Defining genome-wide expression and phenotypic contextual cues in macrophages generated by GM-CSF, M-CSF and heat-killed mycobacteria

Heat-killed (HK) Mycobacterium obuense (NCTC13365) is currently being evaluated in the clinic as an immunotherapeutic agent for cancer treatment. Yet, the molecular underpinnings underlying immunomodulatory properties of HK M. obuense are still largely undefined. To fill this void, we sought to perform immunophenotyping, chemokine/cytokine release analysis and genome-wide characterization of monocyte-derived macrophages (MDM) in which monocytes were originally isolated from healthy donors and differentiated by HK M. obuense (Mob-MDM) relative to macrophage colony-stimulating factor (M-MDM) and granulocyte/macrophage colony-stimulating factor (GM-MDM). Immunophenotyping and cytokine release analysis revealed downregulated surface expression of CD36, decreased spontaneous release of CCL2 and increased spontaneous secretion of CCL5, CXCL8/IL-8, IL-6, and TNF-α in Mob-MDM relative to M-MDM and GM-MDM. Analysis of cytostatic activity showed that Mob-MDM exhibited similar growth inhibitory effects on immortalized and malignant epithelial cells compared with GM-MDM but at an elevated rate relative to M-MDM. To understand global cues in Mob-MDM, we performed comparative RNA-sequencing (RNA-Seq) analysis of Mob-MDM relative to GM-MDM and M-MDM (n = 4 donors). Clustering analysis underscored expression profiles (n = 256) that were significantly modulated in Mob-MDM versus both M-MDM and GM-MDM including, among others, chemokines/cytokines and their receptors, enzymes and transcriptions factors. Topological functional analysis of these profiles identified pathways and gene sets linked to Mob-MDM phenotype including nitric oxide production, acute phase response signaling and microbe recognition pathways as well as signaling cues mediated by the proinflammatory cytokine, interferon-gamma, and the intracellular pattern recognition receptor, nucleotide-binding oligomerization domain-containing protein 2. Taken together, our study highlights molecular immune phenotypes and global signaling cues in Mob-MDM that may underlie immunomodulatory properties of HK M. obuense. Such properties could be of valuable use in immunotherapy approaches such as adoptive cell therapy against cancer

Environmental shaping of codon usage and functional adaptation across microbial communities.

Microbial communities represent the largest portion of the Earth's biomass. Metagenomics projects use high-throughput sequencing to survey these communities and shed light on genetic capabilities that enable microbes to inhabit every corner of the biosphere. Metagenome studies are generally based on (i) classifying and ranking functions of identified genes; and (ii) estimating the phyletic distribution of constituent microbial species. To understand microbial communities at the systems level, it is necessary to extend these studies beyond the species' boundaries and capture higher levels of metabolic complexity. We evaluated 11 metagenome samples and demonstrated that microbes inhabiting the same ecological niche share common preferences for synonymous codons, regardless of their phylogeny. By exploring concepts of translational optimization through codon usage adaptation, we demonstrated that community-wide bias in codon usage can be used as a prediction tool for lifestyle-specific genes across the entire microbial community, effectively considering microbial communities as meta-genomes. These findings set up a 'functional metagenomics' platform for the identification of genes relevant for adaptations of entire microbial communities to environments. Our results provide valuable arguments in defining the concept of microbial species through the context of their interactions within the community

Cytokine/chemokine release patterns and transcriptomic profiles of LPS/IFNγ-activated human macrophages differentiated with heat-killed 'Mycobacterium obuense', M-CSF, or GM-CSF

Macrophages (Mφs) are instrumental regulators of the immune response whereby they acquire diverse functional phenotypes following their exposure to microenvironmental cues that govern their differentiation from monocytes and their activation. The complexity and diversity of the mycobacterial cell wall have empowered mycobacteria with potent immunomodulatory capacities. A heat-killed (HK) whole-cell preparation of Mycobacterium obuense (M. obuense) has shown promise as an adjunctive immunotherapeutic agent for the treatment of cancer. Moreover, HK M. obuense has been shown to trigger the differentiation of human monocytes into a monocyte-derived macrophage (MDM) type named Mob-MDM. However, the transcriptomic profile and functional properties of Mob-MDMs remain undefined during an activation state. Here, we characterized cytokine/chemokine release patterns and transcriptomic profiles of lipopolysaccharide (LPS)/interferon γ (IFNγ)-activated human MDMs that were differentiated with HK M. obuense (Mob-MDM(LPS/IFNγ)), macrophage colony-stimulating factor M-MDM(LPS/IFNγ)), or granulocyte/macrophage colony-stimulating factor (GM-MDM(LPS/IFNγ)). Mob-MDM(LPS/IFNγ) demonstrated a unique cytokine/chemokine release pattern (interleukin (IL)-10low, IL-12/23p40low, IL-23p19/p40low, chemokine (C-x-C) motif ligand (CXCL)9low) that was distinct from those of M-MDM(LPS/IFNγ) and GM-MDM(LPS/IFNγ). Furthermore, M-MDM(LPS/IFNγ) maintained IL-10 production at significantly higher levels compared to GM-MDM(LPS/IFNγ) and Mob-MDM(LPS/IFNγ) despite being activated with M1-Mφ-activating stimuli. Comparative RNA sequencing analysis pointed to a distinct transcriptome profile for Mob-MDM(LPS/IFNγ) relative to both M-MDM(LPS/IFNγ) and GM-MDM(LPS/IFNγ) that comprised 417 transcripts. Functional gene-set enrichment analysis revealed significant overrepresentation of signaling pathways and biological processes that were uniquely related to Mob-MDM(LPS/IFNγ). Our findings lay a foundation for the potential integration of HK M. obuense in specific cell-based immunotherapeutic modalities such as adoptive transfer of Mφs (Mob-MDM(LPS/IFNγ)) for cancer treatment

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Correction to: Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

The original version of this article unfortunately contained a mistake

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Strategies for identification of somatic variants using the Ion Torrent deep targeted sequencing platform

Abstract Background ‘Next-generation’ (NGS) sequencing has wide application in medical genetics, including the detection of somatic variation in cancer. The Ion Torrent-based (IONT) platform is among NGS technologies employed in clinical, research and diagnostic settings. However, identifying mutations from IONT deep sequencing with high confidence has remained a challenge. We compared various computational variant-calling methods to derive a variant identification pipeline that may improve the molecular diagnostic and research utility of IONT. Results Using IONT, we surveyed variants from the 409-gene Comprehensive Cancer Panel in whole-section tumors, intra-tumoral biopsies and matched normal samples obtained from frozen tissues and blood from four early-stage non-small cell lung cancer (NSCLC) patients. We used MuTect, Varscan2, IONT’s proprietary Ion Reporter, and a simple subtraction we called “Poor Man’s Caller.” Together these produced calls at 637 loci across all samples. Visual validation of 434 called variants was performed, and performance of the methods assessed individually and in combination. Of the subset of inspected putative variant calls (n=223) in genomic regions that were not intronic or intergenic, 68 variants (30%) were deemed valid after visual inspection. Among the individual methods, the Ion Reporter method offered perhaps the most reasonable tradeoffs. Ion Reporter captured 83% of all discovered variants; 50% of its variants were visually validated. Aggregating results from multiple packages offered varied improvements in performance. Conclusions Overall, Ion Reporter offered the most attractive performance among the individual callers. This study suggests combined strategies to maximize sensitivity and positive predictive value in variant calling using IONT deep sequencing

Additional file 1 of Strategies for identification of somatic variants using the Ion Torrent deep targeted sequencing platform

Supplementary Materials. (PDF 1208 kb