Pleurotus ostreatus (PO), a common edible mushroom, contains rich nutritional components with medicinal properties. To explore the effect of PO on ameliorating obesity and modulating the gut microbiota, we administered the mice with a low-fat diet or high-fat diet containing different dosages of PO . The body weight, adipose tissue weight, GTT, ITT, blood lipids, serum biomarkers of liver/kidney function, the gut microbiota and function were measured and analyzed after 6 weeks of PO treatment. The results showed PO prevented obesity, maintained glucose homeostasis and beneficially modulated gut microbiota. PO modified the composition and functions of gut microbiota in obese mice and make them similar to those in lean mice, which contributed to weight loss. PO significantly increased the relative abundance of Oscillospira, Lactobacillus group and Bifidobacterium, while decreased the relative abundance of Bacteroides and Roseburia. The prediction of gut microbiota function showed PO upregulated lipid metabolism, carbohydrate metabolism, bile acid biosynthesis, while it downregulated adipocytokine signaling pathway and steroid hormone biosynthesis. Correlation analysis further suggested the potential relationship among obesity, gut microbiota and the function of gut microbiota. In conclusion, all the results indicated that PO ameliorated obesity at least partly by modulating the gut microbiota. So obesity has become a great threat to human health, which is urgent to be resolved worldwide. At present, the main way to lose weight is to reduce energy intake and increase physical activity, but very few people can make it because of the abundant food and little spare time. Therefore, searching for more effective and easier strategies to ameliorate obesity becomes necessary.Nowadays, obesity has become a global pandemic and is still getting worse  because  or more . The wor or more . For obe or more ,6. There or more , includi or more , and inc or more ,10. ActuLactobacillus group, Oscillospira, Bifidobacterium, Saccharomyces, Streptococcus and Enterococcus [More and more studies have confirmed a close relationship between gut microbiota and obesity ,12,13. Srococcus ,20,21. Grococcus  and regurococcus ,23 and mrococcus ,25. Morerococcus  and bilerococcus ,28. Gut Pleurotus ostreatus (PO) is a kind of common gray edible mushroom with rich nutritional components and medicinal properties [Flammulina velutipes, Hypsizygus marmoreus, Lentinusedodes, Grifola frondose, Pleurotus eryngii) suppressed visceral fat accumulation [Pleurotus citrinopileatus has an anti-obesity effect [operties ,30 and ioperties . It has operties ,30,32,33mulation . Our prey effect . Howere,y effect . Besidesy effect , antioxiy effect , enhanciy effect .In this research, we explored the effect of PO on ameliorating obesity induced by a high-fat diet and modulating the composition of gut microbiota. The relationship between the alteration of gut microbiota and weight loss was particularly analyzed. This paper provides a preliminary research basis to curb obesity using PO.The PO was purchased from the Beijing Jingnan mushroom planting base , then it was dried  and ground to powder. The PO powder was added into the high-fat diet pellets. The composition of PO powder was measured according to GB 5009.3-2016, GB 5009.4-2016, GB 5009.5-2016, GB 5009.6-2016, GB 5009.10-2016, NY/T 1676-2008 and the results are shown in All the animal experimental procedures were conducted following the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and approved by Animal Ethics Committee of China Agricultural University, Beijing .n = 40) free from specific pathogens were purchased from Vital River Laboratories  and housed under 22 ± 2 °C temperature and 55% ± 10% humidity with 12 h light/12 h dark cycle in a Specific Pathogen Free (SPF) animal room. After a week of acclimatization with free access to a normal commercial basic diet and sterile water, one group of mice were fed with low-fat diet , while the rest were fed with high-fat diet  (n = 8). After 6 weeks, the mice fed with HFD were randomly divided into 4 groups based on body weight using a stratified randomization procedure: The negative control group  was fed with HFD; Three treated groups were fed with HFD added with PO powder at different mass fractions: 2.5% , 5%  and 10% . In order to fully explore the effects of different dosages of PO, the above three dosages were determined according to relevant studies and our previous studies [Four-week-old C57BL/6J male mice (rom fat) . The mic studies ,34. All The blood was collected from the infraorbital venous plexus of mice and the serum was separated by centrifugation at 3000 rpm for 15 min. The blood biochemical parameters including triglyceride (TG), total cholesterol (CHO), high density lipoprotein (HDL), low density lipoprotein (LDL), blood glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), albumin (ALB), uric acid (UA), blood urea nitrogen (BUN) and creatinine (CRE) were determined using a RA-1000 autoanalyzer .Obesity can lead to an imbalance of glucose homeostasis. To explore the effect of PO on glucose homeostasis, GTT and ITT were measured.In the last week of the experiment, GTT was performed after a 16-h fasting. The mice were intraperitoneally injected with glucose . The blood glucose concentration was measured at 0, 15, 30, 60, 90 and 120 min after injection via tail vein blood using a blood glucose meter .In the last week of the experiment, ITT was performed after a 6-hour fasting. The mice were intraperitoneally injected with insulin , followed by the measurement of blood glucose concentration at 0, 15, 30, 45 and 60 min after injection.The gut microbiota was analyzed at the end of 6 weeks of PO intervention. The fecal samples were collected and sequencing as previously reported . BrieflyAlpha diversity, beta diversity, principal component analysis (PCA) and non-metric multidimensional scaling (NMDS) were performed by Past3 software. Alpha and beta diversity were used to determine the species richness and evenness of gut microbiota. The NMDS analysis was used to explore the differences in microbial community structure among different groups. Heat map was made by Heml software to show the distribution of microbial species in different samples. LEfSe analysis was conducted online , which wt-test, while comparisons among more groups were performed using one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. The significant difference level was p < 0.05.All data were presented as means ± SEM and calculated by GraphPad Prism 5 Software . Comparisons between two groups were performed using a two-tailed Student’s p < 0.01) higher than that (26.2 ± 0.6) in the LFD group. Then the mice were administered with PO at different dosages. After another 6 weeks, PO lowered the body weight in HFD + POL, HFD + POM and HFD + POH  of mice fed with HFD was significantly  were carried out respectively. The taxa from phylum to genus level with significant differences were listed. We analyzed the families and genera whose relative abundances in HFD group were significantly different from those in LFD group or PO treatment groups in particular. By comparison of HFD and LFD, it was found that 20 families and 32 genera were significantly different. Among them, 13 families and 18 genera were significantly higher in LFD group while 7 families and 14 genera were significantly higher in HFD group A,B. CompOscillospira, Lactobacillus