Increased ferroptosis in leukocytes from preeclamptic women involving the long non-coding taurine upregulated gene 1 (TUG1).

BACKGROUND: Ferroptosis plays a key role in placental development and physiology, and abnormal ferroptosis has been implicated in trophoblast injury leading to preeclampsia (PE). We hypothesize that leukocytes isolated from PE exhibit increased ferroptosis and that extracellular vesicles contain long non-coding (lnc) RNA/mRNAs that modulate oxidative stress and iron toxicity in vascular endothelial cells. METHODS: We measured the expression of key regulators of ferroptosis in leukocytes and extracellular vesicles as well as circulating biomarkers of iron homeostasis and oxidative stress in plasma from women with/without PE at different timepoints during pregnancy. For markers that were dysregulated, we assessed their temporal correlation with established markers of disease activity and marker of endothelial activation. For markers dysregulated in early pregnancy, we assessed their ability to predict the development of PE. RESULTS: We found decreased lncRNA/mRNAs in leukocytes, but not extracellular vesicles, in PE that may modulate oxidative stress and iron toxicity. This decrease in anti-ferroptotic markers does not appear to be related to maternal disease activity or plasma oxidative stress status but rather to attenuated anti-inflammatory expression in these cells. Circulating ferritin was elevated in PE, supporting the hypothesis that PE represents a disbalance in iron homeostasis. Low lncRNA taurine upregulated gene 1 RNA levels in leukocytes at 22-24 weeks were strongly associated with the development of PE. CONCLUSIONS: Our findings suggest that maternal leukocytes in PE show decreased anti-ferroptotic activity that correlates with anti-inflammatory expression. Moreover, some of these changes in ferroptotic activity appear to precede the development of PE.

RNA Profiles of Tear Fluid Extracellular Vesicles in Patients with Dry Eye-Related Symptoms.

Currently, diagnosing and stratifying dry eye disease (DED) require multiple tests, motivating interest in a single definitive test. The purpose of this study was to investigate the potential for using tear fluid extracellular vesicle (EV)-RNA in DED diagnostics. With a role in intercellular communication, nanosized EVs facilitate the protected transport of diverse bioactive molecules in biofluids, including tears. Schirmer strips were used to collect tears from 10 patients presenting with dry eye-related symptoms at the Norwegian Dry Eye Clinic. The samples comprised two groups, five from patients with a tear film break-up time (TBUT) of 2 s and five from patients with a TBUT of 10 s. Tear fluid EV-RNA was isolated using a Qiagen exoRNeasy Midi Kit, and the RNA was characterized using Affymetrix ClariomTM D microarrays. The mean signal values of the two groups were compared using a one-way ANOVA. A total of 26,639 different RNA transcripts were identified, comprising both mRNA and ncRNA subtypes. Approximately 6% of transcripts showed statistically significant differential abundance between the two groups. The mRNA sodium channel modifier 1 (SCNM1) was detected at a level 3.8 times lower, and the immature microRNA-130b was detected at a level 1.5 times higher in the group with TBUT 2 s compared to the group with TBUT 10 s. This study demonstrates the potential for using tear fluid EV-RNA in DED diagnostics.

Non-Coding RNA in Salivary Extracellular Vesicles: A New Frontier in Sjögren's Syndrome Diagnostics?

Sjögren's syndrome is an autoimmune rheumatic disease characterized by inflammation of the salivary and lacrimal glands, often manifesting as dry mouth and dry eyes. To simplify diagnostics of primary Sjögren's syndrome (pSS), a non-invasive marker is needed. The aim of the study was to compare the RNA content of salivary extracellular vesicles (EVs) between patients with pSS and healthy controls using microarray technology. Stimulated whole saliva was collected from 11 pSS patients and 11 age-matched controls. EV-RNA was isolated from the saliva samples using a Qiagen exoRNeasy Midi Kit and analyzed using Affymetrix Clariom D™ microarrays. A one-way ANOVA test was used to compare the mean signal values of each transcript between the two groups. A total of 9307 transcripts, coding and non-coding RNA, were detected in all samples. Of these transcripts, 1475 showed statistically significant differential abundance between the pSS and the control groups, generating two distinct EV-RNA patterns. In particular, tRNAs were downregulated in pSS patients, with the transcript tRNA-Ile-AAT-2-1 showing a 2-fold difference, and a promise as a potential biomarker candidate. This study therein demonstrates the potential for using salivary EV-RNA in pSS diagnostics.

