Development of machine learning-based predictors for early diagnosis of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) remains a formidable malignancy that significantly impacts human health, and the early diagnosis of HCC holds paramount importance. Therefore, it is imperative to develop an efficacious signature for the early diagnosis of HCC. In this study, we aimed to develop early HCC predictors (eHCC-pred) using machine learning-based methods and compare their performance with existing methods. The enhancements and advancements of eHCC-pred encompassed the following: (i) utilization of a substantial number of samples, including an increased representation of cirrhosis tissues without HCC (CwoHCC) samples for model training and augmented numbers of HCC and CwoHCC samples for model validation; (ii) incorporation of two feature selection methods, namely minimum redundancy maximum relevance and maximum relevance maximum distance, along with the inclusion of eight machine learning-based methods; (iii) improvement in the accuracy of early HCC identification, elevating it from 78.15 to 97% using identical independent datasets; and (iv) establishment of a user-friendly web server. The eHCC-pred is freely accessible at http://www.dulab.com.cn/eHCC-pred/ . Our approach, eHCC-pred, is anticipated to be robustly employed at the individual level for facilitating early HCC diagnosis in clinical practice, surpassing currently available state-of-the-art techniques.

Experimental and Simulation Studies of Imidazolium Chloride Ionic Liquids with Different Alkyl Chain Lengths for Viscosity Reductions in Heavy Crude Oil: The Effect on Asphaltene Dispersion.

Heavy crude oil poses challenges in terms of extraction and transportation due to its high viscosity. In the pursuit of effective methods to reduce viscosity in heavy crude oil, this study investigates the potential of imidazolium chloride ionic liquids with varying alkyl chain lengths as viscosity reducers. The experimental results demonstrate that the addition of 1-dodecyl-3-methylimidazole chloride ([C12-MIM]Cl) leads to a maximum viscosity reduction of 49.87%. Solubility parameters were calculated based on characterization of the average molecular structure of the asphaltenes. The viscosity reduction effect is enhanced when the solubility parameter of the ionic liquid closely matches that of the asphaltene. The initial asphaltene deposition point of heavy crude oil is increased from 63% to 68% with the addition of 150 mg/L [C12-MIM]Cl. Furthermore, the average particle size of asphaltene deposits decreases from 79.35 µm to 48.54 µm. The viscosity of heavy crude oil is influenced by the aggregation of asphaltenes. The ability of ionic liquids, especially those with longer alkyl chains, to disperse asphaltene molecules and reduce viscosity has been confirmed through molecular dynamics and quantum mechanical simulations.

Colostomy as a definitive treatment in an ALS patient with acute colonic Pseudo-obstruction refractory to medical management, a case report.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease, and ALS patients may experience disturbed gastrointestinal motility often resulting in acute colonic pseudo-obstruction (ACPO). There is currently a paucity in the literature to guide the treatment of patients with both ALS and ACPO. CASE PRESENTATION: Here we describe a 39-year-old male patient with advanced ALS who developed ACPO. His condition was refractory to both medical and procedural managements including polyethylene glycol, senna, and docusate suppository, metoclopramide, linaclotide, erythromycin, prucalopride, neostigmine, and repeated colonoscopies. He ultimately underwent successful colostomy for palliation. Here we report the peri-operative multidisciplinary approach taken with this case, the surgical procedures, the potential risks, and the outcome. CONCLUSION: The patient is delighted with the result and requested publication of this case to raise awareness of constipation in ALS patients and promote the consideration of colostomy as a treatment option for patients with ileus resistant to conservative management. Ultimately, a multidisciplinary team approach is required to properly assess the risks and benefits to achieve good clinical outcomes.

Elevation of fatty acid desaturase 2 in esophageal adenocarcinoma increases polyunsaturated lipids and may exacerbate bile acid-induced DNA damage.

