Nervonic Acid Synthesis Substrates as Essential Components in Profiled Lipid Supplementation for More Effective Central Nervous System Regeneration.

Central nervous system (CNS) damage leads to severe neurological dysfunction as a result of neuronal cell death and axonal degeneration. As, in the mature CNS, neurons have little ability to regenerate their axons and reconstruct neural loss, demyelination is one of the hallmarks of neurological disorders such as multiple sclerosis (MS). Unfortunately, remyelination, as a regenerative process, is often insufficient to prevent axonal loss and improve neurological deficits after demyelination. Currently, there are still no effective therapeutic tools to restore neurological function, but interestingly, emerging studies prove the beneficial effects of lipid supplementation in a wide variety of pathological processes in the human body. In the future, available lipids with a proven beneficial effect on CNS regeneration could be included in supportive therapy, but this topic still requires further studies. Based on our and others' research, we review the role of exogenous lipids, pointing to substrates that are crucial in the remyelination process but are omitted in available studies, justifying the properly profiled supply of lipids in the human diet as a supportive therapy during CNS regeneration.

Bioactive Hydrogel Based on Collagen and Hyaluronic Acid Enriched with Freeze-Dried Sheep Placenta for Wound Healing Support.

In an increasingly aging society, there is a growing demand for the development of technology related to tissue regeneration. It involves the development of the appropriate biomaterials whose properties will allow the desired biological response to be obtained. Bioactivity is strongly affected by the proper selection of active ingredients. The aim of this study was to produce bioactive hydrogel materials based on hyaluronic acid and collagen modified by the addition of placenta. These materials were intended for use as dressings, and their physicochemical properties were investigated under simulated biological environmental conditions. The materials were incubated in vitro in different fluids simulating the environment of the human body (e.g., simulated body fluid) and then stored at a temperature close to body temperature. Using an FT-IR spectrophotometer, the functional groups present in the composites were identified. The materials with the added placenta showed an increase in the swelling factor of more than 300%. The results obtained confirmed the potential of using this material as an absorbent dressing. This was indicated by pH and conductometric measurements, sorption, degradation, and surface analysis under an optical microscope. The results of the in vitro biological evaluation confirmed the cytosafety of the tested biomaterials. The tested composites activate monocytes, which may indicate their beneficial properties in the first phases of wound healing. The material proved to be nontoxic and has potential for medical use.

Clinical and microbiological outcomes of subgingival instrumentation supplemented with high-dose omega-3 polyunsaturated fatty acids in periodontal treatment - a randomized clinical trial.

PURPOSE: This study aimed to evaluate the impact of dietary supplementation with omega-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) combined with scaling and root planing (SRP) in untreated periodontitis stage III and IV. METHODS: Forty patients were randomly assigned to the test group receiving SRP plus omega-3 PUFAs (n = 20) or control group receiving SRP alone (n = 20). Clinical changes of pocket probing depths (PD), clinical attachment level (CAL), bleeding on probing (BOP) and rates of closed pockets (PPD ≤ 4 mm without BOP) were evaluated at baseline and after 3 and 6 months. Phorphyromonas gingivalis, Tanarella forsythia, Treponema denticola and Aggregatibacter actinomycetemcomitans counts were analysed at baseline and at 6 months. Serum was subjected to lipid gas chromatography/mass spectrometry analysis at baseline and at 6 months. RESULTS: Significant improvement of all clinical parameters at 3 and 6 months was observed in both groups. For the primary outcome "change of mean PD," no significant difference was detected between the groups. Patients treated with omega-3 PUFAs demonstrated significantly lower rates of BOP, higher gain of CAL and higher number of closed pockets at 3 months in comparison to the control group. After 6 months, no clinical differences between the groups were found, with the exception of lower BOP rates. Moreover, in the test group, the number of key periodontal bacteria was significantly lower than in the control group at 6 months. Increased proportions of serum n-3 PUFAs and decreased proportions of n-6 PUFAs were detected at 6 months in the patients from the test group. CONCLUSION: High-dose omega-3 PUFA intake during non-surgical treatment of periodontitis results in short-term clinical and microbiological benefits. The study protocol was approved by the ethical committee of Medical University of Lodz (reference number RNN/251/17/KE) and registered at clinicaltrials.gov (NCT04477395) on 20/07/2020.