group, Bifidobacterium, Anaerostipes, Anaerovorax, Anaerofustis, Ruminococcus, Coprococcus, Bilophila and Bacillus) were all enriched in three PO-treated groups compared with HFD group. Among these genera, six genera were more abundant in LFD group than in HFD group  increased by all of the three PO treatments with different dosages, and four of the five families were also more abundant in LFD group than in HFD group A,C–F. ThFD group A,C–H. OnFD group B,I–N. ThTo further explore the relationship between gut microbiota and obesity inhibition, PICRUSt algorithm was used to infer metagenomes based on 16S rRNA gene data and predict the potential function of gut microbiota. PO treatments mainly increased 25 of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The relative reads count of the 25 pathways was also higher in the LFD group than HFD A. Four oOn the other hand, there were 24 KEGG pathways that were depressed by PO treatments. These 24 pathways were also lower in LFD group than those in HFD B. Some dOscillospira, Lactobacillus group, Bifidobacterium, Anaerostipes and Anaerovorax. Primary bile acid biosynthesis and secondary bile acid biosynthesis were positively associated with Lactobacillus group, Bifidobacterium and Anaerofustis. Butanoate metabolism was positively correlated with Oscillospira, Anaerostipes and Anaerovorax. It indicated that these bacteria might promote weight loss. Furthermore, there was a positive correlation between body weight and the decreased bacteria or the decreased pathways. These decreased pathways were positively correlated with the decreased bacteria  and 12 genera (6 increased and 6 decreased). The effect of PO on HFD-induced obesity was probably due to the role of PO in beneficially modulating the composition of gut microbiota, typically involving Oscillospira, Lactobacillus group and Bifidobacterium.In this study, at the phylum level, PO mainly increased Bacillota and TM7, and decreased Pseudomonadota and Bacteroidota. Further analysis showed that PO could beneficially modulate the gut microbiota by enriching six genera. Among the six genera, ght loss ,45,46,47The function of PO in regulating the gut microbiota is mediated by its various bioactive components. Like other mushrooms, PO can play the role of prebiotics, which is mainly responsible by its polysaccharides. Previous studies have shown that the important sources of prebiotics in mushrooms are non-digestible mushroom polysaccharides which can inhibit pathogen proliferation by enhancing the growth of probiotic bacteria in the gut ,49,50. POscillospira, Lactobacillus group and Bifidobacterium might play an important role in increasing lipid metabolism, carbohydrate metabolism, bile acid biosynthesis, and Bacteroides and Roseburia might play an important role in decreasing adipocytokine signaling pathway and steroid hormone biosynthesis. These findings were further confirmed by some existing studies. For example, Oscillospira has been reported to produce SCFAs and prevent the fat accumulation [Lactobacillus group has been reported to be involved in carbohydrate transport and metabolism and can ameliorate obesity [Bifidobacterium has been shown to modulate lipid metabolism [Oscillospira, Lactobacillus group and Bifidobacterium may be mainly responsible for the role of PO in ameliorating obesity, but this still needs to be further verified.To explore whether the function of PO in ameliorating obesity is achieved by regulating the gut microbiota, the functions of gut microbiota were predicted and the correlations between body weight, main differential bacteria and KEGG pathways were analyzed. The synthesis and degradation of ketone bodies and fatty acid metabolism were increased by PO. Ketone bodies are the intermediate metabolites in fat oxidation and metabolism. The increase of synthesis and degradation of ketone bodies and fatty acid metabolism revealed the enhancement of fat motivation. The biosynthesis of primary bile acid and secondary bile acid was both increased in the PO-treated groups. Bile acids can promote energy expenditure through the TGR5–cAMP–D2 signal pathway , mitochomulation . Lactoba obesity ,57, and tabolism . So, OscIn summary, this study proved that PO could prevent obesity and beneficially modulate gut microbiota. All three dosages of PO had significant effects, and the effect was more significant with the increase of PO dosage. The composition and functions of gut microbiota in mice fed with HFD were altered towards those fed with LFD by PO, and the alterations of the composition and functions of gut microbiota caused by PO contributed to weight loss. Therefore, PO ameliorated obesity at least partly by modulating gut microbiota.


The pathophysiology of bipolar disorder remains to be elucidated and there are no diagnostic or prognostic biomarkers for the condition. In this explorative proteomic study, we analyzed 201 proteins in cerebrospinal fluid (CSF) from mood stable bipolar disorder patients and control subjects sampled from two independent cohorts, amounting to a total of 204 patients and 144 controls. We used three Olink Multiplex panels, whereof one specifically targets immune biomarkers, to assess a broad set of CSF protein concentrations. After quality control and removal of proteins with a low detection rate, 105 proteins remained for analyses in relation to case–control status and clinical variables. Only case–control differences that replicated across cohorts were considered. Results adjusted for potential confounders showed that CSF concentrations of growth hormone were lower in bipolar disorder compared with controls in both cohorts. The effect size was larger when the analysis was restricted to bipolar disorder type 1 and controls. We found no indications of immune activation or other aberrations. Growth hormone exerts many effects in the central nervous system and our findings suggest that growth hormone might be implicated in the pathophysiology of bipolar disorder. Bipolar disorder is a lifelong condition characterized by recurrent fluctuations in mood state and energy, affecting more than 1% of the world’s population . MoreoveOne means to investigate brain pathophysiology is the study of proteins in cerebrospinal fluid (CSF). Protein concentrations in CSF might reflect central nervous system (CNS) processes more directly than serum or plasma due to the relative impermeability of the blood–CSF and blood-brain barriers –5. CSF iWe recently analyzed CSF samples from the same two independent bipolar cohorts used in the present study with a multiplex protein assay targeted for CNS processes . Two pron = 134) were recruited from the Northern Stockholm Psychiatric Clinic, Stockholm, Sweden. The SBP-G cohort (n = 70) was recruited from the Bipolar Clinic at the Sahlgrenska University Hospital, Gothenburg, Sweden. Inclusion criteria were patients at least 18 years old meeting the DSM-IV-TR criteria for any bipolar spectrum disorder.The study population is derived from the St. Göran bipolar project (SBP), which comprises two independently collected cohorts of patients with bipolar disorder and controls. The first case–control cohort was collected in Stockholm, Sweden (SBP-S) and the second in Gothenburg, Sweden (SBP-G). Patients in the SBP-S cohort  were randomly selected by Statistics Sweden from the population residing in the same respective catchment areas where patients were recruited. Controls were first screened over phone for