BACKGROUND: The risk of esophageal adenocarcinoma (EAC) is associated with gastro-esophageal reflux disease (GERD) and obesity. Lipid metabolism-targeted therapies decrease the risk of progressing from Barrett's esophagus (BE) to EAC, but the precise lipid metabolic changes and their roles in genotoxicity during EAC development are yet to be established. METHODS: Esophageal biopsies from the normal epithelium (NE), BE, and EAC, were analyzed using concurrent lipidomics and proteomics (n = 30) followed by orthogonal validation on independent samples using RNAseq transcriptomics (n = 22) and immunohistochemistry (IHC, n = 80). The EAC cell line FLO-1 was treated with FADS2 selective inhibitor SC26196, and/or bile acid cocktail, followed by immunofluorescence staining for γH2AX. RESULTS: Metabolism-focused Reactome analysis of the proteomics data revealed enrichment of fatty acid metabolism, ketone body metabolism, and biosynthesis of specialized pro-resolving mediators in EAC pathogenesis. Lipidomics revealed progressive alterations (NE-BE-EAC) in glycerophospholipid synthesis with decreasing triglycerides and increasing phosphatidylcholine and phosphatidylethanolamine, and sphingolipid synthesis with decreasing dihydroceramide and increasing ceramides. Furthermore, a progressive increase in lipids with C20 fatty acids and polyunsaturated lipids with ≥4 double bonds were also observed. Integration with transcriptome data identified candidate enzymes for IHC validation: Δ4-Desaturase, Sphingolipid 1 (DEGS1) which desaturates dihydroceramide to ceramide, and Δ5 and Δ6-Desaturases (fatty acid desaturases, FADS1 and FADS2), responsible for polyunsaturation. All three enzymes showed significant increases from BE through dysplasia to EAC, but transcript levels of DEGS1 were decreased suggesting post-translational regulation. Finally, the FADS2 selective inhibitor SC26196 significantly reduced polyunsaturated lipids with three and four double bonds and reduced bile acid-induced DNA double-strand breaks in FLO-1 cells in vitro. CONCLUSIONS: Integrated multiomics revealed sphingolipid and phospholipid metabolism rewiring during EAC development. FADS2 inhibition and reduction of the high polyunsaturated lipids effectively protected EAC cells from bile acid-induced DNA damage in vitro, potentially through reduced lipid peroxidation.

An open source and convenient method for the wide-spread testing of COVID-19 using deep throat sputum samples.

Importance: The rise of novel, more infectious SARS-CoV-2 variants has made clear the need to rapidly deploy large-scale testing for COVID-19 to protect public health. However, testing remains limited due to shortages of personal protective equipment (PPE), naso- and oropharyngeal swabs, and healthcare workers. Simple test methods are needed to enhance COVID-19 screening. Here, we describe a simple, and inexpensive spit-test for COVID-19 screening called Patient Self-Collection of Sample-CoV2 (PSCS-CoV2). Objective: To evaluate an affordable and convenient test for COVID-19. Methods: The collection method relies on deep throat sputum (DTS) self-collected by the subject without the use of swabs, and was hence termed the Self-Collection of Sample for SARS-CoV-2 (abbreviated PSCS-CoV2). We used a phenol-chloroform extraction method for the viral RNA. We then tested for SARS-CoV-2 using real-time reverse transcription polymerase chain reaction with primers against at least two coding regions of the viral nucleocapsid protein (N1 and N2 or E) of SARS-CoV-2. We evaluted the sensitivity and specificity of our protocol. In addition we assess the limit of detection, and efficacy of our Viral Inactivating Solution. We also evaluated our protocol, and pooling strategy from volunteers on a local college campus. Results: We show that the PSCS-CoV2 method accurately identified 42 confirmed COVID-19 positives, which were confirmed through the nasopharyngeal swabbing method of an FDA approved testing facility. For samples negative for COVID-19, we show that the cycle threshold for N1, N2, and RP are similar between the PSCS-CoV2 and nasopharynx swab collection method (n = 30). We found a sensitivity of 100% (95% Confidence Interval [CI], 92-100) and specifity of 100% (95% CI, 89-100) for our PSCS-CoV2 method. We determined our protocol has a limit of detection of 1/10,000 for DTS from a COVID-19 patient. In addition, we show field data of the PSCS-CoV2 method on a college campus. Ten of the twelve volunteers (N1 < 30) that we tested as positive were subsequently tested positive by an independent laboratory. Finally, we show proof of concept of a pooling strategy to test for COVID-19, and recommend pool sizes of four if the positivity rate is less than 15%. Conclusion and Relevance: We developed a DTS-based protocol for COVID-19 testing with high sensitivity and specificity. This protocol can be used by non-debilitated adults without the assistance of another adult, or by non-debilitated children with the assistance of a parent or guardian. We also discuss pooling strategies based on estimated positivity rates to help conserve resources, time, and increase throughput. The PSCS-CoV2 method can be a key component of community-wide efforts to slow the spread of COVID-19.