Natural fish oil improves the differentiation and maturation of oligodendrocyte precursor cells to oligodendrocytes in vitro after interaction with the blood-brain barrier.

The blood-brain barrier (BBB) tightly controls the microenvironment of the central nervous system (CNS) to allow neurons to function properly. Additionally, emerging studies point to the beneficial effect of natural oils affecting a wide variety of physiological and pathological processes in the human body. In this study, using an in vitro model of the BBB, we tested the influence of natural fish oil mixture (FOM) vs. borage oil (BO), both rich in long-chain polyunsaturated fatty acids (LC-PUFAs) and monounsaturated fatty acids (MUFAs) such as oleic acid (C18:1n9c) or nervonic acid (NA), on human oligodendrocyte precursor cells (hOPCs) during their maturation to oligodendrocytes (OLs) regarding their ability to synthesize myelin peptides and NA. We demonstrated that FOM, opposite to BO, supplemented endothelial cells (ECs) and astrocytes forming the BBB, affecting the function of hOPCs during their maturation. This resulted in improved synthesis of myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), proteolipid protein (PLP), and NA in mature OLs. This effect is probably the result of BBB cell and hOPC stimulation via free fatty acid receptors (FFARs), which increases insulin growth factor-1 (IGF-1), ciliary neurotrophic factor (CNTF), and brain-derived neurotrophic factor (BDNF) and inhibits fibroblast growth factor 2 (FGF-2) synthesis. The unique formula of fish oil, characterized by much more varied components compared to those of BOs, also improved the enhancement of the tight junction by increasing the expression of claudin-5 and VE-cadherin on ECs. The obtained data justify consideration of naturally derived fish oil intake in human diet as affecting during remyelination.

Changes Within H3K4me3-Marked Histone Reveal Molecular Background of Neutrophil Functional Plasticity.

Neutrophils are a heterogenous population capable of both antimicrobial functions and suppressor ones, however, no specific pattern of transcription factors controlling this plasticity has been identified. We observed rapid changes in the neutrophil status after stimulation with LPS, pre-activating concentration of TNF-α, or IL-10. Chromatin immunoprecipitation sequencing (ChIP-Seq) analysis of histone H3K4me3 allowed us to identify various transcriptional start sites (TSSs) associated with plasticity and heterogeneity of human neutrophils. Gene Ontology analysis demonstrated great variation within target genes responsible for neutrophil activation, cytokine production, apoptosis, histone remodelling as well as NF-κB transcription factor pathways. These data allowed us to assign specific target genes positioned by H3K4me3-marked histone with a different pattern of gene expression related to NF-κB pathways, apoptosis, and a specific profile of cytokines/chemokines/growth factors realised by neutrophils stimulated by LPS, IL-10, or TNF-α. We discovered IL-10-induced apoptotic neutrophils being transcriptionally active cells capable of switching the profile of cytokines/chemokines/growth factors desired in resolving inflammation via non-canonical NF-κB pathway with simultaneous inhibition of canonical NF-κB pathway. As apoptotic/suppressive neutrophils induced by IL-10 via positioning genes within H3K4me3-marked histone were transcriptionally active, newly described DNA binding sites can be considered as potential targets for immunotherapy.H3K4me3 histone ChIP-Seq analysis reveals molecular drivers critical for switching neutrophils from their pro- to anti-inflammatory properties.

Effect of Multiple Sclerosis Cerebrospinal Fluid and Oligodendroglia Cell Line Environment on Human Wharton's Jelly Mesenchymal Stem Cells Secretome.

