MYD88 mutation, but patients were treated only in the setting of a diagnostic biopsy. Here we present a case of a patient with inconclusive biopsy results whose CSF ctDNA revealed a MYD88 mutation suggestive of PCNSL and guided her treatment with high-dose methotrexatePrimary central nervous system lymphoma (PCNSL) is a rare tumor that can be difficult to diagnose as it can mimic other CNS diseases. Tissue biopsy is the gold standard for diagnosis, but this is not always feasible if the lesions are in deep structures of the brain. Tissue samples may also be non-diagnostic if steroids have been administered prior to the procedure. Genomic testing of circulating tumor DNA (ctDNA) in the cerebrospinal fluid is a technique that may provide an alternative method for diagnosis. Prior case reports and studies have shown a correlation between PCNSL and .A 65-year-old woman with metastatic malignant peripheral nerve sheath tumor (MPNST) on active treatment with a novel clinical trial drug and no known germline mutations presented with 6 weeks of nausea, worsening gait, and falls. MRI of the brain revealed nonenhancing white matter abnormalities . CerebroOver the next several months, her brain MRI showed worsening fluctuations in enhancing and nonenhancing lesions . Brain bMYD88, CD58, HIST1H1C, KMT2D, and LCK mutations suggestive of a lymphoid malignancy like primary central nervous system lymphoma (PCNSL). Based on these results, she was treated with 8 doses of rituximab and methotrexate with complete radiographic resolution of disease (Given her continued lack of improvement, MSK-IMPACT was performed on the CSF and resulted in the discovery of  disease . She was1 More than 90% of PCNSL lesions enhance on MRI brain, and the gold standard for diagnosis is via biopsy of a lesion.2 In this case, the patient’s treatment with a clinical trial drug with unknown side effects along with her fluctuating symptoms of diplopia and weakness made an autoimmune, inflammatory, or demyelinating process higher on the differential over a second uncommon malignancy with a rare atypical presentation like PCNSL. In addition, her nonenhancing FLAIR hyperintense lesions were in the deep structures of the brain, making biopsy less appealing in the setting of lower clinical suspicion.This case is an example of the importance of CSF ctDNA analysis in the diagnosis of PCNSL in patients who have suspicious lesions in areas that are difficult to biopsy or have nondiagnostic biopsies. PCNSL is a rare type of extranodal non-Hodgkin lymphoma located in the central nervous system that can be difficult to diagnose. Initial clinical presentations and radiographic findings can mimic other disorders such as primary brain tumors, demyelinating diseases, autoimmune or paraneoplastic syndromes, or central nervous system infections.2 In addition, the diagnostic yield in CSF cytology or flow cytometry is often low and do not produce corresponding results. In a prospective study of 123 B-cell lymphoma patients, only 22% of patients had CSF flow cytometry that identified neoplastic B cells, and 8% of patients had conventional CSF cytology with malignant or suspicious cells present.3 Other studies have shown that the presence of monoclonal B cells in the CSF may not be diagnostic of clinically significant CNS involvement by lymphoma, as they can also be seen in inflammatory and demyelinating conditions, making it difficult to interpret CSF flow cytometry results.4 Atypical cells on cytology can also be observed in patients with infections and autoimmune/inflammatory disorders.6 Although this patient had CSF cytology with atypical cells and CSF flow cytometry showing an abnormal B-cell population, it was difficult to interpret these results given that an autoimmune/inflammatory process was high on the differential. In this setting, these results alone could not justify a diagnosis of PCNSL and exposure to high-dose methotrexate with its associated side effects including nephrotoxicity, hepatotoxicity, and myelosuppression.7Clinicians have attempted to rely on CSF for diagnosis of PCNSL as it is less invasive than biopsy, but neither CSF cell count nor protein levels appear to reliably predict the presence of PCNSL.8 The presence of MYD88, CD58, HIST1H1C, KMT2D, and LCK in the patient’s CSF made the molecular diagnosis of PCNSL and guided treatment. Although these mutations have all been linked to hematologic malignancies (MYD88 is of particular interest as it occurs in at least 35% of patients with PCNSL. Fontanilles et al. reported that MYD88 had a diagnostic sensitivity of 24% for PCNSL but a specificity of 100%.9 More recently, Ferreri et al. report a sensitivity of 94% and specificity of 98% for the MYD88 mutation status and IL-10 levels in the CSF.10 Given the specificity of MYD88 reported in the literature at the time the patient was diagnosed, she was treated with high-dose methotrexate and rituximab even though her brain biopsy was inconclusive.The diagnosis was made by performing next-generation molecular testing on ctDNA in the CSF with MSK-IMPACT, a FDA-authorized DNA sequencing panel targeting over 400 oncogenes and tumor suppressor genes identifying mutations, copy number alterations, and gene fusions.gnancies , MYD88 iMYD88 and can be resulted faster, but they can only be utilized if the clinician is highly suspicious of PCNSL.15 In this report, the patient’s diagnosis was unknown in the setting of her MPNST, and the benefit of using a test like MSK-IMPACT is that it targets a wide variety of oncogenes and tumor suppressor genes to help narrow down a diagnosis. This case is an example of how ctDNA can be used to guide treatment of PCNSL in the setting of an inconclusive traditional workup.Although next-generation molecular sequencing allowed for the diagnosis to be made, 1 major drawback is the amount of time it takes to generate the results. Molecular diagnostics can take weeks to months to result whereas frozen pathology from a brain biopsy can confirm the diagnosis before the surgery is finished leading to faster treatment and clinical improvement for the patient. Given how quickly PCNSL patients can deteriorate, rapid diagnosis is paramount to their care. At the time of this diagnosis, MSK-IMPACT took 3 weeks to result, though this has improved over the years. Newer technology such as droplet digital PCR can identify mutations like


Disordered cholesterol metabolism is linked to neurodegeneration. In this study we investigated the profile of cholesterol metabolites found in the cerebrospinal fluid (CSF) of Parkinson’s disease (PD) patients. When adjustments were made for confounding variables of age and sex, 7α,(25R)26-dihydroxycholesterol and a second oxysterol 7α,x,y-trihydroxycholest-4-en-3-one , whose exact structure is unknown, were found to be significantly elevated in PD CSF. The likely location of the additional hydroxy groups on the second oxysterol are on the sterol side-chain. We found that CSF 7α-hydroxycholesterol levels correlated positively with depression in PD patients, while two presumptively identified cholestenoic acids correlated negatively with depression. Parkinson’s disease (PD) is a chronic neurodegenerative disorder of the central nervous system (CNS) that presents with motor deficits, but which also has many non-motor features, including cognitive and neuropsychiatric problems. In PD, the core motor features result mainly from a loss of dopaminergic neurons in the substantia nigra of the midbrain and their projection to the striatum, but more widespread pathology in subcortical and cortical regions, and even outside the CNS, underlies many of the non-motor features.in situ. Excess cholesterol is removed from the brain by the neuron-specific cytochrome P450 (CYP) 46A1- catalyzed metabolism to 24S-hydroxycholesterol  either in combination with gas chromatography (GC)  or with liquid chromatography (LC)  . Most stbisoxocholest-4-en-26-oic acid  and 7α,12α-dihydroxy-3-oxocholeste-4-en-26-oic acid , and scores on the BDI but not other clinical measures. This work highlights the potential clinical significance of the bile acid biosynthesis pathway in PD and defines a methodology that can be used to measure the pathway intermediates within a clinical laboratory setting.In the current study, we analysed “free” non-esterified oxysterols (including cholestenoic acids) in the CSF of PD patients and healthy controls with an aim of identifying metabolites or pathways linked to PD. To achieve the necessary sensitivity, we adopted a two-step derivatisation approach named “enzyme-assisted derivatisation for sterol analysis” (EADSA) in combination with LC-MS , 2017. AThis work was designed in two studies: Study 1 primarily focused on oxysterol and cholestenoic acid identification while Study 2 focused on their quantitation and relationship with a range of PD relevant clinical measures. All patients were recruited from the Parkinson’s Disease Research Clinic at the John van Geest Centre for Brain Repair in Cambridge. The study was approved by the Cambridgeshire 2 Research Ethics Committee (Ref. 08/H0308/331) and written informed consent was obtained from all participants. Controls for Study 2 were carers of patients with PD with no known neurological disease, or patients attending Addenbrooke’s Hospital NHS Neurology clinics for a lumbar puncture to investigate other symptoms (such as headache), but with no known neurodegenerative disease.Lumbar punctures were performed using an aseptic technique as per standard clinical guidelines. 