RNAPhaSep: a resource of RNAs undergoing phase separation.

Liquid-liquid phase separation (LLPS) partitions cellular contents, underlies the formation of membraneless organelles and plays essential biological roles. To date, most of the research on LLPS has focused on proteins, especially RNA-binding proteins. However, accumulating evidence has demonstrated that RNAs can also function as 'scaffolds' and play essential roles in seeding or nucleating the formation of granules. To better utilize the knowledge dispersed in published literature, we here introduce RNAPhaSep (http://www.rnaphasep.cn), a manually curated database of RNAs undergoing LLPS. It contains 1113 entries with experimentally validated RNA self-assembly or RNA and protein co-involved phase separation events. RNAPhaSep contains various types of information, including RNA information, protein information, phase separation experiment information and integrated annotation from multiple databases. RNAPhaSep provides a valuable resource for exploring the relationship between RNA properties and phase behaviour, and may further enhance our comprehensive understanding of LLPS in cellular functions and human diseases.

An integrated pore size distribution measurement method of small angle neutron scattering and mercury intrusion capillary pressure.

Small-angle neutron scattering and high-pressure mercury intrusion capillary pressure testing are integrated to analyze the pore size distribution of the broad sense shale oil reservoir samples of the Permian Lucaogou Formation in the Jimsar Sag, Junggar Basin, China. The results show that, compared with the measurement method integrating gas adsorption and mercury intrusion, combination of small-angle neutron scattering and mercury intrusion can more accurately characterize full-scale pore size distribution. The full-scale pore size distribution curve of the rock samples in the study area includes two types: the declining type and submicron pore-dominated type. The declining type is mainly found with silty mudstone and dolomitic mudstone, and most of its pores are smaller than 80 nm. Silt-fine sandstones and dolarenite are mostly of the submicron pores-dominated type, with most pores smaller than 500 nm. They also present large specific pore volumes and average pore diameters of macropores and are the favorable lithogenous facies for development of high-quality reservoirs.

Distinct properties of Ca2+ efflux from brain, heart and liver mitochondria: The effects of Na+, Li+ and the mitochondrial Na+/Ca2+ exchange inhibitor CGP37157.

Mitochondrial Ca2+ transport is essential for regulating cell bioenergetics, Ca2+ signaling and cell death. Mitochondria accumulate Ca2+ via the mitochondrial Ca2+ uniporter (MCU), whereas Ca2+ is extruded by the mitochondrial Na+/Ca2+ (mtNCX) and H+/Ca2+ exchangers. The balance between these processes is essential for preventing toxic mitochondrial Ca2+ overload. Recent work demonstrated that MCU activity varies significantly among tissues, likely reflecting tissue-specific Ca2+ signaling and energy needs. It is less clear whether this diversity in MCU activity is matched by tissue-specific diversity in mitochondrial Ca2+ extrusion. Here we compared properties of mitochondrial Ca2+ extrusion in three tissues with prominent mitochondria function: brain, heart and liver. At the transcript level, expression of the Na+/Ca2+/Li+ exchanger (NCLX), which has been proposed to mediate mtNCX transport, was significantly greater in liver than in brain or heart. At the functional level, Na+ robustly activated Ca2+ efflux from brain and heart mitochondria, but not from liver mitochondria. The mtNCX inhibitor CGP37157 blocked Ca2+ efflux from brain and heart mitochondria but had no effect in liver mitochondria. Replacement of Na+ with Li+ to test the involvement of NCLX, resulted in a slowing of mitochondrial Ca2+ efflux by ∼70 %. Collectively, our findings suggest that mtNCX is responsible for Ca2+ extrusion from the mitochondria of the brain and heart, but plays only a small, if any, role in mitochondria of the liver. They also reveal that Li+ is significantly less effective than Na+ in driving mitochondrial Ca2+ efflux.

Fyn-tau Ablation Modifies PTZ-Induced Seizures and Post-seizure Hallmarks of Early Epileptogenesis.

