Neisseria meningitidis accumulate in large organs during meningococcal sepsis

BackgroundNeisseria meningitidis (Nm) is the cause of epidemic meningitis and fulminant meningococcal septicemia. The clinical presentations and outcome of meningococcal septic shock is closely related to the circulating levels of lipopolysaccharides (LPS) and of Neisseria meningitidis DNA (Nm DNA). We have previously explored the distribution of Nm DNA in tissues from large organs of patients dying of meningococcal septic shock and in a porcine meningococcal septic shock model.Objective1) To explore the feasibility of measuring LPS levels in tissues from the large organs in patients with meningococcal septic shock and in a porcine meningococcal septic shock model. 2) To evaluate the extent of contamination of non-specific LPS during the preparation of tissue samples.Patients and methodsPlasma, serum, and fresh frozen (FF) tissue samples from the large organs of three patients with lethal meningococcal septic shock and two patients with lethal pneumococcal disease. Samples from a porcine meningococcal septic shock model were included. Frozen tissue samples were thawed, homogenized, and prepared for quantification of LPS by Pyrochrome® Limulus Amoebocyte Lysate (LAL) assay.ResultsN. meningitidis DNA and LPS was detected in FF tissue samples from large organs in all patients with meningococcal septic shock. The lungs are the organs with the highest LPS and Nm DNA concentration followed by the heart in two of the three meningococcal shock patients. Nm DNA was not detected in any plasma or tissue sample from patients with lethal pneumococcal infection. LPS was detected at a low level in all FF tissues from the two patients with lethal pneumococcal disease. The experimental porcine meningococcal septic shock model indicates that also in porcinis the highest LPS and Nm DNA concentration are detected in lungs tissue samples. The quantification analysis showed that the highest concentration of both Nm DNA and LPS are in the organs and not in the circulation of patients with lethal meningococcal septic shock. This was also shown in the experimental porcine meningococcal septic shock model.ConclusionOur results suggest that LPS can be quantified in mammalian tissues by using the LAL assay

Isolated focal lesion in the splenium of the corpus callosum in a patient with acute viral hepatitis A

Mitochondrial dysfunction - Nm vs ctr - FDR5. (XLS 46 kb

Distinct microRNA and protein profiles of extracellular vesicles secreted from myotubes from morbidly obese donors with type 2 diabetes in response to electrical pulse stimulation

Lifestyle disorders like obesity, type 2 diabetes (T2D), and cardiovascular diseases can be prevented and treated by regular physical activity. During exercise, skeletal muscles release signaling factors that communicate with other organs and mediate beneficial effects of exercise. These factors include myokines, metabolites, and extracellular vesicles (EVs). In the present study, we have examined how electrical pulse stimulation (EPS) of myotubes, a model of exercise, affects the cargo of released EVs. Chronic low frequency EPS was applied for 24 h to human myotubes isolated and differentiated from biopsy samples from six morbidly obese females with T2D, and EVs, both exosomes and microvesicles (MV), were isolated from cell media 24 h thereafter. Size and concentration of EV subtypes were characterized by nanoparticle tracking analysis, surface markers were examined by flow cytometry and Western blotting, and morphology was confirmed by transmission electron microscopy. Protein content was assessed by high-resolution proteomic analysis (LC-MS/MS), non-coding RNA was quantified by Affymetrix microarray, and selected microRNAs (miRs) validated by real time RT-qPCR. The size and concentration of exosomes and MV were unaffected by EPS. Of the 400 miRs identified in the EVs, EPS significantly changed the level of 15 exosome miRs, of which miR-1233-5p showed the highest fold change. The miR pattern of MV was unaffected by EPS. Totally, about 1000 proteins were identified in exosomes and 2000 in MV. EPS changed the content of 73 proteins in exosomes, 97 in MVs, and of these four were changed in both exosomes and MV (GANAB, HSPA9, CNDP2, and ATP5B). By matching the EPS-changed miRs and proteins in exosomes, 31 targets were identified, and among these several promising signaling factors. Of particular interest were CNDP2, an enzyme that generates the appetite regulatory metabolite Lac-Phe, and miR-4433b-3p, which targets CNDP2. Several of the regulated miRs, such as miR-92b-5p, miR-320b, and miR-1233-5p might also mediate interesting signaling functions. In conclusion, we have used a combined transcriptome-proteome approach to describe how EPS affected the cargo of EVs derived from myotubes from morbidly obese patients with T2D, and revealed several new factors, both miRs and proteins, that might act as exercise factors

A Transcriptomic Appreciation of Childhood Meningococcal and Polymicrobial Sepsis from a Pro-Inflammatory and Trajectorial Perspective, a Role for Vascular Endothelial Growth Factor A and B Modulation?