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.

Recombinant irisin enhances the extracellular matrix formation, remodeling potential, and differentiation of human periodontal ligament cells cultured in 3D.

BACKGROUND: Irisin is expressed in human periodontal ligament (hPDL), and its administration enhances growth, migration and matrix deposition in hPDL cells cultured in monolayers in vitro. OBJECTIVES: To identify whether irisin affects the gene expression patterns directing the morphology, mechanical properties, extracellular matrix (ECM) formation, osteogenic activity and angiogenic potential in hPDL cell spheroids cultured in 3D. MATERIALS AND METHODS: Spheroids of primary human hPDL cells were generated in a rotational 3D culture system and treated with or without irisin. The gene expression patterns were evaluated by Affymetrix microarrays. The morphology of the spheroids was characterized using histological staining. Mechanical properties were quantified by nanoindentation. The osteogenic and angiogenic potential of spheroids were assessed through immunofluorescence staining for collagen type I, periostin fibronectin and von Willebrand factor (vWF), and mRNA expression of osteogenic markers. The secretion of multiple myokines was evaluated using Luminex immunoassays. RESULTS: Approximately 1000 genes were differentially expressed between control and irisin-treated groups by Affymetrix. Several genes related to ECM organization were differentially expressed, and multiple deubiquitinating enzymes were upregulated in the irisin-exposed samples analyzed. These represent cellular and molecular mechanisms indicative of a role for irisin in tissue remodeling. Irisin induced a rim-like structure on the outer region of the hPDL spheroids, ECM-related protein expression and the stiffness of the spheroids were enhanced by irisin. The expression of osteogenic and angiogenetic markers was increased by irisin. CONCLUSIONS: Irisin altered the morphology in primary hPDL cell-derived spheroids, enhanced its ECM deposition, mechanical properties, differentiation and remodeling potential.

Transcriptomic changes in the large organs in lethal meningococcal shock are reflected in a porcine shock model.

Background: Fulminant meningococcal sepsis with shock and multiple organ failure is associated with a massive systemic inflammatory response involving solid organs. We have previously established a porcine model of the disease to study pathophysiologic and possible therapeutic strategies. Objective: This study examined whether the organ specific gene expression profile in such a large animal model reflects the profile seen in patients with fulminant meningococcal sepsis. Patients and methods: Data from gene expression profiles induced in organs from patients (n=5) and the porcine model (n=8) were imported into the Ingenuity pathway analysis (IPA) software for comparison analysis. The number of meningococci in the organs were quantified by real time-PCR. Results: The all-over transcriptional activation between different organs revealed a striking concordance between the patients and the pigs regarding the pattern of transcriptional activation and activated pathways. Comparison analysis demonstrated similar pattern of upregulation of genes being associated with a large range of inflammatory biofunctions in the patients and the porcine model. Genes associated with biofunctions such as organismal death, morbidity and mortality were similarly downregulated in the patients and the porcine model. Comparison analysis of main predicted canonical pathways also demonstrated a high degree of similarity regarding up- and downregulation in both groups. Core analysis revealed different top-upstream regulators in the different organs in the patients. In the patients pro-inflammatory regulators were most activated in the lungs. In the other organs up-stream factors that regulate signaling pathways involved in development, growth, repair and homeostasis and triglyceride synthesis were most activated. In the porcine model, the top-upstream regulators were pro-inflammatory in all organs. The difference may reflect the shorter duration of the porcine experiment than the duration of the patient's infection before death. Conclusion: The inflammatory responses measured on the transcriptomic level in organs in patients with fulminant meningococcal sepsis is reproduced in the porcine model of the disease, although some differences may exist regarding the top-upregulated factors in individual organs. Thus, this large animal model reproduces important immunological features of meningococcal sepsis and can be a valuable tool in further investigations of inflammatory aspects and possible treatment options.