Both Fyn and tau have been associated with neuronal hyperexcitability and neurotoxicity in many tauopathies, including Alzheimer's disease (AD). Individual genetic ablation of fyn or tau appears to be protective against aberrant excitatory neuronal activities in AD and epilepsy models. It is, however, still unknown whether ablation of both Fyn and tau can likely elicit more profound anti-seizure and neuroprotective effects. Here, we show the effects of genetic deletion of Fyn and/or tau on seizure severity in response to pentylenetetrazole (PTZ)-induced seizure in mouse models and neurobiological changes 24 h post-seizures. We used Fyn KO (fyn -/-), tau KO (tau -/-), double knockout (DKO) (fyn -/- / tau -/-), and wild-type (WT) mice of the same genetic background. Both tau KO and DKO showed a significant increase in latency to convulsive seizures and significantly decreased the severity of seizures post-PTZ. Although Fyn KO did not differ significantly from WT, in response to PTZ, Fyn KO still had 36 ± 8% seizure reduction and a 30% increase in seizure latency compared to WT. Surprisingly, in contrast to WT, Fyn KO mice showed higher mortality in <20 min of seizure induction; these mice had severe hydrocephalous. None of the tau -/- and DKO died during the study. In response to PTZ, all KO groups showed a significant reduction in neurodegeneration and gliosis, in contrast to WT, which showed increased neurodegeneration [especially, parvalbumin (PV)-GABAergic interneurons] and gliosis. DKO mice had the most reduced gliosis. Immunohistochemically, phospho-tau (AT8, pS199/S202), Fyn expression, as well as Fyn-tau interaction as measured by PLA increased in WT post-PTZ. Moreover, hippocampal Western blots revealed increased levels of AT8, tyrosine phospho-tau (pY18), and phosphorylated Src tyrosine family kinases (pSFK) in PTZ-treated WT, but not in KO, compared to respective controls. Furthermore, PV interneurons were protected from PTZ-induced seizure effects in all KO mice. The levels of inwardly rectifying potassium (Kir 4.1) channels were also downregulated in astrocytes in the WT post-PTZ, while its levels did not change in KO groups. Overall, our results demonstrated the role of Fyn and tau in seizures and their impact on the mediators of early epileptogenesis in PTZ model.

Fyn depletion ameliorates tauP301L-induced neuropathology.

The Src family non-receptor tyrosine kinase Fyn has been implicated in neurodegeneration of Alzheimer's disease through interaction with amyloid ß (Aß). However, the role of Fyn in the pathogenesis of primary tauopathies such as FTDP-17, where Aß plaques are absent, is poorly understood. In the current study, we used AAV2/8 vectors to deliver tauP301L to the brains of WT and Fyn KO mice, generating somatic transgenic tauopathy models with the presence or absence of Fyn. Although both genotypes developed tau pathology, Fyn KO developed fewer neurofibrillary tangles on Bielschowsky and Thioflavin S stained sections and showed lower levels of phosphorylated tau. In addition, tauP301L-induced behavior abnormalities and depletion of synaptic proteins were not observed in the Fyn KO model. Our work provides evidence for Fyn being a critical protein in the disease pathogenesis of FTDP-17.

A qualitative transcriptional signature to reclassify histological grade of ER-positive breast cancer patients.

BACKGROUND: Histological grade (HG) is commonly adopted as a prognostic factor for ER-positive breast cancer patients. However, HG evaluation methods, such as the pathological Nottingham grading system, are highly subjective with only 50-85% inter-observer agreements. Specifically, the subjectivity in the pathological assignment of the intermediate grade (HG2) breast cancers, comprising of about half of breast cancer cases, results in uncertain disease outcomes prediction. Here, we developed a qualitative transcriptional signature, based on within-sample relative expression orderings (REOs) of gene pairs, to define HG1 and HG3 and reclassify pathologically-determined HG2 (denoted as pHG2) breast cancer patients. RESULTS: From the gene pairs with significantly stable REOs in pathologically-determined HG1 (denoted as pHG1) samples and reversely stable REOs in pathologically-determined HG3 (denoted as pHG3) samples, concordantly identified from seven datasets, we extracted a signature which could determine the HG state of samples through evaluating whether the within-sample REOs match with the patterns of the pHG1 REOs or pHG3 REOs. A sample was classified into the HG3 group if at least a half of the REOs of the 10 gene pairs signature within this sample voted for HG3; otherwise, HG1. Using four datasets including samples of early stage (I-II) ER-positive breast cancer patients who accepted surgery only, we validated that this signature was able to reclassify pHG2 patients into HG1 and HG3 groups with significantly different survival time. For the original pHG1 and pHG3 patients, the signature could also more accurately and objectively stratify them into distinct prognostic groups. And the up-regulated and down down-regulated genes in HG1 compared with HG3 involved in cell proliferation and extracellular signal transduction pathways respectively. By comparing with existing signatures, 10-GPS was with prognostic significance and was more aligned with survival of patients especially for pHG2 samples. CONCLUSIONS: The transcriptional qualitative signature can provide an objective assessment of HG states of ER-positive breast cancer patients, especially for reclassifying patients with pHG2, to assist decision making on clinical therapy.