This study investigated the temporal dynamics of childhood sepsis by analyzing gene expression changes associated with proinflammatory processes. Five datasets, including four meningococcal sepsis shock (MSS) datasets (two temporal and two longitudinal) and one polymicrobial sepsis dataset, were selected to track temporal changes in gene expression. Hierarchical clustering revealed three temporal phases: early, intermediate, and late, providing a framework for understanding sepsis progression. Principal component analysis supported the identification of gene expression trajectories. Differential gene analysis highlighted consistent upregulation of vascular endothelial growth factor A (VEGF-A) and nuclear factor κB1 (NFKB1), genes involved in inflammation, across the sepsis datasets. NFKB1 gene expression also showed temporal changes in the MSS datasets. In the postmortem dataset comparing MSS cases to controls, VEGF-A was upregulated and VEGF-B downregulated. Renal tissue exhibited higher VEGF-A expression compared with other tissues. Similar VEGF-A upregulation and VEGF-B downregulation patterns were observed in the cross-sectional MSS datasets and the polymicrobial sepsis dataset. Hexagonal plots confirmed VEGF-R (VEGF receptor)–VEGF-R2 signaling pathway enrichment in the MSS cross-sectional studies. The polymicrobial sepsis dataset also showed enrichment of the VEGF pathway in septic shock day 3 and sepsis day 3 samples compared with controls. These findings provide unique insights into the dynamic nature of sepsis from a transcriptomic perspective and suggest potential implications for biomarker development. Future research should focus on larger-scale temporal transcriptomic studies with appropriate control groups and validate the identified gene combination as a potential biomarker panel for sepsis

Advancing sepsis clinical research: harnessing transcriptomics for an omics-based strategy - a comprehensive scoping review

Sepsis continues to be recognized as a significant global health challenge across all ages and is characterized by a complex pathophysiology. In this scoping review, PRISMA-ScR guidelines were adhered to, and a transcriptomic methodology was adopted, with the protocol registered on the Open Science Framework. We hypothesized that gene expression analysis could provide a foundation for establishing a clinical research framework for sepsis. A comprehensive search of the PubMed database was conducted with a particular focus on original research and systematic reviews of transcriptomic sepsis studies published between 2012 and 2022. Both coding and non-coding gene expression studies have been included in this review. An effort was made to enhance the understanding of sepsis at the mRNA gene expression level by applying a systems biology approach through transcriptomic analysis. Seven crucial components related to sepsis research were addressed in this study: endotyping (n = 64), biomarker (n = 409), definition (n = 0), diagnosis (n = 1098), progression (n = 124), severity (n = 451), and benchmark (n = 62). These components were classified into two groups, with one focusing on Biomarkers and Endotypes and the other oriented towards clinical aspects. Our review of the selected studies revealed a compelling association between gene transcripts and clinical sepsis, reinforcing the proposed research framework. Nevertheless, challenges have arisen from the lack of consensus in the sepsis terminology employed in research studies and the absence of a comprehensive definition of sepsis. There is a gap in the alignment between the notion of sepsis as a clinical phenomenon and that of laboratory indicators. It is potentially responsible for the variable number of patients within each category. Ideally, future studies should incorporate a transcriptomic perspective. The integration of transcriptomic data with clinical endpoints holds significant potential for advancing sepsis research, facilitating a consensus-driven approach, and enabling the precision management of sepsis

Effect of Storage Temperature on Key Functions of Cultured Retinal Pigment Epithelial Cells