Dysregulated non-coding telomerase RNA component and associated exonuclease XRN1 in leucocytes from women developing preeclampsia-possible link to enhanced senescence.

Senescence in placenta/fetal membranes is a normal phenomenon linked to term parturition. However, excessive senescence which may be induced by telomere attrition, has been associated with preeclampsia (PE). We hypothesized that the telomerase complex in peripheral blood mononuclear cells (PBMC) and circulating telomere associated senescence markers would be dysregulated in women with PE. We measured long non-coding (nc) RNA telomerase RNA component (TERC) and RNAs involved in the maturation of TERC in PBMC, and the expression of TERC and 5'-3' Exoribonuclease 1 (XRN1) in extracellular vesicles at 22-24 weeks, 36-38 weeks and, 5-year follow-up in controls and PE. We also measured telomere length at 22-24 weeks and 5-year follow-up. The circulating senescence markers cathelicidin antimicrobial peptide (CAMP), ß-galactosidase, stathmin 1 (STMN1) and chitotriosidase/CHIT1 were measured at 14-16, 22-24, 36-38 weeks and at 5-year follow-up in the STORK study and before delivery and 6 months post-partum in the ACUTE PE study. We found decreased expression of TERC in PBMC early in pregnant women who subsequently developed PE. XRN1 involved in the maturation of TERC was also reduced in pregnancy and 5-year follow-up. Further, we found that the senescence markers CAMP and ß-galactosidase were increased in PE pregnancies, and CAMP remained higher at 5-year follow-up. ß-galactosidase was associated with atherogenic lipid ratios during pregnancy and at 5-year follow-up, in PE particularly. This study suggests a potential involvement of dysfunctional telomerase biology in the pathophysiology of PE, which is not restricted to the placenta.

Integrated differential DNA methylation and gene expression of formalin-fixed paraffin-embedded uveal melanoma specimens identifies genes associated with early metastasis and poor prognosis.