inclusion and exclusion criteria by a research nurse. At the subsequent scheduled visit, a psychiatrist or resident in psychiatry conducted an interview using M.I.N.I. to exclude psychiatric disorders. The Alcohol Use Disorders Identification Test (AUDIT) and Drug Use Disorders Identification Test (DUDIT) questionnaires were used to screen for alcohol or drug abuse. Controls underwent somatic examination, lumbar puncture, and blood tests under the same protocol as patients. Exclusion criteria for controls were schizophrenia or bipolar disorder in first-degree relatives, untreated endocrinological disorders, pregnancy, overconsumption of alcohol (defined as elevated concentrations of carbohydrate deficient transferrin or >8 standard drinks per time more than two times per week), any other substance abuse, dementia, neurological conditions other than mild migraines, and any psychiatric condition other than past minor depressive episodes, past isolated episodes of panic disorder, past mild eating or obsessive–compulsive disorder that remitted spontaneously or with brief psychotherapy counseling.The control participants  as judged by the treating physician. Subsyndromal mood symptoms were assessed by the Montgomery-Åsberg Depression Rating Scale (MADRS) and the Young Mania Rating Scale (YMRS). A total of 12 mL CSF was collected, inverted to avoid gradient effects, divided into aliquots, and stored at −80 °C pending analyses. CSF collection from control subjects followed the same procedure. In the SBP-S, no samples were centrifugated except six control samples. In the SBP-G, all samples were centrifugated. All samples in the study were thawed and refrozen once before analysis. In the SBP-S, CSF sampling occurred between 2005 and 2008 for patients and between 2009 and 2011 for controls. In the SBP-G, CSF was collected between 2010 and 2015 for patients and between 2012 and 2014 for controls. Patients remained on their prescribed medication.® Proteomics, Uppsala, Sweden), which has been described in detail elsewhere  = 0.47 0.32–0.60] in the SBP-S and 0.48 [0.33–0.64] in the SBP-G). In principal component analyses , BMI , and CSF/serum albumin ratio , and positively associated with female sex . GH was not associated with nicotine use in either cohort.We conducted p = 0.026), but not in the SBP-G . There was no association with previous (hypo)manic episodes, psychotic symptoms, CGI, GAF-s, or GAF-f. Antipsychotic treatment associated negatively with CSF GH concentrations in the SBP-G , but not in the SBP-S . Treatment with lithium, anticonvulsants, or antidepressants was not associated with CSF GH concentrations. The regression models with CSF GH and clinical features and drugs are shown in Supplementary table p = 0.019), but was no longer significant in the SBP-G  . Finally, we performed a third sensitivity analysis, where we excluded patients with a MADRS score of 13 or higher (26 patients in the SBP-S and nine patients in the SBP-G), and/or YMRS score of 14 or higher (one patient in the SBP-G). Also in this analysis, we found an association between lower CSF GH and bipolar disorder in both cohorts, with larger effect sizes than in the main analyses , placenta growth factor (PGF) (lithium), monocyte chemotactic protein 1 (MCP-1) (lithium), interleukin-8 (IL-8) (lithium), folate receptor alpha  (lithium), and cathepsin D (CTSD) (lithium). Lithium was negatively associated with fibroblast growth factor 19 (FGF-19) in both cohorts. Results from linear regression models are shown in Supplementary table Correlation analyses showed that for each protein separately, the correlations between the CSF concentrations in this study, and the serum concentrations measured in a previous study by us (CSF and blood sampling were performed at the same day) , were geWe conducted an exploratory proteomic study of bipolar disorder using CSF collected in two independent case–control cohorts. This is the second study using the PEA technology to measure CSF protein concentrations in these cohorts . Here, wGrowth hormone is produced by somatotropic cells of the anterior pituitary gland  and exerAlthough no previous study has assessed CSF concentrations of GH in bipolar disorder, there are studies of peripheral GH in psychiatric disorders. One study found higher IGF-1 and slightly lower GH  in serum from bipolar disorder patients compared with controls . FurtherPatients in this study remained on their prescribed medications, which might have influenced CSF protein concentrations. CSF GH concentrations correlated negatively with antipsychotic use in the SBP-G but not in the SBP-S cohort. When excluding patients with antipsychotic treatment, the negative association between CSF GH concentrations and bipolar disorder remained in the SBP-S but fell below the statistical threshold in the SBP-G . Previous studies on the influence of antipsychotics on GH concentrations are scarce and restricted to serum. While one study found a reduced GH peak during sleep for patients on olanzapine , anotherOne of our protein panels targeted immune markers and many of the studied proteins have immunological roles. Notably, no immune marker differences between patients and controls replicated across cohorts. We have previously shown higher CSF concentrations of the immune-related proteins IL-8, MCP-1, and chitinase-3-like protein 1 YKL-40/CHI3L1) in bipolar disorder compared with controls in the SBP-S cohort using other analytical methods (see Supplementary table /CHI3L1 iIt is noteworthy that this proteomic CSF study of bipolar disorder did not reveal any specific aberrations apart from the GH finding. Bipolar disorder hence differs from neurodegenerative CNS disorders such as multiple sclerosis and Alzheimer’s disease, where evident CSF aberrations have been found using the same PEA proteomic technology , 42. BipA main strength of this study is that protein concentrations were measured in CSF instead of in blood which might increase CNS specificity. We showed that the between–fluid correlation for CSF and serum protein concentrations were low for the vast majority of proteins and CSF might reflect brain processes more closely compared with serum. The meticulous clinical examinations ensure diagnostic accuracy and allowed us to control for several potential confounders. Further, we sampled two independent case–control cohorts, which allowed us to replicate—or to not replicate—findings. The issue of low replication rates within the field of CSF studies of bipolar disorder was demonstrated in the previously mentioned review by Knorr et al. Indeed, when we analyzed the two cohorts separately, we found that fourteen and nine proteins differed significantly between patients and controls—but only GH replicated across the two cohorts. Even though the case–control differences seen in only one of the cohorts are not false positives by definition, they are likely to be of less significance. Hence, our use of a replication cohort improved the robustness of results by reducing the risk for false positives.A general limitation of CSF and other biofluid studies is that factors unrelated to bipolar disorder may affect protein concentrations. Even though we adjusted for several potential confounders, there might be residual unmeasured confounding factors. Second, the cross-sectional design hampers causal conclusions. Third, CSF sampling from patients preceded the collection from controls in the SBP-S cohort and long-term storage can affect protein concentrations . HoweverIn conclusion, this exploratory CSF study of two bipolar disorder cohorts demonstrates lower CSF GH concentrations in patients with bipolar disorder compared with controls. Growth hormone exerts many effects in the brain and our findings suggest that GH might be implicated in the pathophysiology of bipolar disorder.Supplemental material 1Supplemental material 2


