Search

Objective: Natural killer (NK) cells are a part of the innate immune system and first-line defense against cancer. Since they possess natural mechanisms to recognize and kill tumor cells, NK cells are considered as a potential option for an off-the-shelf allogeneic cell-based immunotherapy. Here, our objective was to identify the optimal cytokine-based, feeder-free, activation and expansion protocol for cytotoxic NK cells against glioblastoma in vitro. Methods: NK cells were enriched from human peripheral blood and expanded for 16 days with different activation and cytokine combinations. The expansion conditions were evaluated based on NK cell viability, functionality, expansion rate and purity. The cytotoxicity and degranulation of the expanded NK cells were measured in vitro from co-cultures with the glioma cell lines U-87 MG, U-87 MG EGFR vIII, LN-229, U-118 and DK-MG. The best expansion protocols were selected from ultimately 39 different conditions: three magnetic cell-selection steps (Depletion of CD3+ cells, enrichment of CD56+ cells, and depletion of CD3+ cells followed by enrichment of CD56+ cells); four activation protocols (continuous, pre-activation, re-activation, and boost); and four cytokine combinations (IL-2/15, IL-21/15, IL-27/18/15 and IL-12/18/15). Results: The expansion rates varied between 2-50-fold, depending on the donor and the expansion conditions. The best expansion rate and purity were gained with sequential selection (Depletion of CD3+ cells and enrichment of CD56+ cells) from the starting material and pre-activation with IL-12/18/15 cytokines, which are known to produce cytokine-induced memory-like NK cells. The cytotoxicity of these memory-like NK cells was enhanced with re-activation, diminishing the donor variation. The most cytotoxic NK cells were produced when cells were boosted at the end of the expansion with IL-12/18/15 or IL-21/15. Conclusion: According to our findings the ex vivo proliferation capacity and functionality of NK cells is affected by multiple factors, such as the donor, composition of starting material, cytokine combination and the activation protocol. The cytokines modified the NK cells' phenotype and functionality, which was evident in their reactivity against the glioma cell lines. To our knowledge, this is the first comprehensive comparative study performed to this extent, and these findings could be used for upscaling clinical NK cell manufacturing. [ABSTRACT FROM AUTHOR]

The p53 transcription factor plays a key role both in cancer and in the cell-intrinsic response to infections. The ORFEOME project hypothesized that novel p53-virus interactions reside in hitherto uncharacterized, unknown, or hypothetical open reading frames (orfs) of human viruses. Hence, 172 orfs of unknown function from the emerging viruses SARS-Coronavirus, MERS-Coronavirus, influenza, Ebola, Zika (ZIKV), Chikungunya and Kaposi Sarcoma-associated herpesvirus (KSHV) were de novo synthesized, validated and tested in a functional screen of p53 signaling. This screen revealed novel mechanisms of p53 virus interactions and two viral proteins KSHV orf10 and ZIKV NS2A binding to p53. Originally identified as the target of small DNA tumor viruses, these experiments reinforce the notion that all viruses, including RNA viruses, interfere with p53 functions. These results validate this resource for analogous systems biology approaches to identify functional properties of uncharacterized viral proteins, long non-coding RNAs and micro RNAs. Author summary: New viruses are constantly emerging. The ORFEOME project was based on the hypothesis that every virus, regardless of its molecular makeup and biology should encode functions that intersect the p53 signaling network, since p53 guards the cell from genomic insults, of which depositing a foreign, viral nucleic acid is one. The result of the ORFEOME screen of proteins without any known function, of predicted open reading frames and of suspected non-coding RNAs is the identification of two viral proteins that interact with p53. The first one, orf10, is encoded by Kaposi Sarcoma-associated herpesvirus and the second one, NS2A, is encoded by the Zika virus. [ABSTRACT FROM AUTHOR]