PURPOSE: Uveal melanoma (UM) is an aggressive malignancy, in which nearly 50% of the patients die from metastatic disease. Aberrant DNA methylation is recognized as an important epigenomic event in carcinogenesis. Formalin-fixed paraffin-embedded (FFPE) samples represent a valuable source of tumor tissue, and recent technology has enabled the use of these samples in genome-wide DNA methylation analyses. Our aim was to investigate differential DNA methylation in relation to histopathological classification and survival data. In addition we sought to identify aberrant DNA methylation of genes that could be associated with metastatic disease and poor survival. METHODS: FFPE samples from UM patients (n = 23) who underwent enucleation of the eye in the period 1976-1989 were included. DNA methylation was assessed using the Illumina Infinium HumanMethylation450 array and coupled to histopathological data, Cancer Registry of Norway- (registered UM metastasis) and Norwegian Cause of Death Registry- (time and cause of death) data. Differential DNA methylation patterns contrasting histological classification, survival data and clustering properties were investigated. Survival groups were defined as "Early metastasis" (metastases and death within 2-5 years after enucleation, n = 8), "Late metastasis" (metastases and death within 9-21 years after enucleation, n = 7) and "No metastasis" (no detected metastases ≥18 years after enucleation, n = 8). A subset of samples were selected based on preliminary multi-dimensional scaling (MDS) plots, histopathological classification, chromosome 3 status, survival status and clustering properties; "Subset Early metastasis" (n = 4) vs "Subset No metastasis" (n = 4). Bioinformatics analyses were conducted in the R statistical software. Differentially methylated positions (DMPs) and differentially methylated regions (DMRs) in various comparisons were assessed. Gene expression of relevant subgroups was determined by microarray analysis and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). RESULTS: DNA methylation analyses identified 2 clusters that separated the samples according to chromosome 3 status. Cluster 1 consisted of samples (n = 5) with chromosome 3 disomy (D3), while Cluster 2 was comprised of samples (n = 15) with chromosome 3 monosomy (M3). 1212 DMRs and 9386 DMPs were identified in M3 vs D3. No clear clusters were formed based on our predefined survival groups ("Early", "Late", "No") nor histopathological classification (Epithelioid, Mixed, Spindle). We identified significant changes in DNA methylation (beta FC ≥ 0.2, adjusted p < 0.05) between two sample subsets (n = 8). "Subset Early metastasis" (n = 4) vs "Subset No metastasis" (n = 4) identified 348 DMPs and 36 DMRs, and their differential gene expression by microarray showed that 14 DMPs and 2 DMRs corresponded to changes in gene expression (FC ≥ 1.5, p < 0.05). RNF13, ZNF217 and HYAL1 were hypermethylated and downregulated in "Subset Early metastasis" vs "Subset No metastasis" and could be potential tumor suppressors. TMEM200C, RGS10, ADAM12 and PAM were hypomethylated and upregulated in "Subset Early metastasis vs "Subset No metastasis" and could be potential oncogenes and thus markers of early metastasis and poor prognosis in UM. CONCLUSIONS: DNA methylation profiling showed differential clustering of samples according to chromosome 3 status: Cluster 1 (D3) and Cluster 2 (M3). Integrated differential DNA methylation and gene expression of two subsets of samples identified genes associated with early metastasis and poor prognosis. RNF13, ZNF217 and HYAL1 are hypermethylated and candidate tumor suppressors, while TMEM200C, RGS10, ADAM12 and PAM are hypomethylated and candidate oncogenes linked to early metastasis. UM FFPE samples represent a valuable source for methylome studies and enable long-time follow-up.

Uptake of circulating extracellular vesicles from rectal cancer patients and differential responses by human monocyte cultures.

Extracellular vesicles (EVs) released by tumor cells can directly or indirectly modulate the phenotype and function of the immune cells of the microenvironment locally or at distant sites. The uptake of circulating EVs and the responses by human monocytes in vitro may provide new insights into the underlying biology of the invasive and metastatic processes in cancer. Although a mixed population of vesicles is obtained with most isolation techniques, we predominantly isolated exosomes (small EVs) and microvesicles (medium EVs) from the SW480 colorectal cancer cell line (established from a primary adenocarcinoma of the colon) by sequential centrifugation and ultrafiltration, and plasma EVs were prepared from 22 patients with rectal adenoma polyps or invasive adenocarcinoma by size-exclusion chromatography. The EVs were thoroughly characterized. The uptake of SW480 EVs was analyzed, and small SW480 EVs were observed to be more potent than medium SW480 EVs in inducing monocyte secretion of cytokines. The plasma EVs were also internalized by monocytes; however, their cytokine-releasing potency was lower than that of the cell line-derived vesicles. The transcriptional changes in the monocytes highlighted differences between adenoma and adenocarcinoma patient EVs in their ability to regulate biological functions, whereas the most intriguing changes were found in monocytes receiving EVs from patients with metastatic compared with localized cancer.

Cytokine and Gene Expression Profiling in Patients with HFE-Associated Hereditary Hemochromatosis according to Genetic Profile.

BACKGROUND: Hemochromatosis gene (HFE)-associated hereditary hemochromatosis (HH) is characterized by downregulation of hepcidin synthesis, leading to increased intestinal iron absorption. OBJECTIVES: The objectives were to characterize and elucidate a possible association between gene expression profile, hepcidin levels, disease severity, and markers of inflammation in HFE-associated HH patients. METHODS: Thirty-nine HFE-associated HH patients were recruited and assigned to 2 groups according to genetic profile: C282Y homozygotes in 1 group and patients with H63D, as homozygote or in combination with C282Y, in the other group. Eleven healthy first-time blood donors were recruited as controls. Gene expression was characterized from peripheral blood cells, and inflammatory cytokines and hepcidin-25 isoform were quantified in serum. Biochemical disease characteristics were recorded. RESULTS: Elevated levels of interleukin 8 were observed in a significant higher proportion of patients than controls. In addition, compared to controls, gene expression of ζ-globin was significantly increased among C282Y homozygote patients, while gene expression of matrix metalloproteinase 8, and other neutrophil-secreted proteins, was significantly upregulated in patients with H63D. CONCLUSION: Different disease signatures may characterize HH patients according to their HFE genetic profile. Studies on larger populations, including analyses at protein level, are necessary to confirm these findings.

