopreserved. "Classical" DCs were cultured for 8 days in flasks, with one round of medium and cytokine supplementation, and matured with tumor necrosis factor alpha (TNF-α) + prostaglandin E2 (PGE2) during the last 2 days. RESULTS: Four-day MPLA/IFN-γ-matured DCs were superior to 8-day TNF-α/PGE2-matured DCs in terms of yield, co-stimulatory/co-inhibitory molecule expression, resilience to electroporation and cryopreservation and type 1-polarizing cytokine and chemokine release after cell thawing. Electroporated and cryopreserved DCs according to our protocol efficiently present epitopes from tumor antigen-encoding mRNA, inducing a strong expansion of antigen-specific CD8+ T-cells with full cytolytic capacity. CONCLUSION: We demonstrate using a GMP-compliant culture protocol the feasibility of generating high yields of mature DCs in a short time, with a superior immunogenic profile compared with 8-day TNF-α/PGE2-matured DCs, and capable of inducing vigorous cytotoxic T-cell responses to antigen from electroporated mRNA. This method is now being applied in our clinical trial program.

A Statewide Hospital-Based Safe Infant Sleep Initiative: Measurement of Parental Knowledge and Behavior.

Sleep-related infant deaths are a leading cause of infant mortality in Georgia, and these deaths are largely associated with unsafe sleep practices among caregivers. In early 2016, the Georgia Department of Public Health launched the Georgia Safe to Sleep Hospital Initiative, providing hospitals with safe infant sleep information and educational materials to be distributed to families and newborns. This study examined the knowledge and behaviors of a sample of Georgia parents after the implementation of the Hospital Initiative and identified the family characteristics and intervention components most closely associated with the knowledge and practice of safe infant sleep. The primary caretakers of all infants born in Georgia from August to October 2016 were invited to complete a web-based survey 1 month after hospital discharge. The final sample size included 420 parents of newborns, and the primary outcomes assessed included two measures of knowledge and four measures of infant sleep behaviors regarding infant sleep position and location. Most respondents demonstrated knowledge of the correct recommended sleep position (90%) and location (85%). Logistic regression revealed that receipt of information in the hospital was significantly correlated with safe sleep behaviors, and infant sleep habits tended to influence safe sleep practices. Additionally, Medicaid parents receiving bassinets from the hospital were 74% less likely to bed share (OR 0.26; 95% CI 0.007). Implementation of a statewide hospital initiative was associated with high levels of parental knowledge and behavior and may have been successful in reducing the practice of bed sharing among Medicaid parents.

An approach for the identification of exemplar sites for scaling up targeted field observations of benthic biogeochemistry in heterogeneous environments.

Continental shelf sediments are globally important for biogeochemical activity. Quantification of shelf-scale stocks and fluxes of carbon and nutrients requires the extrapolation of observations made at limited points in space and time. The procedure for selecting exemplar sites to form the basis of this up-scaling is discussed in relation to a UK-funded research programme investigating biogeochemistry in shelf seas. A three-step selection process is proposed in which (1) a target area representative of UK shelf sediment heterogeneity is selected, (2) the target area is assessed for spatial heterogeneity in sediment and habitat type, bed and water column structure and hydrodynamic forcing, and (3) study sites are selected within this target area encompassing the range of spatial heterogeneity required to address key scientific questions regarding shelf scale biogeochemistry, and minimise confounding variables. This led to the selection of four sites within the Celtic Sea that are significantly different in terms of their sediment, bed structure, and macrofaunal, meiofaunal and microbial community structures and diversity, but have minimal variations in water depth, tidal and wave magnitudes and directions, temperature and salinity. They form the basis of a research cruise programme of observation, sampling and experimentation encompassing the spring bloom cycle. Typical variation in key biogeochemical, sediment, biological and hydrodynamic parameters over a pre to post bloom period are presented, with a discussion of anthropogenic influences in the region. This methodology ensures the best likelihood of site-specific work being useful for up-scaling activities, increasing our understanding of benthic biogeochemistry at the UK-shelf scale.

Comparing benthic biogeochemistry at a sandy and a muddy site in the Celtic Sea using a model and observations.