Multiple sclerosis (MS) is a neurological disorder of autoimmune aetiology. Experimental therapies with the use of mesenchymal stem cells (MSCs) have emerged as a response to the unmet need for new treatment options. The unique immunomodulatory features of stem cells obtained from Wharton's jelly (WJ-MSCs) make them an interesting research and therapeutic model. Most WJ-MSCs transplants for multiple sclerosis use intrathecal administration. We studied the effect of cerebrospinal fluid (CSF) obtained from MS patients on the secretory activity of WJ-MSCs and broaden this observation with WJ-MSCs interactions with human oligodendroglia cell line (OLs). Analysis of the WJ-MSCs secretory activity with use of Bio-Plex Pro™ Human Cytokine confirmed significant and diverse immunomodulatory potential. Our data reveal rich WJ-MSCs secretome with markedly increased levels of IL-6, IL-8, IP-10 and MCP-1 synthesis and a favourable profile of growth factors. The addition of MS CSF to the WJ-MSCs culture caused depletion of most proteins measured, only IL-12, RANTES and GM-CSF levels were increased. Most cytokines and chemokines decreased their concentrations in WJ-MSCs co-cultured with OLs, only eotaxin and RANTES levels were slightly increased. These results emphasize the spectrum of the immunomodulatory properties of WJ-MSCs and show how those effects can be modulated depending on the transplantation milieu.

Agr Quorum Sensing influences the Wood-Ljungdahl pathway in Clostridium autoethanogenum.

Acetogenic bacteria are capable of fermenting CO2 and carbon monoxide containing waste-gases into a range of platform chemicals and fuels. Despite major advances in genetic engineering and improving these biocatalysts, several important physiological functions remain elusive. Among these is quorum sensing, a bacterial communication mechanism known to coordinate gene expression in response to cell population density. Two putative agr systems have been identified in the genome of Clostridium autoethanogenum suggesting bacterial communication via autoinducing signal molecules. Signal molecule-encoding agrD1 and agrD2 genes were targeted for in-frame deletion. During heterotrophic growth on fructose as a carbon and energy source, single deletions of either gene did not produce an observable phenotype. However, when both genes were simultaneously inactivated, final product concentrations in the double mutant shifted to a 1.5:1 ratio of ethanol:acetate, compared to a 0.2:1 ratio observed in the wild type control, making ethanol the dominant fermentation product. Moreover, CO2 re-assimilation was also notably reduced in both hetero- and autotrophic growth conditions. These findings were supported through comparative proteomics, which showed lower expression of carbon monoxide dehydrogenase, formate dehydrogenase A and hydrogenases in the ∆agrD1∆agrD2 double mutant, but higher levels of putative alcohol and aldehyde dehydrogenases and bacterial micro-compartment proteins. These findings suggest that Agr quorum sensing, and by inference, cell density play a role in carbon resource management and use of the Wood-Ljungdahl pathway as an electron sink.

H3K4me3 Histone ChIP-Seq Analysis Reveals Molecular Mechanisms Responsible for Neutrophil Dysfunction in HIV-Infected Individuals.

Peripheral neutrophils in HIV-infected individuals are characterized by impairment of chemotaxis, phagocytosis, bactericidal activity, and oxidative burst ability regardless of whether patients are receiving antiretroviral therapy or not. Neutrophil dysfunction leads not only to increased susceptibility to opportunistic infections but also to tissue damage through the release of reactive oxygen species (ROS), proteases, and other potentially harmful effector molecules contributing to AIDS progression. In this study, we demonstrated high levels of histone H3 lysine K4 trimethylated (H3K4me3) and dysregulation of DNA transcription in circulating neutrophils of HIV-infected subjects. This dysregulation was accompanied by a deficient response of neutrophils to LPS, impaired cytokine/chemokine/growth factor synthesis, and increased apoptosis. Chromatin immunoprecipitation sequencing (ChIPseq) H3K4me3 histone analysis revealed that the most spectacular abnormalities were observed in the exons, introns, and promoter-TSS regions. Bioinformatic analysis of Gene Ontology, including biological processes, molecular function, and cellular components, demonstrated that the main changes were related to the genes responsible for cell activation, cytokine production, adhesive molecule expression, histone remodeling via upregulation of methyltransferase process, and downregulation of NF-κB transcription factor in canonical pathways. Abnormalities within H3K4me3 implicated LPS-mediated NF-κB canonical activation pathway that was a result of low amounts of κB DNA sites within histone H3K4me3, low NF-κB (p65 RelA) and TLR4 mRNA expression, and reduced free NF-κB (p65 RelA) accumulation in the nucleus. Genome-wide survey of H3K4me3 provided evidence that chromatin modifications lead to an impairment within the canonical NF-κB cell activation pathway causing the neutrophil dysfunction observed in HIV-infected individuals.