Human herpes virus type-6 (HHV-6) is increasingly recognised as a febrile agent in children. However, less is known in sub-Saharan African countries, including Sudan.We investigated the involvement of HHV-6 in paediatric central nervous system (CNS) infections in Khartoum, Sudan.Febrile patients aged up to 15 years with suspected CNS infections at Omdurman Hospital for Children from 01 December 2009 to 01 August 2010 were included. Viral DNA was extracted from leftover cerebrospinal fluid (CSF) specimens and quantitatively amplified by real-time polymerase chain reaction (PCR) at Umeå University in Sweden.5 copies/mL CSF; range: 30–44 × 103 copies/PCR run [12 × 103 – 18 × 106 copies/mL CSF]). All positive patients presented with fever and vomiting; 86.0% had seizures. The male-to-female ratio was 1:1; 50.0% were toddlers, 42.0% infants and 8.0% teenagers. Most (83.0%) were admitted in the dry season and 17.0% in the rainy season. Cerebrospinal fluid leukocytosis was seen in 33.0%, CSF glucose levels were normal in 86.0% and low in 14.0%, and CSF protein levels were low in 14.0% and high in 43.0%.Of 503 CSF specimens, 13 (2.6%) were positive for HHV-6 (33.0% [13/40 of cases with proven infectious meningitis]). The median thermal cycle threshold for all HHV-6-positive specimens was 38 (range: 31.9–40.8). The median number of virus copies was 281.3/PCR run (1 × 10Among children in Sudan with CNS infections, HHV-6 is common. Studies on the existence and spread of HHV-6 chromosomal integration in this population are needed. This interpretation is complicated by the phenomenon of HHV-6 chromosomal integration.17 Increased numbers of white blood cells (˃ 5 cell/mm3) in CSF indicates inflammation; however, normal CSF cellular counts in patients with proven CNS infections have been frequently reported.20 It is therefore recommended not to limit HHV-6 testing to patients with increased CSF cellular counts.21 Most patients recover fully from an HHV-6 infection, but rare complications leading to neurological impairments or death can also occur.23 Little is known on HHV-6 infections in Saharan and sub-Saharan Africa, and the infection has never been investigated in Sudan. We, therefore, intended to identify the involvement of HHV-6 in CNS infections in a large group of children in Khartoum, Sudan. This report is a part of a larger study on the microbial aetiologies of CNS infections in this population.Normal CSF glucose with normal or elevated proteins is the usual finding in viral infections of the CNS.The ethical clearance for conducting this study was obtained from the Ethical Review Board of the National Center for Neurological Sciences in September 2009. Patient consent was determined to be unnecessary and was waived. Patients were not contacted directly; data were obtained from hospital files and were kept anonymous at all stages of the study. Excess specimens were obtained from the hospitals main laboratory after all officially requested tests were applied. Permission to collect data and specimens was granted from hospital authorities verbally  and in writing .®  method.A total of 503 CSF specimens were obtained from febrile (˃ 37 °C) meningitis-suspected attendees of the Omdurman Hospital for Children in Khartoum, Sudan. Patients aged 0 to 15 years who were admitted between 01 December 2009 and 01 August 2010 were included. Clinical and demographic data were obtained from hospital records retrospectively. Routine CSF analyses were performed at the Microbiology Laboratory of Omdurman Hospital. An aliquot of CSF was frozen at −80 °C for further analysis for HHV-6 DNA at the Department of Clinical Microbiology, Umeå University, Umeå, Sweden. Human Herpes Virus type-6 DNA was extracted then quantitatively amplified by real-time polymerase chain reaction (PCR) using TaqMan®, Hilden, Germany) was used for viral DNA extraction in CSF. One mL specimen was used to concentrate viral DNA: buffer AC  was added, shortly incubated, then sediment by low g-force (i.e. 1000–1200 × g) centrifugation  to pellet nucleic acid complexes. The supernatant was discarded; the pellet was re-suspended in buffer AR  and proteinase K and incubated for 10 min at 40°C. Binding conditions were adjusted by adding buffer AB ; the lysate was applied to the silica gel membrane spin column. During brief centrifugation (i.e. 3000–5000 × g), DNA selectively binds to the membrane. Remaining contaminants and enzyme inhibitors were removed by centrifugation (i.e. 3000–5000 × g) in two wash steps using buffer AW1 then buffer AW2. Pure viral nucleic acids were eluted in 30 µL low-salt buffer AVE  twice. Each elute (60 µL) was divided into two aliquots (30 µL each) and preserved at −80 °C.The QIAamp UltraSens Virus Technology Kit  and the TaqMan®  probe (reporter dye FAMTM on 5´ end and quencher dye TAMRATM on 3´ end). The lower assay detection limit is one virus copy per mL specimen. Forward and reverse primers  were diluted to reach final working concentration of 25 µM . Commercially provided oligonucleotide products were diluted to the suitable working solution and the recommendation of QuantiTect®  Q PCR Protocol was followed. A PCR master mix solution was prepared for Harry (HHV-6) primers , 1.0 µL forward primer , 1.0 µL reverse primer , 0.8 µL probe  and 7.2 µL rNase-free water  were added into 2.5 µL template gDNA to complete a total reaction volume of 25.0 µL per single PCR reaction. The mixture was pulse-vortexed  and centrifuged briefly (i.e. 3000 × g). Then, 22.5 µL single reaction mix was transferred into a well of a MicroAmpTM Optical 96-Well Reaction Plate  to which 2.5 µL template gDNA was added to reach total volume of 25.0 µL per one reaction.Viral gDNA amplification and detection was performed by real-time analysis using the Applied Biosystems primers  and prob primers  as folloTM Optical Adhesive Film (Applied Biosystems®), concentrated at the bottom of the plate wells by spinning at low speed in a cold centrifuge (i.e. 1200 × g) using AllegraTM X-12R Centrifuge . Each PCR reaction plate was designed for carrying eight standard dilutions, one negative reverse transcriptase preparation, one non-template control containing ddH2O and duplicates or triplicates of each experimental DNA template.The PCR plates were covered by MicroAmp6 (5E6) virus copies . The first standard preparation was used without dilution, having a concentration of 5E6 copies, followed by 1/10 serial dilutions for the subsequent seven preparations to reach final concentrations of 5E5, 5E4, 5E3, 5E2, 5E1, 5 and 1 virus copy.The standard used for PCR had 5 × 10® DNA Polymerase  at 95 °C for 10 min, denaturation at 94 °C for 15 s and combined annealing and extension at 60 °C for 60 s. The cycle was repeated 45 times taking an approximate duration of 90 min including generation of amplification curve. Data were analysed using the software ABI 7900HT SDS Plate Utility Version 2.4 (Applied Biosystems®).Real-time cycler thermal condition was as follows: heating at 50 °C for 2 min, initial activation of HotStarTaqStatistical package program