This study was conducted to determine the prevalence of respiratory viral infections (RVI) in persons living with HIV (PLH) admitted with a respiratory complaint using real‐time reverse transcription polymerase chain reaction and primer‐independent next‐generation sequencing (NGS). Of 82 subjects, respiratory viruses were the most common pathogen identified in 27 (33%), followed by fungus and bacteria in 8 (10%) and 4 (5%) subjects, respectively. Among subjects with RVI, 11 (41%) required ICU admission and 16 (59%) required mechanical ventilation. The proportion of respiratory viruses identified, and the associated complicated hospital course highlights the significant role that RVIs play in the lung health of PLH. [ABSTRACT FROM AUTHOR]

The 4 Parameter Logistic (4PL) model has been recognized as a major tool to analyze the relationship between doses and responses in pharmacological experiments. A main strength of this model is that each parameter contributes an intuitive meaning enhancing interpretability of a fitted model. However, implementing the 4PL model using conventional statistical software often encounters numerical errors. This paper highlights the issue of convergence failure and presents several causes with solutions. These causes include outliers and a non-logistic data shape, so useful remedies such as robust estimation, outlier diagnostics and constrained optimization are proposed. These features are implemented in a new R package dr4pl (Dose-Response analysis using the 4 Parameter Logistic model) whose code examples are presented as a separate section. Our R package dr4pl is shown to work well for data sets where the traditional dose-response modelling packages drc and nplr fail. [ABSTRACT FROM AUTHOR]

Rationale: The oropharyngeal microbiome is a primary source of lung microbiota, contributes to lower respiratory infection, and is also a driver of oral health.Objectives: We sought to understand oropharyngeal microbial communities in advanced lung disease, community dynamics after lung transplantation, and ecological features of dysbiosis.Methods: Oropharyngeal wash samples were obtained from individuals with end-stage disease awaiting transplantation (n = 22) and longitudinally from individuals at 6 weeks, 3 months, and 6 months after transplantation (n = 33), along with healthy control subjects (n = 14). Bacterial 16S and fungal internal transcribed spacer rRNA regions were deep-sequenced, and bacterial community respiratory patterns were imputed from taxonomic composition.Results: Healthy subjects' oropharyngeal microbiomes showed a gradient of community types reflecting relative enrichment of strictly anaerobic, aerobic, or facultative anaerobic bacteria. Patients with end-stage lung disease showed severe dysbiosis by both taxonomic composition and respiration phenotypes, with reduced richness and diversity, increased facultative and decreased aerobic bacteria, and absence of communities characterized by obligate aerobes. In patients at 6 weeks and 3 months post-transplant, richness and diversity were intermediate between healthy and pretransplant subjects, with near-normal distribution of community types. However, by 6 months post-transplant, oropharyngeal wash resembled the low-diversity facultative-dominated profile of pretransplant subjects. Community ecotype correlated with Candida abundance.Conclusions: End-stage lung disease is associated with marked upper respiratory tract dysbiosis involving both community structure and respiratory metabolism profiles of constituent bacteria. Dynamic changes occur after lung transplantation, with partial normalization early but later appearance of severe dysbiosis similar to pretransplant patients. Aberrant oropharyngeal communities may predispose to abnormal lung microbiota and infection risk both in advanced lung disease and after transplantation. [ABSTRACT FROM AUTHOR]

Extracellular signaling is a mechanism that higher eukaryotes have evolved to facilitate organismal homeostasis. Recent years have seen an emerging interest in the role of secreted microvesicles, termed extracellular vesicles (EV) or exosomes in this signaling network. EV contents can be modified by the cell in response to stimuli, allowing them to relay information to neighboring cells, influencing their physiology. Here we show that the tumor virus Kaposi’s Sarcoma-associated herpesvirus (KSHV) hijacks this signaling pathway to induce cell proliferation, migration, and transcriptome reprogramming in cells not infected with the virus. KSHV-EV activates the canonical MEK/ERK pathway, while not alerting innate immune regulators, allowing the virus to exert these changes without cellular pathogen recognition. Collectively, we propose that KSHV establishes a niche favorable for viral spread and cell transformation through cell-derived vesicles, all while avoiding detection. [ABSTRACT FROM AUTHOR]