2–5 mL of CSF was collected. The CSF was centrifuged at 2,000–3,000 g for 15 min and the supernatant was stored at −80°C prior to analysis.Standard demographic data was collected along with assessments of disease severity including the Movement Disorder Society-Unified Parkinson’s Disease Rating Scale (MDS-UPDRS); neuropsychological assessments including the Addenbrooke’s Cognitive Examination Revised (ACE-R) and semantic fluency and assessment of depression using the BDI.m/z 400) and multistage fragmentation (MSn) for structure determination. Quantification was performed against added isotope-labelled standards. In Study 1 quantification was against 24R/S-hydroxycholesterol ([2H7]24R/S-HC) which has been shown to be an adequate surrogate for side-chain oxysterols and cholestenoic acids  was included to allow quantification of 7α,25-dihydroxycholesterol  and 7α,26-diHC and their 3-oxo analogues  age = 69 (7) years, disease duration = 4 (4) years, MDS-UPDRS motor score on treatment = 31(12), ACE-R = 89 (8), BDI = 6 (6)] with the aim of identifying non-esterified oxysterols present in the CSF. The oxysterols identified in this first study are listed in 5 to Δ4 isomerisation the branches converge at 7αH,3O-CA(25R). We observed each of these metabolites in the CSF and notably the concentration of 7αH,3O-CA was specifically elevated in PD CSF 26-oyl-CoA -CoA, norcholest-4-ene-3,24-dione . In combination, this initial study suggests the acidic pathway is upregulated in the CNS of PD patients.We have previously shown that the acidic pathway of bile acid biosynthesis is at least partially active in the brain . This pa3 spectra, but in the absence of authentic standards, definitive identifications were not made. These partial identifications include 3β,x-dihydroxycholest-5-en-y-one  where x and y may be 22 and 24, or 20 and 22, and 7α,x,y-trihydroxycholest-4-en-3-one , suggesting a cluster of cholesterol metabolites as candidate biomarkers for PD. This data should be treated with caution as the patient and control data were reordered at different times and for samples collected from different hospitals in different countries. Nevertheless, metabolites significant in the univariate analysis  and this yielded a robust model separating PD from controls 26-oic ) and 3β,7α,25-trihydroxycholest-5-en-26-oic  acids from Avanti Polar Lipids Inc., which are easily converted in the laboratory to 7α,24S-dihydroxy-3-oxocholest-4-en-(25R)26-oic ) and 7α,25-dihydroxy-3-oxocholest-4-en-26-oic  acids, respectively, by treatment with cholesterol oxidase enzyme . This al spectra . The locCombining data from Study 1 and Study 2, we have found that the acidic pathway of bile acid biosynthesis is upregulated in the CNS of PD patients .P < 0.05) were found between PD CSF 24S-HC and disease duration , 7α-HC and BDI , 7αH-3,24-diO-CA and BDI  and 7α,12α-diH,3O-CA and BDI . Multivariate regression analysis with 24S-HC as the dependent variable and age and gender as relevant covariates did not confirm the relationship between 24S-HC and disease duration . However, multivariate analyses did confirm the relationships between 7α-HC, 7αH-3,24-diO-CA, 7α,12α-diH,3O-CA, and BDI, with age, gender, and disease duration as relevant confounding covariates  compared to tremor dominant patients . Although the reason for this is not known, it may be the case that PIGD patients are more likely to be given calcium/vitamin D supplements because they are at risk of falls. Vitamin D3 is converted to 25-hydroxyvitamin D3 in the liver and is transported in the blood stream to the kidney where 1α,25-dihydroxyvitamin D3 is formed.Bivariate correlation analyses between each PD CSF oxysterol profile and relevant demographic and clinical variables  were performed. Correlations of significance (at a level of p < 0.05) increase in 24S-HC in PD CSF. In this second study the CSF concentration of 24S-HC was found to correlate with disease progression. These results were suggested to relate to the release of 24S-HC from a subtype of dying neurons in PD, leading to an increase in 24S-HC concentration in the CSF during disease progression  in the CSF of PD patients and controls, concentrations of 24S-HC and 26-HC were found to be elevated in about 10% of PD samples above a cut off defined as the control mean + 3 standard deviations (SD) . Howevergression , 2018. Tgression , 2018.In our current studies, we have measured the biologically more relevant non-esterified molecules. We did not find a statistically significant increase in 24S-HC in CSF from PD patients in either study. 7α,26-diHC, one of the immediate down-stream metabolites of 26-HC , was incvia import across the BBB  are all expressed in human brain  is not expressed in brain , hence tynthesis , one of ynthesis , suggestynthesis . In combynthesis .The levels of the oxysterols 7α-HC, 7αH-3,24-diO-CA, 7α,12α-diH,3O-CA were found to correlate with BDI score  in PD caInterestingly, intermediates in the acidic pathway of bile acid biosynthesis have also been found to be elevated in people suffering from multiple sclerosis but not in those suffering from amyotrophic lateral sclerosis or Alzheimer’s disease , arguingIn conclusion, despite the limitations mentioned around our control CSF sample collection, a number of interesting and novel observations have been made in our study. Our data suggests a cerebral upregulation of the acidic pathway of bile acid biosynthesis in PD. We have also identified a number of cholesterol metabolites whose CSF levels correlate with depression in PD. Further studies are planned utilising greater sample numbers to confirm or refute the current findings.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The studies involving human participants were reviewed and approved by the Cambridgeshire 2 Research Ethics Committee. The patients/participants provided their written informed consent to participate in this study.WG, ST, EA, RB, and YW designed the study. JA-K, PC, EY, SM, RW, DB, and KF performed the study. CW-G, SM, and MT supervised and performed statistical analysis. All authors contributed to writing of the manuscript.The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care.WG, PC, and YW were listed as inventors on the Swansea University patent “Kit and method for quantitative detection of steroids,” US9851368B2, licensed to Avanti Polar Lipids Inc., and Cayman Chemical Company by Swansea University. WG, JA-K, PC, EY, ST, EA, and YW were shareholders in CholesteniX Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Akkermansia mucinphila, Clostridium spp., Lactococcus spp., and Bifidobacterium spp.) and functional metabolites, such as tryptophan, free fatty acids, branched amino acids, and bile acids, which influence adipose tissue and hepatic lipid metabolism, and the level of lipid deposition in tissues and peripheral blood. In conclusion, CIH and CSF adversely affect fecal microbiota composition and function, and host metabolism; these findings provide new insight into the independent and synergistic effects of CIH, CSF, and HFD on lipid disorders.Chronic intermittent hypoxia (CIH) and chronic sleep fragmentation (CSF) are two cardinal pathological features of obstructive sleep apnea (OSA). Dietary obesity is a crucial risk intermediator for OSA and metabolic disorders. Gut microbiota affect hepatic and adipose tissue morphology under conditions of CIH or CSF through downstream metabolites. However, the exact relationship is unclear. Herein, chow and high-fat diet (HFD)-fed mice were subjected to CIH or CSF for 10 weeks each and compared to normoxia (NM) or normal sleep (NS) controls. 16S rRNA amplicon sequencing, untargeted liquid chromatography-tandem mass spectrometry, and histological assessment of liver and adipose tissues were used to investigate the correlations between the microbiome, metabolome, and lipid metabolism under CIH or CSF condition. Our results demonstrated that CIH and CSF regulate the abundance of intestinal microbes (such as  Obstructive sleep apnea (OSA) is a common sleep-disordered breathing disease that affects almost 1 billion adults worldwide . ExploriIn our previous systemic review, we summarized the changes in the metabolome and microbiota that may play an integral role as intermediary factors in the pathophysiology of OSA and related cardiovascular, metabolic, and neurological complications . In rodeIn the present study, we explored the effects of CIH and CSF, with or without dietary intervention, on gut microbial composition, metabolic function, and adipose tissue morphology. We aimed to determine how, and to what extent, CIH and CSF affect hepatic and adipose tissue morphology; changes in gut microbiome and serum metabolites associated with CIH and CSF; and the impact of CIH- and CSF-mediated taxonomic and molecular alterations on hepatic and adipose lipid metabolism.The study protocol was approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, China. All experiments were conducted following the Guide for the Care and Use of Laboratory Animals published by the Animal Welfare Committee of the Agricultural Research Organization. Seventy-two male C57BL/6J mice  were housed in 16 cages (4–5 mice/cage) under standard conditions  with a regular 14‐h light: 10‐h dark cycle (lights on at 7:00 am). After adaptation for 2 weeks, mice (8 weeks old) were randomly divided into eight groups: normoxia , CIH (n = 10), HFD (n = 8), CIH+HFD (n = 10), normal Sleep , CSF (n = 9), NS+HFD (n = 9) and CSF+HFD (n = 9) groups , whereas the HFD group was fed a eutrophic diet with 59% calories derived from coconut oil .2 concentration in the chamber was continuously measured using an O2 analyzer. Hypoxia was induced with 10% O2 for approximately 240 s, followed by 21% O2 for 120 s, as previously described  for 8h/day, from 8:00 am to 4:00 pm, for 10 weeks. The Oescribed . For NM Continuous CSF was induced for 10 weeks by using big large, modified, multiple cages equipped with a fixed platform and twirling pole , controlled using a timer programmed for random rotation . CSF was induced by rotating the pole for 10 s after every 110 s at a speed of 110 rpm for the entire day to disturb sleeping mice .via an overdose of sodium pentobarbital . Then, samples from the liver, inguinal white adipose tissue (IWAT), perirenal white adipose tissue (PWAT), and scapula brown adipose tissue (BAT) were harvested and weighed. Liver tissues were embedded in Tissue-Tek OCT Compound , sliced into cryosections (8-μm-thick) and stained with Oil Red O  to assess the fatty droplet content. The stained liver cells were analyzed using Image Pro-Plus 6.0 . Adipose tissues were fixed in 10% formalin, embedded in paraffin wax, sectioned (5 μm), and stained with hematoxylin and eosin. Hematoxylin and eosin staining was used to evaluate the sizes of subcutaneous and visceral white adipose tissue (WAT) and BAT. Mean cell size and size distribution was determined for WAT. Because it was difficult to isolate the BAT cells due to cytoplasmic lipid accumulation, the lipid area of BAT was quantified. The lipid area and cell size were calculated using MetaMorph imaging software .The body weight of all mice was recorded weekly and blood glucose (BG) levels were measured every 5 weeks after fasting for 12 h using a glucose analyzer  over the tail vein. After the 10-week exposure period and 12-h solid and liquid fast, the mice were sacrificed http://qiime.org/scripts/pick_otus.html) database.Sampling time of intestinal contents was on the morning after 10-week intervention and overnight fasting and water restriction. The intestinal contents were collected from the middle section of colon after anesthesia and laparotomy. Fecal samples were collected from all groups for microbiota 16S rRNA analysis. The microflora were detected by Lianchuan Biotechnology Co., Ltd. . Bacterial genomic DNA was obtained from frozen colon contents using the QIAamp DNA Stool Mini Kit , according to the manufacturer’s instructions. Successful DNA isolation was confirmed by agarose gel electrophoresis. DNA samples were amplified by polymerase chain reaction (PCR) using bar‐coded primers flanking the V3–V4 region of the 16S rRNA gene. PCR was performed using a thermocycler under the following conditions: 1 pre-denaturation cycle at 94°C for 4 min, 25 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 45 s, elongation at 72°C for 30 s, and 1 post-elongation cycle at 72°C for 5 min. The PCR amplicon products were separated on 0.8% agarose gels and extracted. Only PCR products without primer dimers and contaminant bands were used for sequencing by synthesis. High‐throughput pyrosequencing of the PCR products was performed using the Illumina MiSeq platform . Paired‐end reads of the original DNA fragments were excluded from the analysis if they did not well-match a 12‐base Golay barcode (one or no errors), the read overlap was < 35 bases, the overlapped region differed by more than 15%, or there were more than three base calls below Q20. Bacterial operational taxonomic units (OTUs) were created by clustering the reads at 97% identity in the Quantitative Insights into Microbial Ecology  using a Micromass Quattro Micro liquid chromatography (LC)-MS system  equipped with a model HTS-PAL 2777 autosampler , model 1525 high-performance liquid chromatography (HPLC) solvent delivery system , and MassLynx data system . For each batch, periodic analysis of the same pooled quality control (QC) sample was performed to detect variations within and between experiments . The Su* or p < 0.01**.Taxonomy abundance was normalized to the summed OTUs of each sample. Principal coordinate analysis (PCoA) of β-diversity was performed; the (unpaired) Wilcoxon rank-sum test was used to identify the most diverse taxa. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis was applied to metagenome functions predicted from the normalized OTUs for Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologs . The ManAll mice survived and were kept in good condition during the 10-week-long CIH or CSF exposure with or without a HFD  in the NM/NM+HFD (baseline difference), NM/CIH (CIH‐induced changes in chow-fed mice), and NM+HFD/CIH+HFD (CIH-induced changes in HFD mice) groups. Red bars indicate the same direction of log2FC change in the three NM/CIH and NM+HFD/CIH+HFD pairwise group comparisons. The highest log2FC value was observed for Allobaculum spp. Green bars indicate the same direction of log2FC change for the NM/NM+HFD and NM+HFD/CIH+HFD groups; the changes in Ileibacterium spp. were primarily induced by dietary patterns. Blue bars indicate microbes with the same direction of log2FC change for the NM/CIH and NM+HFD/CIH+HFD groups, suggesting that the variation may have been induced by CIH. Importantly, CIH significantly increased the abundances of Desulfovibrio spp., Enterohabdus spp., and Bifidobacterium spp. in chow- and HFD-fed mice . CIH alone significantly increased the abundances of Lactobacillus spp. and Alistipes spp., while decreasing the abundances of Feacalibaculum rodentium and Clostridium spp. . The CIH+HFD intervention down-regulated the abundances of Fournierella massiliensis, Eisenbergiella massiliensis, Intestinimonas spp., and A. muciniphila.We further evaluated differences in the cecal microbial species at the genus level. Twenty-eight microbes were selected, as there was no “0” relative abundance value in any CIH model. The PCoA plot  (CSF stimulation was associated with changes in the abundances of and FFA) 10B.The microbiota composition and host metabolism were affected by CIH- and CSF-induced changes in body weight, fasting BG levels, and the weights of WAT, BAT, and liver. These results suggested that the structure of various fecal microorganisms was influenced by CIH and CSF, which was closely related to host metabolism and hepatic and adipose morphology.In this study, we analyzed OSA-associated pathological changes in intestinal bacteria and metabolites, as well as their correlations with hepatic and adipose tissue morphology and lipid metabolism disorders. The two pathological factors of OSA combined with HFD feeding intervention were used to create the corresponding rodent models. The influence of the above pathological intervention on the composition, abundance, and KEGG-based PICRUSt metagenome functions analyses of intestinal flora was identified by 16S rRNA detection. We also extracted the differential metabolites in serum related to the above pathway through untargeted metabolomics detection data to estimate the impact on the metabolic levels of amino acids, carbohydrates and lipids. Then we conducted correlation analysis on the differential data of the two kinds of omics to see the influence of OSA-related main pathological factors on intestinal bacteria and related metabolites. In the modeling process, we found that CIH, CSF intervention factors obviously caused significant weight loss in mice at the early modeling stage, so we also paid attention to the effects of differential microflora, differential metabolites and lipid deposition in adipose tissue and liver tissue. Therefore, we found that CIH and CSF, with or without HFD-induced dysbiosis of intestinal bacteria and metabolites thereof, may independently or synergistically affect the risk of lipid-related metabolic complications.Adipose tissue and hepatic lipid metabolism involves a dynamic equilibrium between lipogenesis and lipolysis that maintains lipid homeostasis . C57BL/6A. muciniphila  and melatonin  2 pathway . FFAs maIn contrast to BAT, WAT cells contain only one lipid droplet and few mitochondria. Importantly, we did not observe WAT proliferation in response to CIH or CSF exposure alone. However, CIH or CSF combined with HFD led to an apparent increase in lipid deposition in IWAT and PWAT. CIH and CSF increased F/B ratio 5C, consFaecalibacterium prausnitzii, and upregulated Actinobacteria and Desulfovibrio, leading to pro-inflammatory intestinal dysbiosis  cortex , leading process , was dow process 5C, D. S process . Therefo process . The CIH process . In turnE. coprostanoligenes and endogenous vitamin B  through changes in many functional metabolites, such as tryptophan, FFAs, BCAA and Bas. These can affect adipose and hepatic tissue lipid metabolism and deposition . The mechanism of regulation of lipid metabolism by the intestinal microbiota and functional metabolites should be explored in sterile animals, using engineered bacteria (metabolic enzyme knockout or overexpression) and artificial compounds .https://www.ncbi.nlm.nih.gov/, PRJNA781373.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, China.FW, HX, JG, and SY contributed to conception and design of the study. JJZ, FW, WH, XZ, ZW, XL, YL, and JYZ organized the database. FW, JJZ, JYZ, and HZ performed the statistical analysis. FW and JJZ wrote the first draft of the manuscript. HX, HY, FL, and JG wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.This study was supported by grants-in-aid from Shanghai Municipal Commission of Science and Technology (Grant No.18DZ2260200); InnovationProgram of Shanghai Municipal Education Commission (Grant No. 2017-01-07-00-02-E00047); National Natural Science Foundation of China  and Shandong Natural Science Foundation Program (Grant No. ZR201911030252).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This study aimed to evaluate the efficacy of neoadjuvant targeted therapy (TT) in patients with localised clear-cell renal cell carcinoma (RCC).  A special randomised trial was planned and conducted by the Research Department of Plastic and Reconstructive Oncology in the National Cancer Institute of Ukraine for testing the clinical efficacy of neoadjuvant TT in the treatment of clear-cell localised RCC, and the primary endpoint was tumour response evaluation after TT. The secondary endpoints included evaluation of dependence between the use of neoadjuvant TT and the probability of partial nephrectomy and the correlation between tumour size, stage, remaining functioning parenchyma volume, and response to systemic therapy. n = 44 (76.9%) cases. Partial response by the Response Evaluation Criteria in Solid Tumours, version 1.1, was observed in 14 (24.1%) patients and reached a maximum of 60% regression. Tumour reduction in the neoadjuvant TT group allowed kidney preservation in 53 (91.4%) patients. In the control group, the number of organ-sparing procedures was significantly lower . The statistical difference was relevant .  Overall, 118 patients met the inclusion criteria and were randomly assigned to receive combined treatment or surgery alone. The percentage of tumour regression ranged from 0% to 60%, and the median was  20.5 ± 14.3 (16.8–24.3%). Most of the patients had a slightly positive response to TT (3%–29% decrease in tumour size);  The positive results of neoadjuvant TT obtained in our study indicate the clinical validity of combined treatment in patients with localised RCC. Partial nephrectomy is considered to be the surgery of choice in the treatment of localised renal cell carcinoma (RCC) , 2, and Radical nephrectomy is commonly used in clinical practice in cases of renal hilum tumours or large tumours (>4 cm) and significantly impairs patient survival due to a decrease in filtration rate and quality of life –6. In suAccording to modern literature analysis, neoadjuvant TT in localised RCC can be used only in elective clinical cases. The current data suggest performing it only during imperative indications  –12, and A special randomised trial was planned and conducted by the Research Department of Plastic and Reconstructive Oncology at the National Cancer Institute of Ukraine, testing the clinical efficacy of neoadjuvant TT in the treatment of clear-cell localised RCC. The eligibility of participating surgeons included sufficient surgical experience of >100 partial/radical nephrectomies per surgeon annually. The study was approved by the Institutional Review Board and local ethics committee, which was conducted as per with the Declaration of Helsinki and the Good Clinical Practice guidelines. The study flowchart is shown in Lesion larger than 20 mm located in the renal hilumPeripheral or polar RCC extending to kidney sinusRemaining functioning parenchyma volume (RFPV) over 55%The inclusion criteria were clinically and histologically confirmed localised clear-cell RCC (ccRCC) (T1-T2 N0 M0) with intermediate indications for both radical and partial nephrectomy:Patients with chronic kidney disease, expected estimated glomerular filtration rate (eGFR) > 30 mL/min after surgery, coexisting malignant disease, and a history of any other systemic therapy were excluded. It was mandatory to exclude tumour extension and metastatic disease before patient enrolment. A special informed consent form was obtained from the study team and signed along with the National Health Care system form during the investigation. Tumour biopsy to confirm ccRCC was performed routinely, not less than 2 weeks before randomisation.Patients were randomly assigned in a 1 : 