Statistical Package for Social Sciences version 21  was used. Categorical variables were expressed in frequencies and percentages and cross-tabulated and Pearson chi-square tested for statistically significant differences under the 0.05 level. Numerical variables were described using measures of central tendency and of dispersion; Pearson’s correlation and its 95% confidence intervals were calculated.All of the following describe patients with positive CSF HHV-6 DNA: all patients with clinical data  presented with fever (˃ 37 °C) and vomiting and six (86%) with seizures . For the3) with 60% lymphocytosis and the remaining 67% (6/7) showed normal CSF white blood cell count (< 5 cells/mm3). Cerebrospinal fluid glucose levels for most specimens  were normal (45 mg/dL – 100 mg/dL) and one specimen (14%) was low (30 mg/dL). Cerebrospinal fluid proteins levels were high (> 45 mg/dL) in 43% (3/7), normal (14 mg/dL – 45 mg/dL ) in 43% (3/7) and low (11 mg/dL) in 14% (1/7)  infants and 8% (1/12) a 15-year-old teenager. Most patients  were admitted to the hospital in the dry season (December to June) and the remaining 17% (2/12) admitted in the rainy season (July to August). Cerebrospinal fluid specimens from 42% (5/12) were traumatic; cytological and chemical analyses were not performed. The remaining 58% (7/12) were clear; 33% (1/7) showed increased CSF white blood cells (50 cells/mm4% (1/7) . All 13 5 virus copies/mL CSF) with a range of 30 to 44 × 103 copies per PCR run (12 × 103 and 18 × 106 virus copies/mL CSF). Individual PCR data for all 13 positive specimens are shown in Polymerase chain reaction testing for HHV-6 DNA in CSF revealed 13 (2.6%) positive specimens out of a total of 503. Median cycle threshold (Ct) for all HHV-6 positive specimens was 38 with a range of 31.9 to 40.8. Median virus copy was 281.3 per PCR run  of children with aseptic meningitis in Southern Iran. Tavakoli21 reported similar findings of 1.8% (27/1482) in New York, United States. Another study in New York by Yao11 revealed a significantly higher prevalence of 40.0% (14/35) in patients with CNS infections who tested negative for other CNS pathogens. Among well-defined groups in our study, the prevalence was also high, accounting for 33.0% out of 40 cases with proven infectious meningitis  was a 15-year-old teenager. The ratio of boys to girls in this study was 1:1 which agrees with Tavakoli’s findings of 1.3:1.1.It has been reported that primary infection with HHV-6 always occurs in young children (age 0 to 2 years).30 Out of the 24 examined patients, Tavakoli reported fever in 71.0%, altered mental status in 67.0%, headache in 29.0% and seizures in 33.0%. Other reported symptoms were muscle weakness, muscle pain and stiff neck, which are general symptoms for meningitis or encephalitis.21 In the present study, all HHV-6 positive cases presented with fever and vomiting, 86.0% presented with seizures, 14.3% with chills and 14.3% with a stiff neck. None of our patients developed skin rash, which is the only specific – but rare – symptom in HHV-6 meningitis.30 Findings of both Tavakoli and Hosseininasab concur.Clinical signs and symptoms in HHV-6 meningitis are not specific.17 as found in this study, where all specimens showed normal CSF glucose concentration, 33% normal proteins and 67% high proteins. Unfortunately, chemical analysis of the CSF is ineffective in case of viral infections; however, its significance is in distinguishing bacterial from aseptic aetiologies which is a crucial preliminary step in deciding adequate treatment. Cerebrospinal fluid leukocytosis was seen in 33%. Increased CSF white blood cell numbers indicate inflammation; however, normal CSF cellular counts do not rule out viral aetiologies. In fact, normal CSF cellular counts in patients with proven CNS infections were frequently reported.20 Normal CSF profile was reported in 25% of HHV-6 positive cases in Tavakoli’s study21; accordingly, HHV-6 testing should not be limited to patients with abnormal CSF profiles.Normal CSF glucose with normal or elevated proteins is the usual finding in viral infections of the CNS,p = 0.029) inversely correlated  indicating significant variation of viral loads among our patients. Substantial variation among viral loads in patients was also reported by Tavakoli.21Thermal Ct is defined as the number of cycles required for the fluorescent signal to cross the threshold (i.e. exceed background level). Median Ct for our positive cases was 38 with a range of 31.9 to 40.8. In Tavakoli’s study, Ct values ranged from 25.03 to 39.92. In quantitative real-time PCR, Ct values inversely correlate with viral loads; therefore, a low Ct value indicates a high viral load and vice versa. Our Ct values and viral loads were found to be significantly , while a single case (14%) showed an increased CSF cell count. Ward16 elaborated that in order to identify a condition as chromosomal integration, viral load should be high while low virus copies would indicate an infection. They16 reported a significantly lower (i.e. 2.4 log10 copies/mL) CSF HHV-6 DNA concentration in 9 children with primary infection in comparison with 21 patients with viral chromosomal integration (i.e. 4.0 log10 copies/mL). In the present study, the median CSF virus concentration was 1 × 105 copies per mL with a minimum virus concentration of 12 × 103 copies per mL and a maximum of 18 × 106 copies per mL. While Ward16 recommended identifying low virus copies (≤ 103) as an acute HHV-6 infection and high virus copies (≥ 104) as chromosomal integration, Collot31 identified viral integration in approximate concentrations of 103 to 106 copies per mL. In the present study, we identified viral concentrations as low as 104 and as high as 107. Several other studies reported high CSF viral loads in patients with HHV-6 CNS infections.21 In addition, other authors13 insist that chromosomal integration is a rare condition. Accordingly, we assume the detected HHV-6 DNA in our mostly cell-free CSF specimens is more likely to be from free replicating virus than from chromosomally integrated virus.The phenomenon of HHV-6 chromosomal integration is in debate; while some4 Knowing that febrile seizures and vomiting were dominant symptoms among our population and the most frequent age group was children up to the age of 2.3 years, therefore, further supported our assumption. Despite this, and for the sake of scientific relevance, we are not ruling out the possibility of integration among our identified cases. For this reason, studies to identify the prevalence of HHV-6 integration among healthy Sudanese population are warranted.In infants, primary HHV-6 infection is an important cause of febrile seizures with an incidence of 13% in the United States.27 Despite moderate neurological sequel, and less likely death, patients usually recover fully from an HHV-6 infection23, as fortunately observed in this population.Alongside HHV-6, mixed microbial infections were identified in three cases in this study. Several authors also reported mixed viral infections to the CNS.A major limitation in this study, however, is that we were unable to further genotype our identified HHV-6 viral DNA because of a limited CSF volume .Human Herpes Virus type-6 CNS infection is frequent in this population (i.e. identified in one-third of cases with proven infectious meningitis). We recommend studying the existence and spread of HHV-6 chromosomal integration in the healthy Sudanese population.