Loss of tau and Fyn reduces compensatory effects of MAP2 for tau and reveals a Fyn-independent effect of tau on calcium.

Microtubule-associated protein tau associates with Src family tyrosine kinase Fyn and is tyrosine phosphorylated by Fyn. The presence of tyrosine phosphorylated tau in AD and the involvement of Fyn in AD has drawn attention to the tau-Fyn complex. In this study, a tau-Fyn double knockout (DKO) mouse was generated to investigate the role of the complex. DKO mice resembled Fyn KO in novel object recognition and contextual fear conditioning tasks and resembled tau KO mice in the pole test and protection from pentylenetetrazole-induced seizures. In glutamate-induced Ca2+ response, Fyn KO was decreased relative to WT and DKO had a greater reduction relative to Fyn KO, suggesting that tau may have a Fyn-independent role. Since tau KO resembled WT in its Ca2+ response, we investigated whether microtubule-associated protein 2 (MAP2) served to compensate for tau, since the MAP2 level was increased in tau KO but decreased in DKO mice. We found that like tau, MAP2 increased Fyn activity. Moreover, tau KO neurons had increased density of dendritic MAP2-Fyn complexes relative to WT neurons. Therefore, we hypothesize that in the tau KO, the absence of tau would be compensated by MAP2, especially in the dendrites, where tau-Fyn complexes are of critical importance. In the DKO, decreased levels of MAP2 made compensation more difficult, thus revealing the effect of tau in the Ca2+ response.

Tau interacts with SHP2 in neuronal systems and in Alzheimer's disease brains.

Microtubule-associated protein tau, an integral component of neurofibrillary tangles, interacts with a variety of signaling molecules. Previously, our laboratory reported that nerve growth factor (NGF)-induced MAPK activation in a PC12-derived cell line was potentiated by tau, with phosphorylation at T231 being required. Therefore, we sought to identify a signaling molecule involved in the NGF-induced Ras-MAPK pathway that interacted with phospho-T231-tau. Here, we report that the protein tyrosine phosphatase SHP2 (also known as PTPN11) interacted with tau, with phospho-T231 significantly enhancing the interaction. By using proximity ligation assays, we found that endogenous tau-SHP2 complexes were present in neuronal cells, where the number of tau-SHP2 complexes significantly increased when the cells were treated with NGF, with phosphorylation at T231 being required for the increase. The interaction did not require microtubule association, and an association between tau and activated SHP2 was also found. Tau-SHP2 complexes were also found in both primary mouse hippocampal cultures and adult mouse brain. Finally, SHP2 levels were upregulated in samples from patients with mild and severe Alzheimer's disease (AD), and the level of tau-SHP2 complexes were increased in AD patient samples. These findings strongly suggest a role for the tau-SHP2 interaction in NGF-stimulated neuronal development and in AD.This article has an associated First Person interview with the first author of the paper.

Identifying primary site of lung-limited Cancer of unknown primary based on relative gene expression orderings.

BACKGROUND: Precise diagnosis of the tissue origin for metastatic cancer of unknown primary (CUP) is essential for deciding the treatment scheme to improve patients' prognoses, since the treatment for the metastases is the same as their primary counterparts. The purpose of this study is to identify a robust gene signature that can predict the origin for CUPs. METHODS: The within-sample relative gene expression orderings (REOs) of gene pairs within individual samples, which are insensitive to experimental batch effects and data normalizations, were exploited for identifying the prediction signature. RESULTS: Using gene expression profiles of the lung-limited metastatic colorectal cancer (LmCRC), we firstly showed that the within-sample REOs in lung metastases of colorectal cancer (CRC) samples were concordant with the REOs in primary CRC samples rather than with the REOs in primary lung cancer. Based on this phenomenon, we selected five gene pairs with consistent REOs in 498 primary CRC and reversely consistent REOs in 509 lung cancer samples, which were used as a signature for predicting primary sites of metastatic CRC based on the majority voting rule. Applying the signature to 654 primary CRC and 204 primary lung cancer samples collected from multiple datasets, the prediction accuracy reached 99.36%. This signature was also applied to 24 LmCRC samples collected from three datasets produced by different laboratories and the accuracy reached 100%, suggesting that the within-sample REOs in the primary site could reveal the original tissue of metastatic cancers. CONCLUSIONS: The result demonstrated that the signature based on within-sample REOs of five gene pairs could exactly and robustly identify the primary sites of CUPs.