Purpose. Replacement of the diseased retinal pigment epithelium (RPE) with cells capable of performing the specialized functions of the RPE is the aim of cell replacement therapy for treatment of macular degenerative diseases. A storage method for RPE is likely to become a prerequisite for the establishment of such treatment. Herein, we analyze the effect of storage temperature on key functions of cultured RPE cells. Methods. Cultured ARPE-19 cells were stored in Minimum Essential Medium at 4°C, 16°C, and 37°C for seven days. Total RNA was isolated and the gene expression profile was determined using DNA microarrays. Comparison of the microarray expression values with qRT-PCR analysis of selected genes validated the results. Results. Expression levels of several key genes involved in phagocytosis, pigment synthesis, the visual cycle, adherens, and tight junctions, and glucose and ion transport were maintained close to control levels in cultures stored at 4°C and 16°C. Cultures stored at 37°C displayed regulational changes in a larger subset of genes related to phagocytosis, adherens, and tight junctions. Conclusion. RPE cultures stored at 4°C and 16°C for one week are capable of maintaining the expression levels of genes important for key RPE functions close to control levels

Cigarette smoking represses expression of cytokine IL-12 and its regulator miR-21-An observational study in patients with coronary artery disease.

Rationale The heterodimer IL-12 is an inducer of Th1 responses and stimulates INFƴ production. Micro-RNA-21 (miR-21) is described as a key regulator of the pro-inflammatory response and has IL-12p35 mRNA as one of its main targets. The IL-12p40 1188A/C genetic variant located in 3’untranslated region (UTR), thus environmentally exposed, has further been reported to modify IL-12 levels. We have previously reported on the lowering effect of cigarette smoke on circulating IL-12 in patients with coronary artery disease (CAD). Objectives To explore if cigarette smoking affects IL-12p35, IL-12p40, INFƴ and miR-21 gene-expression and further modulates any effect of the IL-12p40 polymorphism on circulating IL-12 levels. Methods and Results The IL-12p40 1188A/C polymorphism was analyzed in 1001 stable CAD patients, of which 330 subjects were included for IL-12p35, IL-12p40 and INFƴ gene-expression analyses in circulating leukocytes and 200 were further selected for plasma miR-21 analysis. Smoking associated with lower expression of miR-21 and its target IL-12p35 mRNA (adjusted p < 0.05, both) whereas the influence on INFƴ expression tended to be high-dose reliant (p = 0.057). The IL-12p40 CC genotype associated with elevated circulating IL-12 levels, however, when stratified according to smoking, only in the non-smoking group (adjusted p < 0.05). Although the markers were mainly downregulated in current smokers, their inter-correlations were potentiated. Conclusion Smoking associated with reduced miR-21 gene-repression and the results can therefore not explain the previously observed reduction in circulating IL-12. Smoking attenuated the IL-12 pro-inflammatory axis in which the investigated IL-12p40 genetic variant may have different clinical impact in smokers vs non-smokers. Opstad, T. B., et al. "Cigarette smoking represses expression of cytokine IL-12 and its regulator miR-21—An observational study in patients with coronary artery disease." Immunobiology 222.2 (2017): 169-175. © 2016. This manuscript version is made available under the CC-BY-NC-ND 4.0 license

Global miRNA expression analysis of serous and clear cell ovarian carcinomas identifies differentially expressed miRNAs including miR-200c-3p as a prognostic marker