1 ratio to receive either a combination of neoadjuvant TT with surgery (main group) or surgery alone (control group). Randomisation was performed using a random number generation method at the randomisation centre of the National Cancer Institute.TT in the main group was performed according to the standard schedule with drugs provided by the National Health Care System: pazopanib 800 mg daily orally for two cycles  or sunitinib 50 mg daily for two cycles . Patients were informed by the investigators about the side effects and oncological efficacy of both drugs before signing the informed consent form and choosing precise medication. In cases of AE greater than level 2, dose reduction was planned, with interruption in patients who might have had life-threatening conditions. Patient safety was the primary goal of the study.Surgical treatment of the main group was planned to start from 2 weeks after neoadjuvant TT cessation or termination for complete excretion of the target drug from the body for better tissue regeneration after surgical treatment. Patients in the control group underwent surgery for 14 days after randomisation. Preference was given for partial nephrectomy in both the groups. The surgical strategy was discussed on a multidisciplinary board. The main indication for kidney preservation was an RFPV over 55%.Treatment efficacy was evaluated based on imaging methods (computed tomography (CT) and magnetic resonance imaging), which were used in the initial examination and in all control examinations. The primary tumour was evaluated using the Response Evaluation Criteria in Solid Tumours, version 1.1 (RECIST) . In the According to the standard of care, the patients did not receive any adjuvant therapy. Follow-up examinations, including CT, were performed every 6 months up to 2 years postoperatively. Thereafter, it was performed once a year.The primary endpoint was tumour response evaluation after TT. The secondary endpoints included evaluation of dependence between the use of neoadjuvant TT and the probability of partial nephrectomy and correlation between tumour size, RFPV, and response to systemic therapy. Recurrence-free survival, cancer-specific survival, and overall survival were not reached at the period of data cutoff.p < 0.05) were considered to be statistically significant differences.The results were statistically analysed using SPSS (version 24.0) software. The distribution of continuous data in the group was estimated using the Kolmogorov–Smirnov criterion. Descriptive statistics included calculating the median with a standard deviation or median of 25–75 percentiles. Quantitative comparisons between groups were conducted using the Mann–Whitney criterion and qualitative comparisons using the Pearson test. A total statistical estimation of regression was performed using the analysis of variance (ANOVA) test. Type I errors less than 5%  patients, whereas surgery alone was performed in 60 (51%) patients.Information on baseline patient characteristics, tumour stage and size, kidney function, and concomitant pathology are summarised in n = 57; 48%) had concomitant pathology in other organs and systems, which affected kidney function both in the preoperative and postoperative periods. The most frequent complications were hypertension in 47 (39.8%) patients, obesity in 41 (34.7%), contralateral kidney cysts in 14 (11.9%), urinary stone disease in 11 (9.3%), diabetes in five (4.2%), contralateral kidney hypoplasia in two (1.7%), and a history of myocardial infarction in three (2.5%) patients.It should be noted that about half of the patients (n = 44 (76.9%) cases. A partial response was observed in 14 patients (24.1%) and reached a maximum of 60% regression. No complete response events were observed in the study cohort. After two blocks of neoadjuvant TT in patients with localised RCC, the mean kidney tumour size reduction was 12.3 mm from (M ± SD (95% CI)) 60.8 ± 19.7 (55.7–66) to 48.5 ± 16.4 (44.2–52.8) mm , which could have influenced surgical tumour complexity.According to the primary endpoint, we estimated the percentage of tumour regression in 58 patients in the neoadjuvant TT group. The degree of tumour regression in each case is shown in U test; p < 0.001) increased the median (Me) RFPV by 21% from  62  mm to 83  mm. We performed a comparative analysis of RFPV before and after neoadjuvant TT in patients with localised RCC. The use of neoadjuvant TT in patients with localised RCC .The distribution of partial and radical nephrectomies in the study groups is shown in r = 0.77613; p < 0.001) of the tumour size before neoadjuvant treatment on the expected outcome. The correlation between tumour size before and after neoadjuvant TT using Pearson correlation analysis revealed a relevant direct correlation  did not respond to neoadjuvant TT at all (0% regression). Similar differences in objective responses were observed in small tumours. η2 = 0.01, with an impact power of 0.1, was not significant (p=0.72). Post hoc analysis using the Newman–Keuls test showed no significant difference between all subgroups of the study. Thus, between T1a and T1b, the confidence indicator was p=0.8 . Therefore, we have not confirmed the theory of greater efficacy of TT in patients with primary small RCC [The evaluation was also performed in terms of comparing the T-stage and regression. Total statistical estimation performed using the ANOVA test revealed no influence of stage on tumour regression. However, the effect mall RCC .Adverse events (AEs) and complication rates are being evaluated by another part of the presented clinical trial and will be further presented. There were no cases of TT interruption, with most patients suffering from AE not higher than Gr 1 during the treatment period. There were no severe surgical complications in either group according to the Clavien–Dindo scoring system that required repeated intervention during the 90-day postoperative period.The results of a randomised prospective study evaluating the efficacy of neoadjuvant TT in the treatment of patients with localised RCC showed promising results.Data from clinical trials indicate a connection between chronic kidney disease and cardiovascular risks. Conservative treatment allows preservation of additional functioning nephrons with reference rates of oncologic survival compared to nephrectomy. Currently, indications for conservative surgical treatment beyond the standard T1a stage are expanding. Particular attention is given to bilateral kidney lesions, single kidney tumours, and RCC with simultaneous opposite kidney pathology, which significantly diminishes its function, while nephrectomy may result in dialysis for such patients. This could induce a problem due to the insufficient number of dialysis beds and their high costs . In thesThe principle of neoadjuvant TT in RCC is based on the concept of increasing procedural safety and improving locoregional control . A potenn = 3) of patients, stable disease in 37% (n = 7), and progression in 47% (n = 9). A reduction in tumour size was observed in 8/19 (42%) patients, with an average of 24% reduction, which allowed nephrectomy in four cases.The positive effect of neoadjuvant TT was first described by Thomas et al. . The stuThe role of neoadjuvant TT is even more significant in increasing the likelihood of conservative treatment for RCC. Silberstein et al. demonstrated TT efficacy in a group of 14 tumours (12 patients) with imperative indications, who showed an average reduction in RCC size of 21.1%. In all cases, a partial nephrectomy with a negative surgical margin was performed. No patients required further haemodialysis . These r2 in the case of nephrectomy. In total, 25 patients were enrolled in the study, 71% of whom showed a decrease in the lesion complexity and 92% showed a decrease in the tumour volume after TT. Kidney preservation was eventually performed in 80% (20/25) of cases with an increased probability of parenchyma preservation from 107 to 173 cm3 (p=0.0015) [Another study by Rini et al. showed the feasibility of pazopanib (800 mg) in optimising parenchyma preservation within high-complexity tumours. A common criterion was that the expected eGFR would be less than 30 mL/min/1.73 m=0.0015) , 20. TheThe results of the study evaluating the efficacy of neoadjuvant TT in the treatment of localised ccRCC have shown promising results. The high sensitivity of localised RCC to neoadjuvant TT with an objective response of 91% prompted the search for surgical strategy optimisation. A hidden benefit is an increase in the possibility of conservative treatment for complex kidney tumours. This might also lead to a reduction in the incidence of chronic kidney disease in this group and induce life expectancy and quality.This approach facilitates the selection of surgical tactics for anatomically complex kidney tumours. The neoadjuvant treatment in oncology aims to reduce side effects and improve the tolerability of the selected therapy. This could be explained by the presence of two functioning kidneys, which significantly increased the body's detoxification capabilities.r = 0.77613; p < 0.001). We could predict the expected tumour volume after neoadjuvant TT based on the initial tumour size according to tomography . This could help predict possible localised RCC regression and shift tactics to conservative surgical treatment. In contrast, we could conclude on its feasibility (in case of unfavourable results) and prevent unnecessary systemic toxicity.Our analysis of the correlation between the kidney tumour size before and after neoadjuvant TT using the Pearson method revealed a reliable direct correlation (The dependence of the correlation between the RCC size and regression degree under the influence of neoadjuvant TT was described by Kroon et al. . The autConsidering the above mentioned, it should be noted that neoadjuvant TT in localised ccRCC is a promising method aimed at improving functional outcomes of treatment, thus decreasing the level of patients with further disability. Additionally, the implementation of the technique aims to facilitate the choice of surgical tactics for anatomically complex kidney tumours.The ongoing study on neoadjuvant TT in localised RCC is aimed at increasing the understanding of anatomical and functional changes in the kidneys affected by TT, evaluation of its efficacy, determination of its indications, prediction of the course of treatment stages, and the choice of surgery type.Neoadjuvant TT in patients with surgically complex localised RCC resulted in clinical tumour regression, which provided a higher rate of conservative surgical treatment compared to similar patients without TT.The positive results of neoadjuvant TT obtained in our study indicate the clinical validity of further investigating preoperative systemic therapy in localised ccRCC.