Results from a 1D setup of the European Regional Seas Ecosystem Model (ERSEM) biogeochemical model were compared with new observations collected under the UK Shelf Seas Biogeochemistry (SSB) programme to assess model performance and clarify elements of shelf-sea benthic biogeochemistry and carbon cycling. Observations from two contrasting sites (muddy and sandy) in the Celtic Sea in otherwise comparable hydrographic conditions were considered, with the focus on the benthic system. A standard model parameterisation with site-specific light and nutrient adjustments was used, along with modifications to the within-seabed diffusivity to accommodate the modelling of permeable (sandy) sediments. Differences between modelled and observed quantities of organic carbon in the bed were interpreted to suggest that a large part (>90%) of the observed benthic organic carbon is biologically relatively inactive. Evidence on the rate at which this inactive fraction is produced will constitute important information to quantify offshore carbon sequestration. Total oxygen uptake and oxic layer depths were within the range of the measured values. Modelled depth average pore water concentrations of ammonium, phosphate and silicate were typically 5-20% of observed values at the muddy site due to an underestimate of concentrations associated with the deeper sediment layers. Model agreement for these nutrients was better at the sandy site, which had lower pore water concentrations, especially deeper in the sediment. Comparison of pore water nitrate with observations had added uncertainty, as the results from process studies at the sites indicated the dominance of the anammox pathway for nitrogen removal; a pathway that is not included in the model. Macrofaunal biomasses were overestimated, although a model run with increased macrofaunal background mortality rates decreased macrofaunal biomass and improved agreement with observations. The decrease in macrofaunal biomass was compensated by an increase in meiofaunal biomass such that total oxygen demand remained within the observed range. The permeable sediment modification reproduced some of the observed behaviour of oxygen penetration depth at the sandy site. It is suggested that future development in ERSEM benthic modelling should focus on: (1) mixing and degradation rates of benthic organic matter, (2) validation of benthic faunal biomass against large scale spatial datasets, (3) incorporation of anammox in the benthic nitrogen cycle, and (4) further developments to represent permeable sediment processes.

A promoter polymorphism in the Per3 gene is associated with alcohol and stress response.

The period homolog genes Per1, Per2 and Per3 are important components of the circadian clock system. In addition to their role in maintaining circadian rhythm, these genes have been linked to mood disorders, stress response and vulnerability to addiction and alcoholism. In this study, we combined high-resolution sequence analysis and quantitative trait locus (QTL) mapping of gene expression and behavioral traits to identify Per3 as a compelling candidate for the interaction between circadian rhythm, alcohol and stress response. In the BXD family of mouse strains, sequence variants in Per3 have marked effects on steady-state mRNA and protein levels. As a result, the transcript maps as a cis-acting expression QTL (eQTL). We found that an insertion/deletion (indel) variant in a putative stress response element in the promoter region of Per3 causes local control of transcript abundance. This indel results in differences in protein binding affinities between the two alleles through the Nrf2 transcriptional activator. Variation in Per3 is also associated with downstream differences in the expression of genes involved in circadian rhythm, alcohol, stress response and schizophrenia. We found that the Per3 locus is linked to stress/anxiety traits, and that the basal expression of Per3 is also correlated with several anxiety and addiction-related phenotypes. Treatment with alcohol results in increased expression of Per3 in the hippocampus, and this effect interacts with acute restraint stress. Our data provide strong evidence that variation in the Per3 transcript is causally associated with and also responsive to stress and alcohol.

Stimulation with type I collagen induces changes in gene expression in peripheral blood mononuclear cells from patients with diffuse cutaneous systemic sclerosis (scleroderma).