Sericin-Induced Melanogenesis in Cultured Retinal Pigment Epithelial Cells Is Associated with Elevated Levels of Hydrogen Peroxide and Inflammatory Proteins.

We previously demonstrated that the silk protein sericin promotes pigmentation of retinal pigment epithelium (RPE) by activating the NF-κB pathway. Among numerous agents, NF-κB can be activated by hydrogen peroxide. In the present study, we explored possible associations between reactive oxygen species and sericin-induced melanogenesis in RPE. The proteome of human fetal RPE cultured for seven days with or without 1% sericin was analyzed using ingenuity pathway analysis (IPA). The proteomic data was verified by immunofluorescence and immunoblotting. Light microscopy and scanning electron microscopy were used to assess morphology. Dihydroethidium (DHE) and dihydrorhodamine (DHR) assays were used to measure superoxide and hydrogen peroxide species. Expression levels of proteins related to inflammation, differentiation, cell survival and cell adhesion were higher in cells cultured in Dulbecco's Modified Eagle Medium (DMEM) with 1% sericin, whereas cells cultured in DMEM alone showed higher expression levels of proteins associated with Bruch's membrane and cytoskeleton. Despite upregulation of inflammatory proteins, sericin co-cultured RPE yielded significantly higher cell viability compared to cells cultured without sericin. Addition of sericin to culture media significantly increased hydrogen peroxide-levels without significantly affecting superoxide-levels. We suggest that sericin-induced melanogenesis in cultured RPE is associated with elevated levels of superoxide dismutase, hydrogen peroxide and inflammatory proteins.

Lacrimal Gland Myoepithelial Cells Are Altered in a Mouse Model of Dry Eye Disease.

The purpose of this study was to determine the pathogenic changes that occur in myoepithelial cells (MECs) from lacrimal glands of a mouse model of Sjögren syndrome. MECs were cultured from lacrimal glands of C57BL/6J [wild type (WT)] and thrombospondin 1 null (TSP1-/-, alias Thbs1-/-) mice and from mice expressing α-smooth muscle actin-green fluorescent protein that labels MECs. MECs were stimulated with cholinergic and α1-adrenergic agonists, vasoactive intestinal peptide (VIP), and the purinergic agonists ATP and UTP. Then intracellular [Ca2+] was measured using fura-2, and contraction was observed using live cell imaging. Expression of purinergic receptors was determined by Western blot analysis, and mRNA expression was analyzed by microarray. The increase in intracellular [Ca2+]I with VIP and UTP was significantly smaller in MECs from TSP1-/- compared with WT mice. Cholinergic agonists, ATP, and UTP stimulated contraction in MECs, although contraction of MECs from TSP1-/- mice was reduced compared with WT mice. The amount of purinergic receptors P2Y1, P2Y11, and P2Y13 was significantly decreased in MECs from TSP1-/- compared with WT mice, whereas several extracellular matrix and inflammation genes were up-regulated in MECs from TSP1-/- mice. We conclude that lacrimal gland MEC function is altered by inflammation because the functions regulated by cholinergic agonists, VIP, and purinergic receptors are decreased in TSP1-/- compared with WT mice.

Postmenopausal osteoporosis is a musculoskeletal disease with a common genetic trait which responds to strength training: a translational intervention study.