Omega-3 Polyunsaturated Fatty Acids EPA and DHA as an Adjunct to Non-Surgical Treatment of Periodontitis: A Randomized Clinical Trial.

Periodontitis is a chronic multifactorial inflammatory disease that leads to the loss of supportive tissues around the teeth with gradual deterioration of masticatory function and esthetics, resulting eventually in the decrease of the life quality. Host immune response triggered by bacterial biofilm is responsible for the chronic periodontal inflammation and ongoing tissue loss. Omega-3 polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have anti-inflammatory properties, thus may be used for the treatment of chronic inflammatory diseases. In this study, we aimed to evaluate the effect of dietary supplementation with omega-3 PUFA in the patients with stage III and IV periodontitis. Thirty otherwise healthy patients were treated with scaling and root planning (SRP). In the test group (n = 16), patients were additionally supplemented with 2.6 g of EPA and 1.8 g of DHA. In the control group (n = 14), patients received only SRP. Periodontal examination was performed at baseline and three months following initial therapy. Salivary samples were taken twice at baseline and at the end of the experiment. We found that there was a statistically significant reduction in the bleeding on probing (BOP) and improvement of clinical attachment loss (CAL) at three months in the test group compared to the control group. Moreover, a statistically significant higher percentage of closed pockets (probing depth ≤ 4 mm without BOP) was achieved in the test group vs. control group after three months of treatment. Accordingly, the levels of pro-inflammatory cytokines/chemokines interleukin (IL)-8 and IL-17 were markedly lower, while the level of anti-inflammatory IL-10 was significantly higher in the salivary samples of the patients supplemented with omega-3 PUFA at three months in comparison to the patients treated with SRP alone. Our findings demonstrate that dietary intervention with high-dose of omega-3 PUFA during non-surgical therapy may have potential benefits in the management of periodontitis.

Multiple Sclerosis CD49d+CD154+ As Myelin-Specific Lymphocytes Induced During Remyelination.

Multiple sclerosis (MS) is a demyelinating autoimmune disease of the central nervous system (CNS) mediated by autoreactive lymphocytes. The role of autoreactive lymphocytes in the CNS demyelination is well described, whereas very little is known about their role in remyelination during MS remission. In this study, we identified a new subpopulation of myelin-specific CD49d+CD154+ lymphocytes presented in the peripheral blood of MS patients during remission, that proliferated in vitro in response to myelin peptides. These lymphocytes possessed the unique ability to migrate towards maturing oligodendrocyte precursor cells (OPCs) and synthetize proinflammatory chemokines/cytokines. The co-culture of maturing OPCs with myelin-specific CD49d+CD154+ lymphocytes was characterized by the increase in proinflammatory chemokine/cytokine secretion that was not only a result of their cumulative effect of what OPCs and CD49d+CD154+ lymphocytes produced alone. Moreover, maturing OPCs exposed to exogenous myelin peptides managed to induce CD40-CD154-dependent CD49d+CD154+ lymphocyte proliferation. We confirmed, in vivo, the presence of CD49d+CD154+ cells close to maturating OPCs and remyelinating plaque during disease remission in the MS mouse model (C57Bl/6 mice immunized with MOG35-55) by immunohistochemistry. Three weeks after an acute phase of experimental autoimmune encephalomyelitis, CD49d+/CD154+ cells were found to be co-localized with O4+ cells (oligodendrocyte progenitors) in the areas of remyelination identified by myelin basic protein (MBP) labelling. These data suggested that myelin-specific CD49d+CD154+ lymphocytes present in the brain can interfere with remyelination mediated by oligodendrocytes probably as a result of establishing proinflammatory environment.