Fecal microbiota transplantation (FMT) is a therapy that involves the transplantation of healthy human fecal microorganisms into the gut of patients to rebuild or consolidate the intestinal microecology. It has been utilized in many diseases. However, FMT had a limited effect on patients with small intestinal diseases because of the unique ecological characteristics of the microorganisms. Thus, we proposed a new microecology transplantation therapy called human intestinal fluid transplantation (HIFT). Human intestinal fluid can be collected through a nasojejunal tube and be made into capsules using the freeze-dried powder method. In addition, strict standards for donor screening and management have been established. We are currently developing a high-standard HIFT preparation system and conducting high-quality clinical studies to validate the safety and efficacy of HIFT combined with FMT. Fecal microbiota transplantation (FMT) is a new therapy that involves the transplantation of healthy human fecal microorganisms into the gut of patients to rebuild or consolidate the intestinal microecology . Many diGiven that FMT mainly treats the colon flora, the 4-meter-long small intestine is not given the priority it deserves. Small intestine is one of the most important organs in human beings, which has lots of digestive enzymes, microorganisms, immunoglobulins and other vital substances –11. It iHIFT is a new therapy without a standardized methodology yet. To ensure the safety and efficacy of HIFT –19, the Donor screening criteria and management criteria:Objective criteria2.– Age 18–30, male or female, body mass index of 18.5–22.9 kg/m– Normr negative hematology tests: blood routine test, hepatic and renal function, electrolytes and c-reactive protein, infectious hepatitis, HIV, syphilis, Epstein-Barr virus, cytomegalovirus, nematode, amoeba, and other pathogens.Clostridium Difficile, Campylobacteria, Salmonella, Shigella, Shiga-toxigenic Escherichia coli, worm eggs, vesicles, parasites, spores, norovirus, rotavirus, and multiple drug resistance genes .– Normal or negative stool tests: feces regular test, occult blood test, Psychological evaluation– Assessed as having a good psychological state by a cardiologist or psychological consultant.– Normal scores in Self-rating Depression Scale, Self-rating Anxiety Scale, and Pittsburgh Sleep Quality Index.History of diseases– Past History: without gastrointestinal symptoms in recent 2 weeks, no antibiotics, acid inhibitors, immunosuppressants, or chemotherapeutic drug use in the last 3 months, no chronic pain symptoms, no history of digestive surgery, no history of infection and infectious disease exposure, no allergic disease, no autoimmune disease, no metabolic disease, no cardiovascular and cerebrovascular diseases, no neuropathy, no psychosis, no malignancy, no growth hormone, insulin, coagulation factors, or other injection treatment.– Personal history: have a regular routine, healthy diet and harmonious family, and no bad sexual history; no smoking, drinking, and drug addiction history; no vaccination, drug trial, skin damage, and contact with tropical areas in the last 6 months.– Family history: no family history of gastrointestinal diseases, malignant tumors, or infectious diseases.– Others: not pregnant, not in menstrual period.Archive and follow-up system establishment– Standard donor file establishment, including recording each inspection result, HIF donation, and related treatment.– Follow-up system establishment, to ensure that the donors regularly complete and pass the physical examination and donation requirements.Donor management group establishment– The donor management should be in charge of the full-time donor managers of the HIFT center, including at least 1 principal and 2 assistants.– The donor managers should maintain regular communication with the donors, establish a good trust relationship, and carry out necessary management and intervention on the donor's lifestyle and diet structure. They should immediately correct the unhealthy lifestyle and diet structure, and eliminate unqualified donors according to the follow-up results.Informed consent of the donors– Donor candidates should be fully informed and signed the informed consent before screening.– The HIFT donor should be fully informed and signed the informed consent for nasointestinal tube catheterization before donation.HIFT Donor donation requirements– The donors shall ensure the continuity of the donation. Each donation can be made for 3–7 consecutive days, once every 1–2 months.– The amount of HIF donation should be no <350 mL per day after filtering, and the color of the HIF must be golden yellow.The above items should be reviewed every 2 months. Only ~2% of the population can be screened as ideal donors. After screening, HIFT donors provided informed consent for the donation and nasointestinal tube catheterization during the HIF collection period. Every donor should obey the criteria of donor management to ensure the stabilization and safety of HIF. Stool and HIF samples of each donor were saved for 16s rRNA sequencing and composition analysis to ensure the basic stability of the bacterial community and biological components, and to allow tracing when recipients have adverse effects. In addition, some donors should restrict certain types of food for 5 days prior to the donation of HIF when recipients have food allergy or food intolerance symptoms.Donors underwent nasojejunal tube catheterization by using a modified Flocare nasogastric feeding tube, with a depth of 175 cm away from the nose. The modified disposable sterile negative pressure collecting devices were connected to the catheter for continuous drainage, which was controlled below 6.7 kPa. The collecting devices were changed every 2 h because the HIF may be metamorphic outside. After collection, HIF was successively filtered through screen cloths of 2.0, 1.0, and 0.5 mm. Then, 10% glycerin was added to the filtrate as a cryoprotectant and HIF was turned into the lyophilized powder by lyophilizer , 21, keeThe entire preparation process required information registration, HIF identification, weighing, and testing. A 2 mL HIF sample from each donation must be set aside for at least 6 months to allow tracing in case of adverse events. Donor information code, donation date, production date, expiration date, dose, and storage temperature were recorded.Although the efficiency of FMT for rCDI is 90% , 23, the5 cfu/ml. 1 cfu is almost several numbers of bacteria. However, with the application of 16S rRNA and Trypan Blue test, the number of bacteria recorded in HIF is >3.0 ×108/ml. There is a significant difference between these two values, and many new microorganisms have been identified  and its management varies from country to country, the risks of its clinical use must be carefully considered . The mos5 cfu/ml  and Shanghai Action Plan on Science, Technology and Innovation (21Y11908300).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.