An autoantigenic role for collagen type I (CI) has been suggested previously in diffuse cutaneous systemic sclerosis (dcSSc). Whether CI is indeed capable of affecting the immune system in dcSSc is not known. Patients with early (3 years or less) or late (>3 years) dcSSc and healthy controls donated blood. Peripheral blood mononuclear cells (PBMC) were cultured with or without CI, and expression of genes known for their involvement in autoimmune and inflammatory processes was assessed using cDNA arrays; results were confirmed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay for selected genes. Patients with early and late dcSSc were similarly different from healthy controls in basal gene expression. When cultured with CI, PBMC from patients with early dcSSc differed from healthy controls in expression of 34 genes, whereas PBMC from patients with late dcSSc differed from healthy controls in expression of only 29 genes. Direct comparisons of matched PBMC samples cultured with and without CI revealed differences in expression of eight genes in healthy controls, of five genes in patients with early dcSSc, and no differences in patients with late dcSSc. Thus, PBMC from patients with dcSSc respond differently than do PBMC from healthy controls when cultured with CI. Exposure to CI in culture of PBMC from patients in the early stage of dcSSc in contrast to PBMC from patients with late-stage dcSSc evokes a greater degree of activation of immune-related genes, suggesting that CI is more dominant as an autoantigen in early versus late dcSSc.

Pamidronate infusion in patients with systemic sclerosis results in changes in blood mononuclear cell cytokine profiles.

A single infusion of pamidronate was given to patients with systemic sclerosis (scleroderma, SSc) to assess effects on cytokine production by peripheral blood mononuclear cells (PBMC) and lymphocyte subsets. Eighteen patients with SSc received a single intravenous dose of 60 mg of pamidronate and were followed for 6 months. Assessment of cytokine production [interferon (IFN)-gamma, interleukin (IL)-10, transforming growth factor (TGF)-beta1, tumour necrosis factor (TNF)-alpha and IL-4] by PBMC and lymphocyte subsets by flow cytometry was carried out before and after the pamidronate infusion. Unstimulated PBMC produced increased amounts of IFN-gamma and TNF-alpha and reduced levels of TGF-beta1 for up to 24 weeks after the infusion. gammadelta T cells from patients with SSc were activated in vitro and produced increased IFN-gamma. The effects of pamidronate on modulation of cytokine profiles in patients with SSc may merit future study.

Gamma irradiation of HIV-1.

The potential for transmission of human immunodeficiency virus (HIV) type 1 has created serious concern for the continued clinical use of bone and soft-tissue allografts. Tissue banks have employed 1.5-2.5 Mrad for sterilization of bone and tendon allografts, which, according to the current literature, approaches the level at which the tissue quality is adversely affected for implantation. Our working hypothesis was that gamma irradiation at increasing doses can proportionately inactivate HIV type 1. The objective of this study was to inactivate HIV type 1 by irradiation, as determined by its capacity to infect human T-lymphocytes and established cell lines in vitro. The replicative competence of HIV type 1 was also assessed by the presence of reverse transcriptase activity, enzyme-linked immunoadsorbent assay (ELISA), immunofluorescence assays for p24 viral core antigen, and the formation of syncytia induced by HIV type 1 in the cultures inoculated with irradiated virus. The results demonstrated the presence of active viral replication in previously noninfected cells in the supernatant samples that were exposed to as much as 5.0 Mrad. The data for the 10-Mrad sample were indeterminate due to cellular damage. These data suggest that gamma irradiation (1.5-2.5 Mrad) does not constitute a virucidal dose for HIV type 1. Current technologies for screening have greatly improved, and the surgeon should rely on tissue bank screening procedures and other methods of preparation rather than sterilization by gamma radiation techniques in choosing allograft material.

Opportunistic adaptations to man-induced habitat changes by some South American caprimulgidae.

We describe the opportunistic adaptation to man-induced changes in the habitats of six neotropical Caprimulgidae. Habitats created by those changes, and similar to their original and usual ones, are readily occupied by these nightjars. The occasional invasion of urban environments ("urbanization") is the most recent and most remarkable behavioural adaptation.

Acute phosphine poisoning? A case report and review.

A case compatible with an acute phosphine poisoning after inhalation is presented. The observation was notably characterized by necrosis of the nasal mucosa, the delayed onset of a pulmonary edema and myocardial injury with multisystemic involvement. A review of literature, not very prolific in this field, is included in the discussion.

Liquid helium cryopump and reliable opening device for a balloon-borne mass spectrometer.

The design, technical characteristics, and test and flight results of a liquid helium cryopump and an opening device operating on board a balloon-borne mass spectrometer combining a cryopump and a quadrupole mass filter are reported. The gas inlet of this mass spectrometer is opened through a simple and reliable remote-controlled system, which is also described.