1α,25-dihydroxyvitamin D3 and resolvin D1 retune the balance between amyloid-ß phagocytosis and inflammation in Alzheimer's disease patients.

As immune defects in amyloid-ß (Aß) phagocytosis and degradation underlie Aß deposition and inflammation in Alzheimer's disease (AD) brain, better understanding of the relation between Aß phagocytosis and inflammation could lead to promising preventive strategies. We tested two immune modulators in peripheral blood mononuclear cells (PBMCs) of AD patients and controls: 1α,25(OH)2-vitamin D3 (1,25D3) and resolvin D1 (RvD1). Both 1,25D3 and RvD1 improved phagocytosis of FAM-Aß by AD macrophages and inhibited fibrillar Aß-induced apoptosis. The action of 1,25D3 depended on the nuclear vitamin D and the protein disulfide isomerase A3 receptors, whereas RvD1 required the chemokine receptor, GPR32. The activities of 1,25D3 and RvD1 commonly required intracellular calcium, MEK1/2, PKA, and PI3K signaling; however, the effect of RvD1 was more sensitive to pertussis toxin. In this case study, the AD patients: a) showed significant transcriptional up regulation of IL1RN, ITGB2, and NFκB; and b) revealed two distinct groups when compared to controls: group 1 decreased and group 2 increased transcription of TLRs, IL-1, IL1R1 and chemokines. In the PBMCs/macrophages of both groups, soluble Aß (sAß) increased the transcription/secretion of cytokines (e.g., IL1 and IL6) and chemokines (e.g., CCLs and CXCLs) and 1,25D3/RvD1 reversed most of the sAß effects. However, they both further increased the expression of IL1 in the group 1, sß-treated cells. We conclude that in vitro, 1,25D3 and RvD1 rebalance inflammation to promote Aß phagocytosis, and suggest that low vitamin D3 and docosahexaenoic acid intake and/or poor anabolic production of 1,25D3/RvD1 in PBMCs could contribute to AD onset/pathology.

Neuronal phagocytosis by inflammatory macrophages in ALS spinal cord: inhibition of inflammation by resolvin D1.

Although the cause of neuronal degeneration in amyotrophic lateral sclerosis (ALS) remains hypothetical, there is evidence of spinal cord infiltration by macrophages and T cells. In post-mortem ALS spinal cords, 19.8 ± 4.8 % motor neurons, including caspase-negative and caspase-positive neurons, were ingested by IL-6- and TNF-α-positive macrophages. In ALS macrophages, in vitro aggregated superoxide dismutase-1 (SOD-1) stimulated in ALS macrophages expression of inflammatory cytokines, including IL-1ß, IL-6, and TNF-α, through activation of cyclooxy-genase-2 (COX-2) and caspase-1. The lipid mediator resolvin D1 (RvD1) inhibited IL-6 and TNF-α production in ALS macrophages with 1,100 times greater potency than its parent molecule docosahexaenoic acid. ALS peripheral blood mononuclear cells (PBMCs) showed increased transcription of inflammatory cytokines and chemokines at baseline and after stimulation by aggregated wild-type SOD-1, and these cytokines were down regulated by RvD1. Thus the neurons are impacted by macrophages expressing inflammatory cytokines. RvD1 strongly inhibits in ALS macrophages and PBMCs cytokine transcription and production. Resolvins offer a new approach to suppression of inflammatory activation in ALS.

Tocilizumab attenuates inflammation in ALS patients through inhibition of IL6 receptor signaling.