Idiopathic chronic enterocolitis (ICE) is one of the most commonly encountered and difficult to manage diseases of captive rhesus macaques (Macaca mulatta). The etiology is not well understood, but perturbations in gut microbial communities have been implicated. Here we evaluated the effects of a 14-day course of vancomycin, neomycin, and fluconazole on animals affected with ICE, comparing treated, untreated, and healthy animals. We performed microbiome analysis on duodenal and colonic mucosal samples and feces in order to probe bacterial and/or fungal taxa potentially associated with ICE. All treated animals showed a significant and long-lasting improvement in stool consistency over time when compared to untreated and healthy controls. Microbiome analysis revealed trends associating bacterial community composition with ICE, particularly lineages of the Lactobacillaceae family. Sequencing of DNA from macaque food biscuits revealed that fungal sequences recovered from stool were dominated by yeast-derived food additives; in contrast, bacteria in stool appeared to be authentic gut residents. In conclusion, while validation in larger cohorts is needed, the treatment described here was associated with significantly improved clinical signs; results suggested possible correlates of microbiome structure with disease, though no strong associations were detected between single microbes and ICE. [ABSTRACT FROM AUTHOR]

The article analysis the relation between disease severity, gut dysbiosis, and bacterial production of free amino acids in Crohn's disease patients. Topics discussed include role of intestinal inflammation in developing dysbiosis and chronic alteration of the gut microbiota; association of amino acids in fecal microbiota with disease activity; and association of fecal amino acids with proteobacteria in pediatric patients with Crohn's disease.

Insufficient sleep increasingly characterizes modern society, contributing to a host of serious medical problems. Loss of sleep is associated with metabolic diseases such as obesity and diabetes, cardiovascular disorders, and neurological and cognitive impairments. Shifts in gut microbiome composition have also been associated with the same pathologies; therefore, we hypothesized that sleep restriction may perturb the gut microbiome to contribute to a disease state. In this study, we examined the fecal microbiome by using a cross-species approach in both rat and human studies of sleep restriction. We used DNA from hypervariable regions (V1-V2) of 16S bacteria rRNA to define operational taxonomic units (OTUs) of the microbiome. Although the OTU richness of the microbiome is decreased by sleep restriction in rats, major microbial populations are not altered. Only a single OTU, TM7-3a, was found to increase with sleep restriction of rats. In the human microbiome, we find no overt changes in the richness or composition induced by sleep restriction. Together, these results suggest that the microbiome is largely resistant to changes during sleep restriction. [ABSTRACT FROM AUTHOR]

We have recently reported that Altered Schaedler Flora (ASF) can be used to durably engineer the gut microbiota to reduce ammonia production as an effective modality to reduce morbidity and mortality in the setting of liver injury. Here we investigated the effects of a low protein diet on ASF colonization and its ability to engineer the microbiota. Initially, ASF inoculation was similar between mice fed a normal protein diet or low protein diet, but the outgrowth of gut microbiota differed over the ensuing month. Notable was the inability of the dominant Parabacteroides ASF taxon to exclude other taxa belonging to the Bacteroidetes phylum in the setting of a low protein diet. Instead, a poorly classified yet highly represented Bacteroidetes family, S24-7, returned within 4 weeks of inoculation in mice fed a low protein diet, demonstrating a reduction in ASF resilience in response to dietary stress. Nevertheless, fecal ammonia levels remained significantly lower than those observed in mice on the same low protein diet that received a transplant of normal feces. No deleterious effects were observed in host physiology due to ASF inoculation into mice on a low protein diet. In total, these results demonstrate that low protein diet can have a pronounced effect on engineering the gut microbiota but modulation of ammonia is preserved. [ABSTRACT FROM AUTHOR]