Living kidney donation is the most common type of living-donor transplant. Italian guidelines allow the living donations from emotionally related donors only after clear and voluntary consent expressed by both the donor and the recipient involved. Living donation raises ethical and legal issues because donors voluntarily undergo a surgical procedure to remove a healthy kidney in order to help another person. According to the Italian standards, the assessment of living donor-recipient pair has to be conducted by a medical “third party”, completely independent from both the patients involved and the medical team treating the recipient. Starting from the Hospital “Città della Salute e della Scienza” of Turin  experience, including 116 living kidney donations, the Authors divided the evaluation process performed by the “Third-Party” Commission into four stages, with a particular attention to the potential donor. Living donation procedures should reflect fiduciary duties that healthcare providers have toward their patients, originating from the relationship of trust between physician and patient. In addition to that, the social implications are enormous if one considers the worldwide campaigns to promote public awareness about organ donation and transplantation, and to encourage people to register their organ donation decisions. The systematic process proposed here can be a tool that proactively reduces and controls the risks of coercion, organ trafficking, vitiated consent, insufficient weighting of donative choice, that could arise especially in donors involved in living kidney donation. Due to the dramatically increased number of transplant candidates listed for deceased donor transplantation over the years, in Europe patients wait on average 3–5 years for a deceased donor kidney , 2.Brain-death donors are the most common source of organs, and are the core of the national organ transplant programs –5. The cLiving kidney donation is the most common type of living-donor transplant , 11. TheSeveral factors account for LOD increase , 13–15:- the effectiveness of the organ procurement system,- the progress in transplant surgery,- the better graft and patient survival when compared with cadaver donor transplantation,- the surgery planned in advance when a living donor is available with reduction or prevention of the need for dialysis,- the growing need for organs,- the shortage of available dead donor organs.There are different types of living donation for kidney transplantation , 11, 16:- directed donation: genetically-related donation , emotionally-related donation ; paired-donation ; pooled donation (the pair is matched with other donors and recipients from a pool of pairs in similar situations);- non-directed altruistic donation or good Samaritan donation: the donor does not have any relationship with the recipient.Italian guidelines allow the living donations from emotionally related donors only after explicit and voluntary consent to the donation expressed by the donor and recipient involved .So, in addition to the clinical and immunological assessments and a psychological exam, living donors are also assessed regarding their reasons for donating, their understanding of the potential risks and of the real likelihood of the transplant being successful in terms of graft and patient survival, the bond of affection between the donor and recipient, and the honesty of their free and informed consent –20.In 2020, 129,681 solid organs were transplanted annually, with a 17.6% decrease over 2019 . Among 80,926 kidney transplants performed worldwide, 31.07% were from live donors .In 2020, the Covid-19 pandemic led to an overall decrease in the Italian transplant activity of <10%, compared to 2019. A decrease of −19.4% in transplants from living donors was registered compared to 2019. As a result, in 2020 patients waited for 2.07 years on average for a deceased donor kidney . The redn = 169) were performed by A.O.U. Città della Salute e della Scienza of Turin, followed by A.O. of Padua (n = 139) and Ospedale Civile Maggiore of Verona (n = 96)  .The Member States of the European Union are active in the field of living organ donation starting from the Directive 2010/53/EU, also known as The European Organs Directive  stating that the handling and disposal of human organs should respect the fundamental rights and the human body . Thus, iThe evaluation, of the donor in particular, must comply with a rigorous process. In 2004, the Consensus Statement of the Amsterdam Forum on the Care of the Live Kidney Donor developed an international standard of care with a position statement of the Transplantation Society regarding the responsibility of the community for the live kidney donor . It was https://www.iss.it/web/iss-en/transplants). The Italian CNT supports the correct information on the issues of donation and transplantation through the promotion of information, education and cultural growth of the general public especially in the field of living transplantation.In Italy, Law n. 91 dated 1st of April 1999 introduced the National Transplant Center (CNT) as the technical body of the Ministry of Health  focuses on the improvements of the quality and safety of healthcare as key elements in the preservation of the constitutionally protected right to enjoy good health , 29.Living donation raises ethical and legal issues because donating an organ exposes a healthy person to the risk of and recovery from unnecessary major surgery, with a permanent injury of psycho-physical integrity , 30.The organs and tissues donation system is a complex process. In living donation, the most important issue is a free and informed consent correctly obtained from the donor and the recipient. The donors must be aware of their donation act and must express their will in serenity and without any coercion, considering that the donation cannot and must not represent any opportunity for profit , 31, 32.This is well summarized in the definition of organ donation provided by the European Parliamentary Research Service, as “the act of giving one or more organs (or parts thereof), without compensation, for transplantation into someone else” .It is therefore clear that in the context of living kidney transplantation it is more than ever mandatory to protect the donor's health and safety and to demonstrate to the community the effectiveness and transparency of the service during the whole process.The procedure should be documentable and transparent in the light of a free and informed consent, given by a healthy subject, without coercion and/or disinformation, and in the absence of any interest and/or utility. Healthcare safety is also based on these principles .The article 1 of Law 24/2017 stated that safety is guaranteed through proper prevention tools and healthcare risk management, in connection with the most effective use of structural, technological and organizational resources available , 29.In living donation, the risks for the donor and recipient can come from missed surveillance as imposed by the guidelines. This could affect patients' trust in healthcare professionals and transplantation Organizations concerning living donation.The biggest risks, regarding the safety of the living donation process, affect the donor, as resumed in the following , 31, 34:- the consent is absent;- the consent is not informed or not fully informed;- the consent is coerced or secondary to practices such as organ trafficking or commercialization;the consent is invalid , the donor is not genetically, legally or emotionally related to the recipient.In addition to the clinical and immunological assessments, the living donors have to be assessed with regard to their reasons for donating, their understanding of information provided by physicians, the nature of the relationship between the donor and recipient, and the validity of the consent.For this purpose, according to national guidelines on kidney transplantation from living donors, donors and recipients are assessed by a medical “Third-Party” Commission (TPC), independent from the transplantation team. The TPC was instituted by the Italian National Transplant Center (NTC) in living-donor kidney transplantation guidelines released in 2002 , 17.The Decree n. 116/2010 by the Minister of Health, providing the regulation of living organ (kidney and liver) donor transplantation, underlined the dual role of TPC of verifying and monitoring the process. This evaluation takes place at the last stage of the donation process. Only after the successful conclusion of this process, the transplant can occur.With particular attention to the donor as the most exposed person to the risks, it is requested:1) the verification of informed, free and conscious consent through verbal and written information, and of the relationship between donor and recipient ;2) the control of the procedure to prevent risks of organ trafficking or commercialization or coercion.The tools of TPC are:- medical records, identity documents (objective assessment);- structured interview with the potential donors.At last, TPC expresses a joint opinion.analytical stage; 2° or listening stage; 3° stage of the legal requirements; 4° stage of traceability.The evaluation process performed by TPC can be divided into four stages: 1° or According to the national guidelines, in 2010 the Hospital “Città della Salute e della Scienza” of Turin, Italy, established a TPC for kidney transplantation, including a forensic pathologist, a nephrologist and a medical director, all independent from the transplantation team.Between April 2011 and December 2020, TPC assessed 116 potential donor-recipient pairs, examining their medical records, and interviewed potential donors and recipients, recording on evaluation reports and eligibility determination forms. Donors and recipients were assessed separately; the Commissioners, then, expressed a joint opinion.The mean time between identifying a living donor and donation is 6–12 months.The data are described in aggregate form. In total, 232 subjects were examined , consistThe relationship between donor and recipient included genetically-related donation (58%) and non-genetically-related donation (42%). Among the genetically-related donation, parents were 63% (mostly the mothers), brothers/sisters were 34%, and other relatives 3%. Among non-genetically-related donations, married couples were 75%, unmarried couples 10% and other 15% .The recipients' kidney pathologies, for which the living-donor transplant was proposed, included, for the most cases, acquired glomerular/tubular/interstitial nephropathy and congenital cystic nephropathy.The donation was prompted by the donor in 80% of cases; the living donation was suggested by the physicians of the recipient in most cases.For most donors, the decision arose from the desire to help a loved one to improve their quality of life and to avoid dialysis, which was perceived as a very stressful family situation. Several donors made their decision after rationally considering the pros and cons for the recipient's life in the absence of alternatives for relief. Those donors were unable to accept the recipient's risks deriving from cadaver transplantation and a too long waiting list. In some cases, the reason for the donation was to improve the familiar setting which was severely affected by the recipient's pathology. Thus, an active gesture “had” to be made, especially if alternative donor options were lacking. On the other hand, the recipients' reactions were happiness with no attempt to influence the donor's decision, ranging to the concern for the consequences for the donors with initial opposition to the donor's decision. In no cases were the donors' and recipients' consents