Orally administered, small-molecule anticancer drugs with tumor-specific cellular protein targets (OACD) have revolutionized oncological pharmacotherapy. Nevertheless, the differences in exposure to these drugs in the systemic circulation and extravascular fluid compartments have led to several cases of therapeutic failure, in addition to posing unknown risks of toxicity. The therapeutic drug monitoring (TDM) of OACDs in therapeutically relevant peripheral fluid compartments is therefore essential. In this work, the available knowledge regarding exposure to OACD concentrations in these fluid spaces is summarized. A review of the literature was conducted by searching Embase, PubMed, and Web of Science for clinical research articles and case reports published between 10 May 2001 and 31 August 2022. Results show that, to date, penetration into cerebrospinal fluid has been studied especially intensively, in addition to breast milk, leukocytes, peripheral blood mononuclear cells, peritoneal fluid, pleural fluid, saliva and semen. The typical clinical indications of peripheral fluid TDM of OACDs were (1) primary malignancy, (2) secondary malignancy, (3) mental disorder, and (4) the assessment of toxicity. Liquid chromatography–tandem mass spectrometry was most commonly applied for analysis. The TDM of OACDs in therapeutically relevant peripheral fluid spaces is often indispensable for efficient and safe treatments. These synthetic molecules bind to proteins that are expressed excessively or even exclusively in cancer cells, resulting in the inhibition of the functions of cancer cells with a limited impact on non-malignant cells. Most OACDs are found in the subgroup L01E of the Anatomical Therapeutic Chemical (ATC) classification system , and are further classified at level 4 as BCR-ABL tyrosine kinase inhibitors, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, B-raf serine-threonine kinase (BRAF) inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, mitogen-activated protein kinase (MEK) inhibitors, cyclin-dependent kinase (CDK) inhibitors, mammalian target of rapamycin (mTOR) kinase inhibitors, human epidermal growth factor receptor 2 (HER2) tyrosine kinase inhibitors, Janus-associated kinase (JAK) inhibitors, vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors, Bruton’s tyrosine kinase (BTK) inhibitors, phosphatidylinositol-3-kinase (Pi3K) inhibitors, fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitors, and “other” protein kinase inhibitors. Further, four OACDs are listed under level 3 code “Other antineoplastic agents” (ATC code: L01X) including histone deacetylase (HDAC) inhibitors, hedgehog pathway inhibitors, and poly-ADP-ribose polymerase inhibitors.The past two decades have seen the rise of a new era of targeted oncological pharmacotherapy. The novel treatment options have led to a tremendous increase in success rates since the first market approval of the now generic imatinib  to provide a comprehensive overview of the available knowledge regarding the distribution of OACDs to the peripheral fluid spaces, and (2) to explore the methodological approaches employed for the clinical monitoring of the concentrations of OACDs in peripheral fluid spaces.Due to the types of publications available, a systematic review could not be conducted; however, adhered to the applicable items of the PRISMA Guidelines . The revA search of the following databases was performed:https://pubmed.ncbi.nlm.nih.gov), keyword combination:  AND  AND (concentration OR chromatography OR mass spectrometry OR therapeutic drug monitoring OR levels).1. The database of the National Library of Medicine, National Center for Biotechnology Information  keyword combination:  AND  AND (concentration OR chromatography OR ’mass spectrometry’ OR ’therapeutic drug monitoring’ OR levels).2. The Web of Science (Clarivatehttps://embase.com), keyword combination:  AND  AND (concentration OR chromatography OR mass spectrometry OR therapeutic drug monitoring OR levels).3. The Embase database .In addition, a manual Google search was conducted using the following query terms: “name of drug” AND “therapeutic drug monitoring”, “name of drug” + “milk”, “name of drug” + “liquor”, “name of drug” + “cerebrospinal fluid”, “name of drug” + “semen”, and “name of drug” + “liquid chromatography mass spectrometry”. Each database record was evaluated by two reviewers (Z.K. and G.B.K.) who also conducted the manual research. Duplicate publications were removed by Z.K. before screening. No automation tools were employed for evaluating the eligibility of the records.The workflow of retrieving research articles for full evaluation is shown in First, duplicates of the PubMed records were removed from the results of the Embase and Web of Science database search. The remaining records were subsequently assessed individually for meeting basic requirements. Only peer-reviewed full manuscripts written in English, assigned an individual digital object identifier, and made available online by the publisher within the searched period were considered for further screening. Level 2 screening was based on the contents of the title and the abstract. Only records with an explicit evidence of ineligibility were removed at this level. The type of the article was the first object of assessment. Articles presenting randomized and nonrandomized registered clinical studies, non-registered, researcher-initiated clinical studies, retrospective observational studies, case series , and individual case reports were included for further evaluation. Book chapters, comment articles, editorials, meta-analyses, practical guidelines, research protocols, scoping reviews, and systematic reviews were not considered. Second, articles describing experiments in which the subjects were not humans, i.e., in vitro experiments or in vivo animal studies, were removed. Subsequently, studies performed with the participation of human subjects, but without the aim to evaluate or to support decisions related to their medical treatment, i.e., without direct therapeutic relevance , or including medical intervention which, by current understanding, would not be part of the clinical practice  were eliminated. Finally, studies explicitly performed without the monitoring of any of the drugs listed in In the phase of full manuscript screening, the first object of assessment was the ethical review board approval. Case reports and case series were exempt from this requirement. Articles continued to be retained if explicit evidence was found in the main text confirming that the study had been performed with direct therapeutic relevance, as described for level 2 screening. The presentation of the results of monitoring at least one drug displayed in publ/nyear, where npubl is the number of included publications on the drug, and nyear is the number of years the drug had been available on the market. The latter was defined as the period starting with the day of the first approval by the American Food and Drug Administration, and ending on 31 August 2022. Visualization was carried out using Microsoft Office applications.All descriptive information on the database records and the contents of the manuscripts found were stored and processed using Microsoft Excel. The year-normalized number of publications on each drug was calculated as nThe database search yielded a set of 1503 potentially relevant articles . Four-hundred and seventy-four duplicates were removed. Of the remaining 1029 papers, 258 were presentation abstracts, and 31 were not written in English. In a