BACKGROUND: Clinical evidence suggests that body muscle mass is positively associated with bone mass, of significance for the elderly population at risk of osteoporosis (OP). Furthermore, muscle and bone interact mechanically and functionally, via local interactions as well as remotely via secreted components. Thus, it was of interest to compare muscle transcriptomes in postmenopausal OP and healthy women, and study effects of strength training on the muscle transcriptome, muscle stress proteins and bone mineral density (BMD). METHODS: Skeletal muscle histological and genetic properties were compared in postmenopausal healthy (n = 18) and OP (n = 17) women before and after heavy-load strength training for 13-15 weeks. The cohorts were of similar age and body mass index without interfering diseases, medication or difference in lifestyle factors. Muscle biopsies obtained before and after intervention were studied histologically, and stress proteins and transcriptomes analyzed. RESULTS: The OP women showed distinct muscle transcription profiles when compared with healthy women and had higher levels of the stress proteins HSP70 and α-ß-crystalline. A set of 12 muscle transcripts, including ACSS3, FZD4, GNAI1 and IGF1, were differentially expressed before and after intervention (false discovery rate ⩽0.10, p ⩽0.001), and their corresponding bone transcripts were associated with BMD. Experimental data underline and describe the functionality of these genes in bone biology. OP women had 8% (p <0.01) higher proportion of type I fibres, but muscle fibre cross-sectional area did not differ. Muscle strength increased in both groups (p <0.01). CONCLUSIONS: Postmenopausal healthy and OP women have distinct muscle transcriptomes [messenger ribonucleic acids (mRNAs) and microRNAs] that are modulated by strength training, translating into key protein alterations and muscle fibre changes. The function of common skeletal muscle and bone genes in postmenopausal OP is suggestive of a shared disease trait.

Vitamin D metabolites influence expression of genes concerning cellular viability and function in insulin producing ß-cells (INS1E).

BACKGROUND: Studies have shown that vitamin D can enhance glucose-stimulated insulin secretion (GSIS) and change the expression of genes in pancreatic ß-cells. Still the mechanisms linking vitamin D and GSIS are unknown. MATERIAL AND METHODS: We used an established ß-cell line, INS1E. INS1E cells were pre-treated with 10 nM 1,25(OH)2vitamin D or 10 nM 25(OH)vitamin D for 72 h and stimulated with 22 mM glucose for 60 min. RNA was extracted for gene expression analysis. RESULTS: Expression of genes affecting viability, apoptosis and GSIS changed after pre-treatment with both 1,25(OH)2vitamin D and 25(OH)vitamin D in INS1E cells. Stimulation with glucose after pre-treatment of INS1E cells with 1,25(OH)2vitamin D resulted in 181 differentially expressed genes, whereas 526 genes were differentially expressed after pre-treatment with 25(OH)vitamin D. CONCLUSION: Vitamin D metabolites may affect pancreatic ß-cells and GSIS through changed gene expression for genes involved in ß-cell function and viability.

Extensive Changes in Transcriptomic "Fingerprints" and Immunological Cells in the Large Organs of Patients Dying of Acute Septic Shock and Multiple Organ Failure Caused by Neisseria meningitidis.