MS CD49d+CD154+ Lymphocytes Reprogram Oligodendrocytes into Immune Reactive Cells Affecting CNS Regeneration.

The critical aspect in multiple sclerosis (MS) progression involves insufficient regeneration of CNS resulting from deficient myelin synthesis by newly generated oligodendrocytes (OLs). Although many studies have focused on the role of autoreactive lymphocytes in the inflammatory-induced axonal loss, the problem of insufficient remyelination and disease progression is still unsolved. To determine the effect of myelin-specific lymphocytes on OL function in MS patients and in a mouse model of MS, we cultured myelin induced MS CD49d+CD154+ circulating lymphocytes as well as Experimental Autoimmune Encephalomyelitis (EAE) mouse brain-derived T and memory B cells with maturing oligodendrocyte precursor cells (OPCs). We found that myelin-specific CD49d+CD154+ lymphocytes affected OPC maturation toward formation of immune reactive OLs. Newly generated OLs were characterized by imbalanced myelin basic protein (MBP) and proteolipid protein (PLP) production as well as proinflammatory chemokine/cytokine synthesis. The analysis of cellular pathways responsible for OL reprogramming revealed that CD49d+CD154+ lymphocytes affected miRNA synthesis by dysregulation of polymerase II activity. miR-665 and ELL3 turned out to be the main targets of MS myelin-specific lymphocytes. Neutralization of high intracellular miR-665 concentration restored miRNA and MBP/PLP synthesis. Together, these data point to new targets for therapeutic intervention promoting CNS remyelination.

Naturally Occurring Nervonic Acid Ester Improves Myelin Synthesis by Human Oligodendrocytes.

The dysfunction of oligodendrocytes (OLs) is regarded as one of the major causes of inefficient remyelination in multiple sclerosis, resulting gradually in disease progression. Oligodendrocytes are derived from oligodendrocyte progenitor cells (OPCs), which populate the adult central nervous system, but their physiological capability to myelin synthesis is limited. The low intake of essential lipids for sphingomyelin synthesis in the human diet may account for increased demyelination and the reduced efficiency of the remyelination process. In our study on lipid profiling in an experimental autoimmune encephalomyelitis brain, we revealed that during acute inflammation, nervonic acid synthesis is silenced, which is the effect of shifting the lipid metabolism pathway of common substrates into proinflammatory arachidonic acid production. In the experiments on the human model of maturating oligodendrocyte precursor cells (hOPCs) in vitro, we demonstrated that fish oil mixture (FOM) affected the function of hOPCs, resulting in the improved synthesis of myelin basic protein, myelin oligodendrocyte glycoprotein, and proteolipid protein, as well as sphingomyelin. Additionally, FOM reduces proinflammatory cytokines and chemokines, and enhances fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF) synthesis by hOPCs was also demonstrated. Based on these observations, we propose that the intake of FOM rich in the nervonic acid ester may improve OL function, affecting OPC maturation and limiting inflammation.

C5a-Preactivated Neutrophils Are Critical for Autoimmune-Induced Astrocyte Dysregulation in Neuromyelitis Optica Spectrum Disorder.

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune neuroinflammatory disease. In contrast to multiple sclerosis, autoantibodies against aquaporin-4 (AQP4) expressed on astrocytic end-feet have been exclusively detected in sera of NMOSD patients. Several lines of evidence suggested that anti-AQP4 autoantibodies are pathogenic, but the mechanism triggering inflammation, impairment of astrocyte function, and the role of neutrophils presented in NMOSD cerebrospinal fluid remains unknown. In this study, we tested how human neutrophils affect astrocytes in the presence of anti-AQP4 Ab-positive serum derived from NMOSD patients. An in vitro model of inflammation consisted of human astrocyte line, NMOSD serum, and allogenic peripheral blood neutrophils from healthy individuals. We showed evidence of pathogenicity of NMOSD serum, which by consecutive action of anti-AQP4 Abs, complement system, and neutrophils affected astrocyte function. Anti-AQP4 Ab binding astrocytes initiated two parallel complementary reactions. The first one was dependent on the complement cytotoxicity via C5b-9 complex formation, and the second one on the reverse of astrocyte glutamate pump into extracellular space by C5a-preactivated neutrophils. As a consequence, astrocytes were partially destroyed; however, a major population of astrocytes polarized into proinflammatory cells which were characterized by pathological glutamate removal from extracellular space.