Background Improved insight into the molecular characteristics of the different ovarian cancer subgroups is needed for developing a more individualized and optimized treatment regimen. The aim of this study was to a) identify differentially expressed miRNAs in high-grade serous ovarian carcinoma (HGSC), clear cell ovarian carcinoma (CCC) and ovarian surface epithelium (OSE), b) evaluate selected miRNAs for association with clinical parameters including survival and c) map miRNA-mRNA interactions. Methods Differences in miRNA expression between HGSC, CCC and OSE were analyzed by global miRNA expression profiling (Affymetrix GeneChip miRNA 2.0 Arrays, n = 12, 9 and 9, respectively), validated by RT-qPCR (n = 35, 19 and 9, respectively), and evaluated for associations with clinical parameters. For HGSC, differentially expressed miRNAs were linked to differentially expressed mRNAs identified previously. Results Differentially expressed miRNAs (n = 78) between HGSC, CCC and OSE were identified (FDR < 0.01%), of which 18 were validated (p < 0.01) using RT-qPCR in an extended cohort. Compared with OSE, miR-205-5p was the most overexpressed miRNA in HGSC. miR-200 family members and miR-182-5p were the most overexpressed in HGSC and CCC compared with OSE, whereas miR-383 was the most underexpressed. miR-205-5p and miR-200 members target epithelial-mesenchymal transition (EMT) regulators, apparently being important in tumor progression. miR-509-3-5p, miR-509-5p, miR-509-3p and miR-510 were among the strongest differentiators between HGSC and CCC, all being significantly overexpressed in CCC compared with HGSC. High miR-200c-3p expression was associated with poor progression-free (p = 0.031) and overall (p = 0.026) survival in HGSC patients. Interacting miRNA and mRNA targets, including those of a TP53-related pathway presented previously, were identified in HGSC. Conclusions Several miRNAs differentially expressed between HGSC, CCC and OSE have been identified, suggesting a carcinogenetic role for these miRNAs. miR-200 family members, targeting EMT drivers, were mostly overexpressed in both subgroups, among which miR-200c-3p was associated with survival in HGSC patients. A set of miRNAs differentiates CCC from HGSC, of which miR-509-3-5p and miR-509-5p are the strongest classifiers. Several interactions between miRNAs and mRNAs in HGSC were mapped

Use of Robotized DNA Isolation and Real-Time PCR To Quantify and Identify Close Correlation between Levels of Neisseria meningitidis DNA and Lipopolysaccharides in Plasma and Cerebrospinal Fluid from Patients with Systemic Meningococcal Disease

The present study, using robotized DNA isolation and quantitative PCR based on the Neisseria meningitidis-specific capsular transport A gene, demonstrates the ease, rapidity, specificity, and sensitivity of quantifying neisserial DNA in plasma (n = 65) and cerebrospinal fluid (CSF) (n = 12) from patients with systemic meningococcal disease. We found a close correlation between the levels of neisserial DNA and lipopolysaccharides in plasma (r = 0.905) and in CSF (r = 0.964). The median concentration of neisserial DNA in plasma in 23 patients with persistent shock was 2 × 10(7) copies/ml, versus <10(3) copies/ml in 42 nonshock patients. Furthermore, quantitative PCR made possible estimates of the total number of meningococci in plasma, as opposed to conventional blood cultures, suggesting about 1,000 dead meningococci for every viable bacterium. Finally, with logistic regression analyses, neisserial DNA may predict a patient's disease severity and outcome at hospital admission. The number of meningococci in plasma and CSF appears to be the main determinant of the lipopolysaccharide levels, clinical presentation, and outcome

Urine β-2-Microglobulin, Osteopontin, and Trefoil Factor 3 May Early Predict Acute Kidney Injury and Outcome after Cardiac Arrest

Purpose. Acute kidney injury (AKI) is a common complication after out-of-hospital cardiac arrest (OHCA), leading to increased mortality and challenging prognostication. Our aim was to examine if urine biomarkers could early predict postarrest AKI and patient outcome. Methods. A prospective observational study of resuscitated, comatose OHCA patients admitted to Oslo University Hospital in Norway. Urine samples were collected at admission and day three postarrest and analysed for β-2-microglobulin (β2M), osteopontin, and trefoil factor 3 (TFF3). Outcome variables were AKI within three days according to the Kidney Disease Improving Global Outcome criteria, in addition to six-month mortality and poor neurological outcome (PNO) (cerebral performance category 3–5). Results. Among 195 included patients (85% males, mean age 60 years), 88 (45%) developed AKI, 88 (45%) died, and 96 (49%) had PNO. In univariate analyses, increased urine β2M, osteopontin, and TFF3 levels sampled at admission and day three were independent risk factors for AKI, mortality, and PNO. Exceptions were that β2M measured at day three did not predict any of the outcomes, and TFF3 at admission did not predict AKI. In multivariate analyses, combining clinical parameters and biomarker levels, the area under the receiver operating characteristics curves (95% CI) were 0.729 (0.658–0.800), 0.797 (0.733–0.861), and 0.812 (CI 0.750–0.874) for AKI, mortality, and PNO, respectively. Conclusions. Urine levels of β2M, osteopontin, and TFF3 at admission and day three were associated with increased risk for AKI, mortality, and PNO in comatose OHCA patients. This trail is registered with NCT01239420