Background: Patients developing meningococcal septic shock reveal levels of Neisseria meningitidis (106-108/mL) and endotoxin (101-103 EU/mL) in the circulation and organs, leading to acute cardiovascular, pulmonary and renal failure, coagulopathy and a high case fatality rate within 24 h. Objective: To investigate transcriptional profiles in heart, lungs, kidneys, liver, and spleen and immunostain key inflammatory cells and proteins in post mortem formalin-fixed, paraffin-embedded (FFPE) tissue samples from meningococcal septic shock patients. Patients and Methods: Total RNA was isolated from FFPE and fresh frozen (FF) tissue samples from five patients and two controls (acute non-infectious death). Differential expression of genes was detected using Affymetrix microarray analysis. Lung and heart tissue samples were immunostained for T-and B cells, macrophages, neutrophils and the inflammatory markers PAI-1 and MCP-1. Inflammatory mediators were quantified in lysates from FF tissues. Results: The transcriptional profiles showed a complex pattern of protein-coding and non-coding RNAs with significant regulation of pathways associated with organismal death, cell death and survival, leukocyte migration, cellular movement, proliferation of cells, cell-to-cell signaling, immune cell trafficking, and inflammatory responses in an organ-specific clustering manner. The canonical pathways including acute phase response-, EIF2-, TREM1-, IL-6-, HMBG1-, PPAR signaling, and LXR/RXR activation were associated with acute heart, pulmonary, and renal failure. Fewer genes were regulated in the liver and particularly in the spleen. The main upstream regulators were TNF, IL-1ß, IL-6, RICTOR, miR-6739-3p, and CD3. Increased numbers of inflammatory cells (CD68+, MPO+, CD3+, and CD20+) were found in lungs and heart. PAI-1 inhibiting fibrinolysis and MCP-1 attracting leukocyte were found significantly present in the septic tissue samples compared to the controls. Conclusions: FFPE tissue samples can be suitable for gene expression studies as well as immunostaining of specific cells or molecules. The most pronounced gene expression patterns were found in the organs with highest levels of Neisseria meningitidis DNA. Thousands of protein-coding and non-coding RNA transcripts were altered in lungs, heart and kidneys. We identified specific biomarker panels both protein-coding and non-coding RNA transcripts, which differed from organ to organ. Involvement of many genes and pathways add up and the combined effect induce organ failure.

Healthy Nordic Diet Modulates the Expression of Genes Related to Mitochondrial Function and Immune Response in Peripheral Blood Mononuclear Cells from Subjects with Metabolic Syndrome-A SYSDIET Sub-Study.

SCOPE: To explore the effect of a healthy Nordic diet on the global transcriptome profile in peripheral blood mononuclear cells (PBMCs) of subjects with metabolic syndrome. METHODS AND RESULTS: Subjects with metabolic syndrome undergo a 18/24 week randomized intervention study comparing an isocaloric healthy Nordic diet with an average habitual Nordic diet served as control (SYSDIET study). Altogether, 68 participants are included. PBMCs are obtained before and after intervention and total RNA is subjected to global transcriptome analysis. 1302 probe sets are differentially expressed between the diet groups (p-value < 0.05). Twenty-five of these are significantly regulated (FDR q-value < 0.25) and are mainly involved in mitochondrial function, cell growth, and cell adhesion. The list of 1302 regulated probe sets is subjected to functional analyses. Pathways and processes involved in the mitochondrial electron transport chain, immune response, and cell cycle are downregulated in the healthy Nordic diet group. In addition, gene transcripts with common motifs for 42 transcription factors, including NFR1, NFR2, and NF-κB, are downregulated in the healthy Nordic diet group. CONCLUSION: These results suggest that benefits of a healthy diet may be mediated by improved mitochondrial function and reduced inflammation.

Differential expression of vitamin D associated genes in the aorta of coronary artery disease patients with and without rheumatoid arthritis.

BACKGROUND: Vitamin D has an important role in the immune system, and has been linked to rheumatoid arthritis (RA) and coronary artery disease (CAD). The exact mechanisms by which vitamin D is involved in these processes are still unclear. Therefore, we wanted to search for differences in expression of genes involved in the vitamin D receptor (VDR) activation pathway and genes that are known to alter upon vitamin D stimulation, in the aortic adventitia of CAD patients with and without RA. METHODS: Affymetrix microarray was used to determine gene expression profile in surgical specimens from the adventitia of the ascending aorta of CAD patients with RA (n = 8) and without RA (n = 8) from the Feiring Heart Biopsy Study. RESULTS: We identified three vitamin D associated genes that were differentially expressed between RA and non-RA patients: Growth arrest and DNA-damage-inducible protein 45 alpha (GADD45A) (FC = 1.47; p = 0.006), Nuclear Receptor Co-repressor 1 (NCOR1) (FC = 1,21; p = 0.005) and paraoxonases 2 (PON2) (FC = -1.37; p = 0.01). High expression of GADD45A in RA tissues was confirmed by real-time qRT-PCR. GADD45A expression correlated with plasma levels of 1,25(OH)2D3 (rs = 0.69; p = 0.003). CONCLUSIONS: Microarray analyses revealed higher expression of GADD45A and NCOR1; and lower expression of PON2 in the aortic adventitia of RA than non-RA patients. Further studies are needed to elucidate if and how GADD45A, NCOR1 and PON2 are involved in the development of accelerated atherosclerosis in RA. In theory, some of these factors might have proatherogenic effects whereas others might reflect an underlying vascular pathology promoting atherogenesis (such as vascular stress).