The family of resistance gene analogues (RGAs) with a nucleotide-binding site (NBS) domain accounts for the largest number of disease resistance genes and is one of the largest gene families in plants. We have identified 868 RGAs in the genome of the apple (Malus × domestica Borkh.) cultivar ‘Golden Delicious’. This represents 1.51% of the total number of predicted genes for this cultivar. Several evolutionary features are pronounced in M. domestica, including a high fraction (80%) of RGAs occurring in clusters. This suggests frequent tandem duplication and ectopic translocation events. Of the identified RGAs, 56% are located preferentially on six chromosomes (Chr 2, 7, 8, 10, 11, and 15), and 25% are located on Chr 2. TIR-NBS and non-TIR-NBS classes of RGAs are primarily exclusive of different chromosomes, and 99% of non-TIR-NBS RGAs are located on Chr 11. A phylogenetic reconstruction was conducted to study the evolution of RGAs in the Rosaceae family. More than 1400 RGAs were identified in six species based on their NBS domain, and a neighbor-joining analysis was used to reconstruct the phylogenetic relationships among the protein sequences. Specific phylogenetic clades were found for RGAs of Malus, Fragaria, and Rosa, indicating genus-specific evolution of resistance genes. However, strikingly similar RGAs were shared in Malus, Pyrus, and Prunus, indicating high conservation of specific RGAs and suggesting a monophyletic origin of these three genera. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is constantly evolving. Prior studies focused on high-case-density locations, such as the northern and western metropolitan areas of the United States. This study demonstrates continued SARS-CoV-2 evolution in a suburban southern region of the United States by high-density amplicon sequencing of symptomatic cases. 57% of strains carry the spike D614G variant, which is associated with higher genome copy numbers, and its prevalence expands with time. Four strains carry a deletion in a predicted stem loop of the 3′ UTR. The data are consistent with community spread within local populations and the larger continental United States. The data instill confidence in current testing sensitivity and validate "testing by sequencing" as an option to uncover cases, particularly nonstandard coronavirus disease 2019 (COVID-19) clinical presentations. This study contributes to the understanding of COVID-19 through an extensive set of genomes from a non-urban setting and informs vaccine design by defining D614G as a dominant and emergent SARS-CoV-2 isolate in the United States. • NGS of SARS-CoV-2 from a rural/suburban area shows local spread as an epidemic driver • The D614G spike mutant is observed in >50% of cases • Deletion in the 3′ UTR of SARS-CoV-2 is identified • Targeted NGS has 100% specificity and is as sensitive as qPCR McNamara et al. use next-generation sequencing (NGS) with a high-density tiling array across SARS-CoV-2 to find a deletion and document how the D614G spike protein mutation rapidly swept through a rural/suburban population. D614G is associated with slightly higher viral loads. [ABSTRACT FROM AUTHOR]

Gene expression plasticity is central for macrophages' timely responses to cues from the microenvironment permitting phenotypic adaptation from pro-inflammatory (M1) to wound healing and tissue-regenerative (M2, with several subclasses). Regulatory macrophages are a distinct macrophage type, possessing immunoregulatory, anti-inflammatory, and angiogenic properties. Due to these features, regulatory macrophages are considered as a potential cell therapy product to treat clinical conditions, e.g., non-healing diabetic foot ulcers. In this study we characterized two differently manufactured clinically relevant regulatory macrophages, programmable cells of monocytic origin and comparator macrophages (M1, M2a and M0) using flow-cytometry, RT-qPCR, phagocytosis and secretome measurements, and RNA-Seq. We demonstrate that conventional phenotyping had a limited potential to discriminate different types of macrophages which was ameliorated when global transcriptome characterization by RNA-Seq was employed. Using this approach we confirmed that macrophage manufacturing processes can result in a highly reproducible cell phenotype. At the same time, minor changes introduced in manufacturing resulted in phenotypically and functionally distinct regulatory macrophage types. Additionally, we have identified a novel constellation of process specific biomarkers, which will support further clinical product development. [ABSTRACT FROM AUTHOR]