Patients with amyotrophic lateral sclerosis (ALS) have evidence of chronic inflammation demonstrated by infiltration of the gray matter by inflammatory macrophages, IL17A-positive T cells, and mast cells. Increased serum levels of IL6 and IL17A have been detected in sporadic ALS (sALS) patients when compared to healthy controls. Herein we investigate, in peripheral blood mononuclear cells (PBMCs), the baseline transcription of genes associated with inflammation in sALS and control subjects and the impact of the IL6 receptor (IL6R) antibody (tocilizumab) on the transcription and/or secretion of inflammation factors (e.g. cytokines) stimulated by the apo-G37R superoxide dismutase (SOD1) mutant. At baseline, PBMCs of four sALS patients (Group 1) showed significantly increased expression of TLR2 and CD14; ALOX5, PTGS2 and MMP1; IL1α, IL1ß, IL6, IL36G, IL8 and TNF; CCL3, CCL20, CXCL2, CXCL3 and CXCL5. In four other sALS patients (Group 2), most of the genes just mentioned were expressed at near control levels and a significant decrease in the expression of PPARG, PPARA, RARG, HDAC4 and KAT2B; IL6R, IL6ST and ADAM17; TNFRSF11A; MGAT2 and MGAT3; PLCG1; CXCL3 were detected. Apo-G37R SOD1 up regulated the transcription of cytokines (e.g. IL1α/ß, IL6, IL8, IL36G), chemokines (e.g. CCL20; CXCL3, CXCL5), and enzymes (e.g. PTGS2 and MMP1). In vitro, tocilizumab down regulated the transcription of many inflammatory cytokines, chemokines, enzymes, and receptors, which were up regulated by pathogenic forms of SOD1. Tocilizumab also reduced the secretion of the pro-inflammatory cytokines IL1ß, IL6, TNFα, GM-CSF, IFNγ, and IL17A by Group 1 PBMCs. Finally, sALS patients had significantly higher concentrations of IL6, sIL6R and C-reactive protein in the cerebrospinal fluid when compared to AD patients. This pilot study demonstrates that in vitro tocilizumab suppresses many factors that drive inflammation in sALS patients, with possible increased efficacy in Group 1 ALS patients.

IL-17A is increased in the serum and in spinal cord CD8 and mast cells of ALS patients.

The contribution of inflammation to neurodegenerative diseases is increasingly recognized, but the role of inflammation in sporadic amyotrophic lateral sclerosis (sALS) is not well understood and no animal model is available. We used enzyme-linked immunosorbent assays (ELISAs) to measure the cytokine interleukin-17A (IL-17A) in the serum of ALS patients (n = 32; 28 sporadic ALS (sALS) and 4 familial ALS (fALS)) and control subjects (n = 14; 10 healthy subjects and 4 with autoimmune disorders). IL-17A serum concentrations were 5767 ± 2700 pg/ml (mean ± SEM) in sALS patients and 937 ± 927 pg/ml in fALS patients in comparison to 7 ± 2 pg/ml in control subjects without autoimmune disorders (p = 0.008 ALS patients vs. control subjects by Mann-Whitney test). Sixty-four percent of patients and no control subjects had IL-17A serum concentrations > 50 pg/ml (p = 0.003 ALS patients vs. healthy subjects by Fisher's exact test). The spinal cords of sALS (n = 8), but not control subjects (n = 4), were infiltrated by interleukin-1ß- (IL-1ß-), and tumor necrosis factor-α-positive macrophages (co-localizing with neurons), IL-17A-positive CD8 cells, and IL-17A-positive mast cells. Mononuclear cells treated with aggregated forms of wild type superoxide dismutase-1 (SOD-1) showed induction of the cytokines IL-1ß, interleukin-6 (IL-6), and interleukin-23 (IL-23) that may be responsible for induction of IL-17A. In a microarray analysis of 28,869 genes, stimulation of peripheral blood mononuclear cells by mutant superoxide dismutase-1 induced four-fold higher transcripts of interleukin-1α (IL-1α), IL-6, CCL20, matrix metallopeptidase 1, and tissue factor pathway inhibitor 2 in mononuclear cells of patients as compared to controls, whereas the anti-inflammatory cytokine interleukin-10 (IL-10) was increased in mononuclear cells of control subjects. Aggregated wild type SOD-1 in sALS neurons could induce in mononuclear cells the cytokines inducing chronic inflammation in sALS spinal cord, in particular IL-6 and IL-17A, damaging neurons. Immune modulation of chronic inflammation may be a new approach to sALS.