withdrawn, and no pressures or obligation from the outside were mentioned by the pairs or perceived by the Commission.Living-donor transplantation represents an alternative—not a substitution—to the waiting list for a deceased donor organ. Nevertheless, living transplant often is the only treatment option to improve or save the life of patients with an end-stage kidney failure . It is wLiving organ donors are a unique group of patients because they voluntarily undergo a surgical procedure to remove a perfectly healthy kidney to help another person. Their evaluation has to take into account all the physical, psychological, and social risks to the individual donor , 36. Alsprohibits any actions on one's own body when causing a permanent damage to physical integrity or when violating Law, public order or decency. In fact, the Law n. 458 of June 26, 1967, allows the living donor kidney transplantation. The regulatory framework of living donation includes the Italian Code of Medical Deontology (last edition in 2014) where the article 39 states that living organ and tissue donations allowed only for diagnostic, therapeutic, or scientific research purposes and if it does not cause permanent damages to donor's physical or psychic integrity, according to the Law. The donation must be a no-profit and no-commercial activity, a written, free and informed consent must be given by the donor.In Italy, the donation from living people is allowed notwithstanding the Art.5 of Civil Code, which The inspiring principles are the same of those underlined in the Amsterdam Forum .The selection and the assessment of donor eligibility must follow appropriate standards. These include a specific and informed consent process which should make the potential donors aware of the risks of their upcoming procedure, as well as future prospects of a life with only one kidney. Consent may be withdrawn at any time before the transplant, and this should be clearly written and explained , 31, 34.The importance of the evaluation of motivations, in addition to the clinical, immunological and psychological assessments, arises from the peculiar nature of unrelated living donors in kidney transplantation, which is much debated. The idea behind the PTC is the creation of an independent committee that assesses the donor's motivation and the understanding of the process, and that confirms lack of coercion or commercial activity, even if this proves difficult at times.Starting from the experience of the Hospital “Città della Salute e della Scienza” of Turin, Italy, in living kidney donation, the Authors divided the evaluation process performed by TPC into four stages, with a particular attention to the potential donor who is the one most exposed to ethical risks.Evaluation of the medical records  with regard to the donor's age, the relationship with the recipient, risks and potential consequences of the donation . The previous psychological assessments are evaluated to better understand the reasons for donation, the pair's emotional relationship, the support of the pairs' family, the expectations regarding the health of the couple and to understand if the potential donor is mentally fit for the procedure. Any coercion, unethical or financial practices between donor and recipient could be ruled out at this point.Structured interviews are carried out with the donor and recipient, regarding the whole donation's process, and the information received by the donor about his/her health, the surgical risks, and the potential consequences of the donation. The consistency of donor and recipient's interviews is assessed.The final donor's consent must be free and informed, and both donor and recipient have the option to withdraw at any time from the donation process. The Commission definitely excludes any coercion and opportunity of profit and/or other utility from donation, evaluates the impartiality of the records and the transparency of the process. TPC expresses a written joint opinion, and the outcome can be positive or negative. In the latter case, the Commission could suggest supplementary assessments or more time to make the decision filling any gap in the informed consent process.It includes the recording of the eligibility determination forms of the potential donor and recipient, and the conclusive evaluation report which all Commissioners must sign. A copy of the conclusive evaluation is provided to the donor/recipient pair and exhibited to the judicial authority that ultimately authorizes the donation.This systematic process of the TPC proposed here could be a tool that proactively reduces and controls the risks of coercion, organ commercialization, lack of information, insufficient weighting of donative choice, that can arise in living kidney donation.All living donation procedures should reflect fiduciary obligations that healthcare providers have toward their patients, deriving from the relationship of trust between physician and patient. The social implications are also wide if one considers the worldwide campaigns to promote public awareness about organ donation and transplantation, and to encourage people to register their organ donation decision.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.LT and DS conceived and designed the study. LT, DS, GD, IR, and AS contributed to the writing of the manuscript. GD made the critical revision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor DF declared a past co-authorship with one of the author LT at the time of review.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Opioid use disorder (OUD) is a serious health problem that has dramatically increased over the last decade. Although current therapies for the management of OUD can be effective, they have limitations. The complementary strategy to combat the opioid crisis is the development of a conjugate vaccine to generate high affinity antibodies in order to neutralize opioids in circulation before reaching the brain. The components of an opioid vaccine include an opioid hapten (6-AmHap) that is conjugated to a carrier protein (tetanus toxoid) with the addition of adjuvants . There is no consensus in the literature as to whether preexisting immunity to the carrier protein may impact the immunogenicity of the conjugate vaccine by inducing an enhanced or suppressed immune response to the hapten. Here, we investigated whether pre-exposure to tetanus toxoid would affect the immunogenicity and efficacy of the heroin vaccine, TT-6-AmHap. Mice were primed with diphtheria, tetanus, and acellular pertussis (DTaP) vaccine at weeks -4 and -2, then immunized with TT-6-AmHap vaccine at weeks 0, 3, and 6. Using ELISA and behavioral assays, we found that preexisting immunity to tetanus toxoid had no influence on the immunogenicity and efficacy of the TT-6-AmHap vaccine. Opioid use disorder (OUD) is a serious public health concern in the United States and around the world. The rate of opioid addiction and overdose-related deaths has increased exponentially over the last decade ,2, with Vaccines against drugs of abuse must induce high affinity antibodies that bind the drugs in the systemic circulation and prevent them from crossing the blood–brain barrier, thereby preventing the drug’s binding to and activation of the μ-opioid receptors (MOR) in the brain and their subsequent physiological effects. Opioids, such as heroin, are small molecules that lack the ability to elicit T-cell-dependent immune responses, which are characterized by immunological memory and potent antibody responses. To overcome this problem, a heroin hapten, 6-AmHap was developed. This hapten is conjugated to an immunogenic carrier protein, tetanus toxoid (TT), which can induce strong immune responses.197) are three commonly used carrier proteins in licensed conjugate vaccines [Haemophilus influenzae type b (Hib) disease [Tetanus toxoid (TT), diphtheria toxoid (DT) and cross-reacting material of diphtheria toxin with amino acid 197 substitution  vaccine or through continuing boosting with adult tetanus, diphtheria, and pertussis (Tdap) or TT alone, it is important to know if preexisting immunity to TT would impact the efficacy of the heroin vaccine. Here, we investigated whether priming with TT through administration of DTaP would affect the immunogenicity and efficacy of the heroin conjugate vaccine TT-6-AmHap in mice. Notably, our results revealed that antibodies generated to the priming agent, TT, had no impact on the ability of the TT-6-AmHap vaccine to induce high titer anti-hapten antibodies that blocked the effects of heroin.sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), monophosphoryl lipid A (PHAD®), and semi-synthetic cholesterol were purchased from Avanti Polar Lipids . Bovine Serum Albumin (BSA), which was used in the blocking buffer for ELISA. was purchased from Sigma-Aldrich . Heroin HCl was purchased from Lipomed Inc. . Tetanus toxoid (TT) was purchased from Statens Serum Institut . DTaP (Infanrix®) was purchased from GlaxoSmithKline Biologicals . BSA, SM(PEG)2 linker, and bicinchoninic acid (BCA) protein assay kit, which were used for the coupling reactions, were purchased from Pierce Protein Research/Thermo FisherScientific . Immunolon 2HB flat-bottom ELISA plates were purchased from Thermo Scientific . Diphtheria toxoid (DT), pertussis toxin (PT), and TT used as coating antigens for ELISA were purchased from List Biological Laboratories, Inc. . Peroxidase-linked sheep anti-mouse IgG (γ-chain specific) was purchased from The Binding Site . Peroxidase substrate solution was purchased from KPL, Inc. . Alhydrogel was purchased from Brenntag . BD Insulin Syringes (0.5 mL) used for vaccine injection were purchased from FisherScientific . Pipettes and tips were purchased from Mettler and Toledo .Liposomal lipids consisting of 1,2-dimyristoyl-2 cross-linker at a molar ratio of 1:1600 for 2 h at room temperature. Excess linker was removed by spin desalting column and the protein content was quantified by BCA protein assay. The hapten, 6-AmHap, was added dropwise to a stirring solution of TT−SM-(PEG)2 intermediate at a molar ratio of 1:300 and was incubated at room temperature for 2 h. Overnight dialysis against PBS, pH 7.4, at 4 °C was used to remove excess haptens. The TT−hapten conjugates were quantified using the BCA protein assay. The number of heroin haptens attached to the TT molecule was assessed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry  carrier protein as described ,16. Brietrometry . The 1:12 linker in PBS at a molar ratio of 1:10 for 2 h at room temperature. Excess linker was removed by spin desalting column. A 6-AmHap was added dropwise to a stirring solution of BSA−SM-(PEG)2 intermediate at a molar ratio of 1:300 and was incubated at room temperature for 2 h, followed by excess 6-AmHap removal by overnight dialysis against PBS, pH 7.4, at 4 °C. The BSA-6-AmHap conjugate was membrane filtered and quantified by BCA assay. The 1:10 BSA:SM-(PEG)2 ratio yielded approximately 5 haptens per molecule [BSA was coupled to 6-AmHap by incubating BSA and SM-(PEG)molecule .