Whole genome sequence and manual annotation of Clostridium autoethanogenum, an industrially relevant bacterium.

BACKGROUND: Clostridium autoethanogenum is an acetogenic bacterium capable of producing high value commodity chemicals and biofuels from the C1 gases present in synthesis gas. This common industrial waste gas can act as the sole energy and carbon source for the bacterium that converts the low value gaseous components into cellular building blocks and industrially relevant products via the action of the reductive acetyl-CoA (Wood-Ljungdahl) pathway. Current research efforts are focused on the enhancement and extension of product formation in this organism via synthetic biology approaches. However, crucial to metabolic modelling and directed pathway engineering is a reliable and comprehensively annotated genome sequence. RESULTS: We performed next generation sequencing using Illumina MiSeq technology on the DSM10061 strain of Clostridium autoethanogenum and observed 243 single nucleotide discrepancies when compared to the published finished sequence (NCBI: GCA_000484505.1), with 59.1 % present in coding regions. These variations were confirmed by Sanger sequencing and subsequent analysis suggested that the discrepancies were sequencing errors in the published genome not true single nucleotide polymorphisms. This was corroborated by the observation that over 90 % occurred within homopolymer regions of greater than 4 nucleotides in length. It was also observed that many genes containing these sequencing errors were annotated in the published closed genome as encoding proteins containing frameshift mutations (18 instances) or were annotated despite the coding frame containing stop codons, which if genuine, would severely hinder the organism's ability to survive. Furthermore, we have completed a comprehensive manual curation to reduce errors in the annotation that occur through serial use of automated annotation pipelines in related species. As a result, different functions were assigned to gene products or previous functional annotations rejected because of missing evidence in various occasions. CONCLUSIONS: We present a revised manually curated full genome sequence for Clostridium autoethanogenum DSM10061, which provides reliable information for genome-scale models that rely heavily on the accuracy of annotation, and represents an important step towards the manipulation and metabolic modelling of this industrially relevant acetogen.

Six-minute walk test distance and resting oxygen saturations but not functional class predict outcome in adult patients with Eisenmenger syndrome.

BACKGROUND: Eisenmenger syndrome (ES) represents the extreme manifestation of pulmonary arterial hypertension in patients with congenital heart disease, associated with significant exercise intolerance and mortality. Even though of six-minute-walk-test (6MWT) is routinely used in these patients, little is known about its prognostic value in comparison to functional class. METHODS AND RESULTS: We included 210 adult patients with ES who underwent a total of 822 6MWTs. Median walking distance (6MWD) was 330 m [IQR 260-395], oxygen saturation (SO2) at baseline 86% [IQR 82-91%] and SO2 at peak-exercise 69% [IQR 60-80%]. In patients commenced on advanced therapy for pulmonary hypertension, but not in the reminder, there was a significant improvement in walking distance (297±97 m vs. 325±87 m,P=0.0019), SO2 at rest (84.9±7.1 vs. 86.8±5.9%,P=0.003), SO2 at peak exercise (69.1±12.7 vs. 72.3±12.2%,P=0.04) and NYHA functional class (P=0.0047). During a follow up of 3.3 years, 29 patients died. On time-dependent Cox analysis, 6MWD (HR 0.94 per 10 m, 95%CI: 0.91-0.97,P<0.001) and baseline SO2 (HR 0.90, 95%CI:0.86-0.94,P<0.0001) were predictors of death. In contrast, age, functional class, peak-exercise SO2 and SO2 change were not related to mortality. A three-fold increased risk of death was identified in patients not reaching a 6MWD of 350 m or with baseline SO2 below 85%. CONCLUSIONS: The 6MWD and resting SO2, but not functional class were predictive of outcome in this contemporary cohort of Eisenmenger patients and should be incorporated in both risk stratification and management algorithms for these patients.