Transcription and microRNA Profiling of Cultured Human Tympanic Membrane Epidermal Keratinocytes.

The human tympanic membrane (TM) has a thin outer epidermal layer which plays an important role in TM homeostasis and ear health. The specialised cells of the TM epidermis have a different physiology compared to normal skin epidermal keratinocytes, displaying a dynamic and constitutive migration that maintains a clear TM surface and assists in regeneration. Here, we characterise and compare molecular phenotypes in keratinocyte cultures from TM and normal skin. TM keratinocytes were isolated by enzymatic digestion and cultured in vitro. We compared global mRNA and microRNA expression of the cultured cells with that of human epidermal keratinocyte cultures. Genes with either relatively higher or lower expression were analysed further using the biostatistical tools g:Profiler and Ingenuity Pathway Analysis. Approximately 500 genes were found differentially expressed. Gene ontology enrichment and Ingenuity analyses identified cellular migration and closely related biological processes to be the most significant functions of the genes highly expressed in the TM keratinocytes. The genes of low expression showed a marked difference in homeobox (HOX) genes of clusters A and C, giving the TM keratinocytes a strikingly low HOX gene expression profile. An in vitro scratch wound assay showed a more individualised cell movement in cells from the tympanic membrane than normal epidermal keratinocytes. We identified 10 microRNAs with differential expression, several of which can also be linked to regulation of cell migration and expression of HOX genes. Our data provides clues to understanding the specific physiological properties of TM keratinocytes, including candidate genes for constitutive migration, and may thus help focus further research.

Proliferative Cells Isolated from the Adult Human Peripheral Retina only Transiently Upregulate Key Retinal Markers upon Induced Differentiation.

Purpose/Aim: The adult human retina has limited regenerative potential, and severe injury will result in permanent damage. Lower vertebrates handle retinal injury by activating neural stem cells (NSCs) in the ciliary marginal zone (CMZ). Müller glia-like cells expressing markers of NSCs are also present in the peripheral retina (PR) of the adult human eye, leading to the hypothesis that a CMZ-like zone might exists also in humans. In order to shed further light on this hypothesis we investigated the in vitro differentiation potential of proliferative cells isolated from the adult human PR towards a retinal phenotype. MATERIALS AND METHODS: Proliferative cells were isolated from the peripheral retina of human eyes (n = 6) within 24 to 48 hours post mortem and further expanded for 2 or 3 passages before being differentiated for 1-3 weeks. Gene expression was analyzed by microarray and qRT-PCR analysis, while protein expression was identified by immunocytochemistry. RESULTS: A high density of cells co-staining with markers for progenitor cells and Müller glia was found in situ in the PR. Cells isolated from this region and cultured adherently showed fibrillary processes and were positive for the immature marker Nestin and the glial marker GFAP, while a few co-expressed PAX6. After 7 days of differentiation, there was a transient upregulation of early and mature photoreceptor markers, including NRL, CRX, RHO and RCVRN, as well as the Müller cell and retinal pigmented epithelium (RPE) marker CRALBP, and the early RPE marker MITF. However, the expression of all these markers dropped from Day 14 and onwards. CONCLUSIONS: Upon exposure of proliferating cells from the adult human PR to differentiating conditions in culture, there is a widespread change in morphology and gene expression, including the upregulation of key retinal markers. However, this upregulation is only transient and decreases after 14 days of differentiation.