®) as adjuvant mixed with 6-AmHap conjugated to TT (TT-6-AmHap). ALFA was prepared as previously described [® were dissolved in solvents that were removed by rotary evaporation, hydrated in water and microfluidized. The molar ratio of DMPC:cholesterol:DMPG was 9:7.5:1, and the molar ratio of phospholipid:PHAD® was 220:1. The multilamellar vesicles (MLVs) were lyophilized. The preformed multilamellar lyophilized liposomes contained 50 mM phospholipids which had been mixed 1:1 with TT-6-AmHap and then bound to Alhydrogel® to give a final vaccine concentration of 10 μg TT, 30 μg aluminum hydroxide in Alhydrogel®, and 20 μg PHAD® per dose of 50 μL in PBS, pH 7.4. The approximately 32 haptens/TT molecule ratio corresponds to approximately 0.8–0.9 µg of 6-AmHap/dose. The vaccine was made prior to each immunization. The TT-6-AmHap prepared in the engineering run for the GMP manufacture of TT-6-AmHap was stable at the most recent testing time point of 9 months from the date of manufacture.The formulation of TT-conjugated vaccine consisted of Army Liposome Formulation adsorbed to aluminum hydroxide (ALFA) and contained monophosphoryl lipid A   were immunized via the intramuscular (IM) route in alternate rear thighs at weeks -4 and -2 with 0.1 µL, 1 µL, 10 µL, or 25 µL of DTaP vaccine diluted in saline to a final volume of 50 µL for all groups. On weeks 0, 3, and 6, mice were immunized by the IM route in alternate rear thighs with 50 µL of TT-6-AmHap vaccine. DTaP doses were drawn using pipettes with varying sizes. Both DTaP and TT-6-AmHap vaccines were injected using 0.5 mL insulin syringes. All animals were bled prior to each immunization with TT-6-AmHap vaccine.Female BALB/c mice  for 5 min. Following the subcutaneous administration of heroin (1 mg/kg), the mice were placed in the chamber and the movement monitored for 5 min. Data were analyzed using the EthoVision XT software  to calculate the total distance traveled (cm) for each animal using the following equation: Total Distance Traveled (cm) = Distance after heroin challenge − Distance before heroin challenge.Prior to the testing day, mice were acclimated to the locomotion chamber for 30 min on at least two occasions. The locomotion assay was performed immediately following the hot plate pre- and post-challenge tests. Baseline motor activity was recorded by an overhead camera  and incubated overnight at 4 °C. The ELISA was performed the next day as previously described . Brieflyp ≤ 0.05. All values represent the mean ± S.E.M.GraphPad Prism 8  was used for statistical analyses. Antibody titers are expressed as endpoint titers which represent the dilution of sera where the absorbance is twice that found in the background. Data were analyzed using one- or two-way ANOVA as indicated in the figure legends. A post hoc test was performed using Tukey’s multiple comparisons test to identify differences between groups relative to the control. Differences were considered significant if To determine if preexisting immunity to TT would interfere with TT-6-AmHap immune responses, mice were primed with increasing doses of the DTaP vaccine ranging from 1/5000th dose of DTaP (0.1 µL DTaP) to 1/20th dose of DTaP (25 µL DTaP) at weeks -4 and -2. Immunization with TT-6-AmHap induced high antibody titers to 6-AmHap. DTaP-primed animals had equivalent 6-AmHap antibody titers to those obtained from animals that received only the TT-6-AmHap vaccine b. FurtheThe efficacy of the TT-6-AmHap vaccine was tested by challenging the mice with 1 mg/kg heroin SC at week 10 followed by assessment using the hot plate and locomotion assays. Heroin-induced antinociception was significantly reduced in the TT-6-AmHap vaccine-only group and DTaP-primed groups compared to the naive control mice indicated by the low % maximum possible effect (% MPE) values a. There In the present study, we sought to determine the effects of preexisting immunity to tetanus toxoid on the efficacy of a heroin-conjugated vaccine. Specifically, we determined the immunogenicity and efficacy of the TT-6-AmHap vaccine in mice pre-immunized with TT using ELISA and behavioral assays. We found that priming with increasing doses of DTaP did not interfere with the generation of hapten-specific antibodies, nor did it change the efficacy of the vaccine . To the We found that priming with TT through DTaP administration enhanced TT immune responses in the vaccinated animals with TT-6-AmHap a, demonsWhile others have reported that preexisting immunity to the priming agent suppressed hapten-specific antibody responses ,26, we h®) and aluminum hydroxide. We have previously demonstrated the superiority of TT over CRM197 conjugate vaccines in terms of inducing potent inhibition of heroin-induced antinociception [These conflicting findings in the literature could be due to several factors, including the type of carrier protein used, the concentration of the carrier protein, the hapten–carrier molar ratio, and the choice of adjuvants. Our heroin vaccine contained 10 μg of TT as a carrier with approximately 32 6-AmHap molecules per TT and was adjuvanted with liposomes containing monophosphoryl lipid A  between the 1 µL and 10 µL DTaP groups in the locomotion assay (In addition to not having any influence on anti-6-AmHap immune responses, pre-immunization with TT did not impact the efficacy of the heroin vaccine. The vaccine showed an equal protection against the heroin challenge in the antinociceptive and the locomotion assays in all groups . Althougon assay b; howevePreexisting immunity to tetanus toxoid had no effect on the antibody titer or efficacy from the heroin challenge induced by immunization with the 6-AmHap heroin vaccine. Neither an enhancement nor a suppressive effect on the efficacy of the TT-6-AmHap conjugate vaccine was observed. Considering most people have a preexisting immunity to TT, the data from this study suggest that this will not reduce the efficacy of the TT-6-AmHap heroin vaccine.


P = 0.953) and post-remission. In this non-randomized study, there were no significant differences in 4-year event-free  and overall  survival in the LDC/G-CSF and SDC cohorts, respectively. After the first course of induction, recovery of white blood cell (WBC) and platelet counts were significantly faster in patients receiving LDC/G-CSF than in those receiving SDC (11.5 vs. 18.5 d for WBCs (P < 0.001); 15.5 vs. 22.0 d for platelets (P < 0.001)). To examine the quality of molecular response, targeted deep sequencing was performed. Of 137 mutations detected at diagnosis in 20 children who attained hematological CR after two courses of LDC/G-CSF (n = 9) or SDC (n = 11), all of the mutations were below the reference value  after two courses, irrespective of the treatment group. In conclusion, children with AML receiving LDC/G-CSF appear to have similar outcomes and mutation clearance levels, but significantly lower toxicity than those receiving SDC. Thus, LDC/G-CSF should be further evaluated as an effective alternative to remission induction in pediatric AML.Standard chemotherapy regimens for remission induction of pediatric acute myeloid leukemia (AML) are associated with significant morbidity and mortality. We performed a cohort study to determine the impact of reducing the intensity of remission induction chemotherapy on the outcomes of selected children with AML treated with a low-dose induction regimen plus granulocyte colony stimulating factor (G-CSF) (low-dose chemotherapy (LDC)/G-CSF). Complete response (CR) after two induction courses was attained in 87.0% (40/46) of patients receiving LDC/G-CSF. Post-remission therapy was offered to all patients, and included standard consolidation and/or stem cell transplantation. During the study period, an additional 94 consecutive children with AML treated with standard chemotherapy (SDC) for induction (80/94 (85.1%) of the patients attained CR after induction II,  Malignant tumors, including acute myeloid leukemia (AML), are widely considered to have a genetically heterogeneous cell population ,2. RecenWith the pre-existence of small drug-resistant subclones, the elimination of a dominant drug-sensitive clone by current chemotherapy, usually given at the maximum tolerated dose, might allow the competitive release of resistant subclones to undergo accelerated growth in a resource-rich environment, resulting in more rapid disease progression or relapse ,9. ThesePediatric AML is generally managed with two courses of near-myeloablative therapy to induce remission, followed by two-to-three additional courses of chemotherapy or hematopoietic cell transplantation (HCT) in selected patients for post-remission therapy ,14. Howede novo AML.A low-dose chemotherapy (LDC) regimen featuring one-tenth of the standard dose of chemotherapeutic drug cytarabine and one-half of the dose of anthracycline in conjunction with granulocyte colony stimulating factor (G-CSF), was used in induction and consolidation therapies for elderly patients with AML who were medically unfit to receive intensive chemotherapy . ApproxiGiven the efficacy and good tolerance of LDC/G-CSF regimens in older patients, we report here a cohort study of children with newly diagnosed AML treated with the LDC/G-CSF regimen. The clinical and biological features, and outcomes of children with AML receiving at least one induction cycle of LDC/G-CSF were compare with those receiving standard-dose chemotherapy (SDC), to investigate whether LDC could achieve similar clinical efficacy as SDC.de novo AML were treated at our hospital. For induction I, 46 received LDC/G-CSF and 94 received SDC. Median age of 82 males and 58 females was 81 months . Median white blood cell (WBC) count was 16.5 × 109/L . According to World Health Organization (WHO) criteria, 85 patients had AML with recurrent cytogenetic abnormalities and 55 had AML that was not otherwise specified  with P = 0.014) and had lower initial WBC counts  vs. 22.1 × 109/L (0.4–606.0 × 109/L); P = 0.002) than those treated with SDC. There were no significant differences between other presenting clinical or biological features between both groups  vs. 93 months ; Thirty-four of 46 (73.9%) children who received the first course of LDC/G-CSF (induction I) attained remission. Failure to attain remission was observed in 12 (26.1%) patients because of no response (NR) in 7 and partial response (PR) in five patients Table . A seconOverall, after inductions I and II, 40/46 (87.0%) of patients originally assigned to the LDC/G-CSF group attained remission. Persistent disease was the most common cause of failure in achieving CR after induction II Table . No toxiSixty-four of 94 children assigned to the SDC group 68.1%) attained remission after induction I. Failure to attain remission was observed in 30 patients (NR in 13 and PR in 17 patients). After induction II, 80/94 (85.1%) of the patients attained remission. Causes of failure included persistent disease (12 cases) and treatment-related death (two cases)  and 71/94 (75.5%) initially in the LDC/G-CSF and SDC groups, respectively, were alive without evidence of disease. At a median follow-up of 2.5 years , 118 (84.3%), 80 (57.1%), 56 (40.0%) and 30 (21.4%) patients were followed for at least 1, 2, 3 or 4 years, respectively. The 4-year OS and event-free survival (EFS) for 140 children with AML were 73.5 ± 3.9% and 67.7 ± 4.3%, respectively Fig. , and 4-yP = 0.69 and P = 0.99, respectively, log-rank test stratified by AML risk; There were no significant differences in OS and EFS between the LDC/G-CSF and SDC groups (P = 0.91) or EFS . High-risk AML was significantly associated with OS  and EFS . Even when gender, age, initial WBC and HCT were added as covariates, chemotherapy dose was not significantly associated with OS or EFS (Multivariate Cox regression with chemotherapy dose groups (LDC/G-CSF and SDC) and the AML risk group  as covariates  showed nS or EFS .P = 0.94; Fig. P = 0.49; Similarly, 4-year CIR rates in the LDC/G-CSF and SDC groups were not significantly different . AML  and bone marrow (BM) transplant  were significant factors for death (P = 0.46). High-risk AML  was significantly associated with EFS. Similar results were obtained for risk of relapse  of 0.5 × 10P < 0.001). Pulmonary infection and sepsis were the most common complications. Non-hematologic toxicities were mild (mostly grade I or II) in both groups.Grade III or IV infectious complications occurred in 4 (8.7%) and 23 (24.4%) patients receiving LDC/G-CSF and SDC regimens, respectively (n = 9) or SDC (n = 11) regimens. There were no significant differences in clinical and cytogenetic characteristics by treatment group . A total of 149 mutations identified by exome sequencing, including 137 found in diagnosis samples and 12 identified in relapsed samples, were analyzed (Whole exome sequencing data not shown), and mean coverage was 14 010.99 × . At diagnosis, the average number of mutations per patient for the LDC/G-CSF and SDC groups was 7.6 (range 4–13) and 6.3 (range 2–13), respectively. The average variant allelic frequency (VAF) at diagnosis was 29.4 and 35.3% for the LDC/G-CSF and SDC groups, respectively .P = 0.876 (Wilcoxon rank-sum test); Fig. 0 samples were cleared in the LDC/G-CSF group  , as repo0 samples, only one mutation remained in follow-up analyses by targeted deep sequencing  was detected during the 11-month off-therapy visit of a child receiving SDC; this patient relapsed 4 months later. BM samples from 19 CR patients during follow-up (0–19 months off therapy) harbored no detectable mutations (VAF < 1%). For 3/20 patients for whom samples were collected after HCT, no mutation was detectable 6–19 months after transplantation.Of all mutations detected in Ding Fig. . This peIn our study, the CR, EFS, OS and CIR rates of 46 children with AML initially given a low-intensity regimen for the first induction remission were comparable to those of 94 children admitted to the same hospital during the study period and given the SDC regimen. Moreover, chemotherapy dose during induction was not associated with CR, EFS, OS or CIR. AML risk of relapse and HCT were independently associated with outcome. Given that ∼90% of relapses in pediatric AML occur within 2 years of diagnosis , our resObserved clinical outcomes were supported by mutation clearance patterns during and after completion of therapy. Residual AML detected by next-generation sequencing is an independent prognostic factor and predictor of relapse in AML ,23, and 9/L during induction I. Also, the frequency of severe infections in children given LDC/G-CSF was significantly lower than for children given SDC. This finding is unexpected, because 28.2% of patients treated with LDC/G-CSF because they had active infection and were not candidates for SDC. Infection was managed simultaneously with AML treatment and no patient died from toxicity. Reduced duration of neutropenia and thrombocytopenia, and fewer infectious complications, suggest that the integrity of the hematopoietic system was preserved during hematologic remission in children receiving LDC/G-CSF. These observations challenge the concept [Intensive chemotherapy regimens for pediatric AML are associated with high morbidity and mortality . Intensi concept  that neaAlthough mechanisms underlying the clinical efficacy of LDC/G-CSF are currently unknown, it is possible that G-CSF makes leukemia cells more vulnerable to chemotherapy through sensitization or priming ,33. G-CS9/L did not receive LDC/G-CSF in our study. We found no evidence of increased expansion of leukemia cells in the peripheral blood of children receiving LDC/G-CSF during first induction remission (data not shown). Importantly, as the initial WBC count is considered to be one of the most important influencers on the outcome of AML, we added initial WBC as covariates and found that chemotherapy dose was not significantly associated with OS or EFS in our cohort. Also, the OS and EFS were not significantly different between the LDC/G-CSF group and a SDC subgroup with WBC count <70 × 109/L. Nevertheless, the efficacy of the LDC/G-CSF regimens in pediatric AML patients with hyperleukocytosis remains to be investigated.Because G-CSF can potentially stimulate leukemia cell proliferation , children = 46) and possible selection bias (WBC counts <70 × 109/L). Randomized multicenter clinical trials are needed to further investigate the clinical efficacy of the LDC/G-CSF regimen as induction remission therapy.Our initial rationale for offering a low-intensity regimen for children with AML was based on the high risk of toxicity-related death or treatment abandonment due to economic reasons. Hence, our results have major implications for children in limited-resource countries. Furthermore, low-intensity treatment as induction remission therapy might impart a reduced selection pressure on the leukemic genome and clonal structure, while use of G-CSF will concurrently sensitize leukemic cells to achieve efficient mutation clearance. However, our study has some limitations including that it was non-randomized, recruited a relatively small number of patients  with newly diagnosed AML, as defined by WHO criteria . The stuAML diagnosis was based on morphologic, immunophenotypic, karyotyping and molecular genetic studies. Patients with promyelocytic leukemia, AML arising after the diagnosis of myelodysplastic syndrome, treatment-related AML or AML of Down syndrome were not included in this study. AML was classified as low, intermediate or high risk according to National Comprehensive Cancer Network criteria .9/L were not eligible for LDC/G-CSF. Post-remission therapy was the same for all patients and administered in the following sequence: (1) cytarabine and mitoxantrone, (2) etoposide and cytarabine, and (3) high-dose cytarabine and L-asparaginase .Targeted deep sequencing of mutations detected at diagnosis was performed in 9 patients who had received LDC/GCSF for induction I and II, and 11 patients who had received SDC for both induction courses. These 20 patients were selected based on sample availability. Sampling time points for sequencing samples are given in P<0.05 was considered statistically significant. Time to failure is defined in the Exact Pearson's chi-squared test was used to compare categorical variables between two or more groups. Wilcoxon rank-sum test was used to compare continuous variables between two groups. Risk group was used in survival analyses as a stratification factor or a covariate. Kaplan–Meier analysis was used to estimate survival functions. Stratified log-rank test and Cox regression model were applied to compare OS and EFS between the LDC/G-CSF and SDC groups. CIR or refractory AML was estimated by the Kalbfleisch–Prentice method, accounting for competing risk . Gray's nwz006_Supplemental_FileClick here for additional data file.


Immunoglobulin gamma autoantibodies directed against myelin oligodendrocyte glycoprotein (MOG-IgG) are associated with specific neurological syndromes, most frequently acute disseminated encephalomyelitis, optic neuritis and longitudinally extensive transverse myelitis.We audited the database of MOG-IgG cell-based assay (CBA) requests from the Oxford Autoimmune Neurology Diagnostic Laboratory between 2011 and 2019. These data encompassed all samples referred to the centre for MOG-IgG testing by physicians over this time period. Serum samples were available from 272 centres, mostly within the UK, and CSF from 97 centres without clear referral bias. MOG-IgG testing was performed using a cell-based assay as previously described, modified to detect MOG-IgG1 since 2014.For patients with negative serum and positive CSF MOG-IgG results, the patient’s general practitioner and referring neurologists supplied case notes and test results from which pre-specified clinical and paraclinical parameters were extracted . Data were compared with a published cohort.Overall, 118/533 (22.1%) patients with paired samples showed MOG-IgG in either serum or CSF of which 66/118 (55.9%) were only positive in the serum and 48/118 (40.7%) were positive in both compartments. We identified only 4/118  patients with MOG-IgG exclusively detected in the CSF with semi-quantitative visually assessed CBA scores  which were not significantly different to the CBA scores of CSF samples from patients with both serum and CSF MOG-IgG positivity .Clinical details were located on three of the four patients with exclusively CSF MOG-IgG. One patient was diagnosed with pneumococcal meningitis, and neither treated for nor thought to have MOG-IgG-associated disease . In the remaining two patients (CSF CBA scores 4 and 1), MRI spine results showed longitudinally extensive transverse myelitis . In one patient, CSF showed a lymphocytic pleocytosis with slightly elevated protein. CSF oligoclonal bands were negative in both patients. Both patients were treated with a short course of steroids and subsequently with either intravenous immunoglobulins or plasma exchange. We compared their clinical characteristics and outcome measures with a published Oxford cohort of MOG-IgG serum positive patients and found no clearly distinguishing features .Our study represents an unbiased assessment of all consecutive samples referred to a national laboratory using a highly specific CBA, over 8 years. It shows that MOG-IgG CSF+/serum– patients are detected at a frequency of 0.8% of all paired CSF-serum samples from the UK’s national centre for MOG-IgG testing. Although limited by small numbers, our comparison identified no distinctive clinical features in patients with LETM, suggesting a disease continuum regardless of compartment positivity.Our findings suggest that, were MOG-IgG testing on CSF to become routine, the number needed to test (NNT) to identify one extra MOG-IgG patient would be 133, assuming the same rate of positivity as seen in the patients with matched serum and CSF results. In actuality, the proportion of positive tests in the cohort with only serum available for testing n=21 731) was 8.7%, suggesting that the pre-test probability of unselected patients is likely to be lower than for those who underwent paired serum and CSF sampling. Similarly, for patients with only CSF available, the proportion of CSF positive results  was lower than for patients with paired samples (9.8%). Assuming this lower pre-test probability, the NNT to detect one extra case may be as high as 328 if all patients referred for MOG-IgG testing underwent lumbar puncture. Additionally, we identified one patient with pneumococcal meningitis, for whom CSF MOG-IgG was felt to be clinically unrelated to the presentation. This means that the significance of MOG-IgG CSF+/serum– findings must be interpreted within the context of the clinical presentation.1 731 wasLimitations include a change in the detection method of MOG-IgG versus IgG1 during the study period  and both clinically-relevant CSF+/serum– MOG-IgG patients having monophasic disease after 3–4 years of follow-up. The majority of paired samples were taken on the same day (74%) but it is possible that CSF MOG-IgG positivity could reduce over time. The rate of CSF MOG-IgG positivity in our cohort was lower than that reported in previous studies .Based on our results, extending CSF testing to all patients is likely to only capture a small number of additional patients with additional difficulties in the interpretation of CSF+/serum– MOG-IgG results. Therefore, we suggest CSF MOG-IgG testing should be guided by each individual’s presentation and undertaken when clinically indicated. The benefits of rarely detecting additional MOG-IgG CSF+/serum– patients, with implications for the duration of immunosuppression, would need to be balanced against the small but non-negligible risks of post-lumbar puncture complications. However, it will be important to understand how the specific method used to measure CSF MOG-IgG determines sensitivity and specificity in different patient cohorts. Further research should focus on studying paired serum and CSF samples from patients and controls including individuals with clinically definite multiple sclerosis.