single case, a record was listed with false authors. These records were also excluded. The manual search yielded five additional hits which were subsequently found in the PubMed database, but had not been listed by the automatic search. The assessment of the remaining 739 articles based on title and abstract resulted in the exclusion of further 546 publications. Forty-three articles were excluded based on their type. Three hundred and ninety works described in vitro experiments or in vivo animal studies, and 35 were conducted in humans, but without a direct therapeutic goal. Seventy-eight studies were excluded based on evidence retrieved from the title and/or the abstract that drug concentrations were not monitored in any peripheral fluid space.All of the 193 publications retained for full evaluation could be retrieved from the websites of the publishers. An in-depth study of these manuscripts resulted in the elimination of 113 publications. The authors of two papers failed to present evidence of the approval of an ethical review board for conducting research on humans. Thirty-five studies were not performed in humans, and five were conducted without direct therapeutic relevance. Seventy-one works were excluded because drug concentrations were not monitored in any peripheral fluid space. The remaining 80 publications were selected for the detailed review , Table 2Thirty-two small-molecule, orally taken anticancer medications with specific cellular protein targets were monitored with a clinical indication in at least one peripheral liquid space on at least one occasion in the investigated period. This comprises 38.6% of the OACDs which had an ATC code on 31 August 2022. CSF was the most frequently monitored peripheral space . The share of manuscripts on all other peripheral spaces —2%) was low. In 61% of the cases, the indication for monitoring an OACD in a peripheral fluid compartment was to control a secondary malignancy. Other indications were the treatment of a primary malignancy (19%), controlling toxicity (14%), and treatment of a mental disorder (6%). In a single manuscript, an additional indication was the prevention of graft rejection . Registe®) triggered the largest interest, followed by erlotinib (Tarceva®), ribociclib , imatinib, and osimertinib (Tagrisso®), while the OACDs receiving the least attention were lapatinib (Tyverb®), vorinostat (Zolinza®) and sunitinib  in pediatric recurrent or refractory medulloblastoma [O-demethylated, pharmacokinetically active metabolite OSI-420 in plasma and in CSF. Six time-matched pairs of specimens were collected. The CSF/total plasma concentration ratio (CSF-TPR) of erlotinib was 7.0%, while the ratio of drug exposure was 6.9% based on 24-h areas under the concentration-time curves. This evaluation was based on total plasma levels. Since the fraction of erlotinib bound to plasma proteins is approximately 93%, it is reasonable to assume that the unbound fraction equilibrated between plasma and CSF at a 1:1 ratio [Currently, the most common indication of monitoring OACDs in a peripheral fluid space is to improve the treatment of primary and secondary CNS tumors in adults and in pediatric patients by performing measurements in the CSF. Early examples for such efforts included the assessment of erlotinib in pediatric glioblastoma and in CNS hemangioblastoma with von Hippel–Lindau disease, and of vismodegib  as well as its active N-oxide and demethylated N-oxide products were assessed in recurrent malignant glioma. All three substances attained detectable levels in CSF. While the concentration values were not explicitly provided by the authors, visual plots showed that the CSF-TPR’s were 0.01 or higher. Approximately 99.5% of circulating regorafenib is bound to proteins, indicating that the CSF levels exceeded unbound plasma concentrations [The monitoring of OACDs in this context has gained more attention only very recently. The concentrations of regorafenib (Stivargatrations .®) and ribociclib in patients diagnosed with recurrent glioblastoma was performed [The monitoring of ceritinib , imatinib, nintedanib (Ofev®), panobinostat (Farydak®), regorafenib, ribociclib, and vorinostat were assayed in 42 CSF samples obtained from nine pediatric brain tumor patients. Nintedanib and panobinostat were undetectable in the samples. There was a correlation between blood protein levels and imatinib concentrations. In addition, imatinib and regorafenib proved to bind to CSF proteins as well, resulting in unbound fractions of 88% and 65%, respectively. These data indicate that both plasma and CSF protein concentrations may have an impact on detectable drug levels, and that the elevation of drug availability can be expected in CSF when the blood–brain barrier is not intact and CSF protein levels increase [Dasatinib (Sprycelincrease .®) was measured in CSF in primary CNS lymphomas [Ibrutinib  concentrations were assayed in 23 time-matched plasma and CSF samples of 13 patients, 8 of whom were diagnosed with primary CNS lymphoma, and 5 with diffuse large B-cell lymphoma. The CSF-TPR was 2.39±1.71%. With an assumed 94% protein binding rate, the authors calculated CSF-UPR’s of 42.7±27.7%, and concluded that zanubrutinib was successfully transported through the blood–brain barrier [Zanubrutinib (Brukinsa barrier .®) and Trametinib (Mekinist®) did not reach detectable levels in CSF in patients diagnosed with V600e positive glioma [Dabrafenib  chronic myeloid leukemia (CML), non-small cell lung cancer, and gastrointestinal stromal tumors.Imatinib concentrations were monitored in patients diagnosed with Ph + CML. In a follow-up study conducted with 15 adult patients, Nambu et al. found a weak correlation between imatinib levels determined in leukocytes (buffy coat cells) and in plasma (r = 0.281). While the intracellular concentrations of the drug were not associated with the cytogenic response, there was a significant difference between groups of patients with different genotypes (SLCO1B3 334TT and 334 TG/GG) . In anotMalignant pleural effusion is a severe condition developing as a complication of lung or breast cancer in women . Masago The involvement of the CNS presents a major challenge in the therapy of leukemias. Adult patients present with CNS leukemia in approximately 5% of acute leukemia cases, while CNS involvement occurs in about every third pediatric patient presenting with a relapse . The risThe poor penetration of the blood–brain barrier by imatinib, the first marketed tyrosine kinase inhibitor drug, was first mentioned in 2002 ,16. The On five separate days in an 11-day period, measurable imatinib concentrations were found in the CSF and plasma samples of a male Ph + CML patient who was in a lymphoid blast crisis after achieving complete cytogenic remission in the bone marrow following more than eight months of imatinib therapy, but had developed an isolated neoplastic meningitis. The authors concluded that imatinib CSF concentrations were not sufficient to inhibit 50% of BCR/ABL tyrosine kinase, and assume that the reason underlying the poor penetration of imatinib is its affinity to p-glycoprotein, a protein responsible for multi-drug resistance. Nevertheless, total imatinib concentrations were evaluated, and by calculating its unbound plasma concentrations, imatinib CSF-UPR’s can be established as 7.7–56.2% . FurtherDasatinib concentrations were below the detection limit in the CSF of a female adult patient treated with Ph + ALL and an extramedullary and meningeal relapse following bone marrow transplantation. The trough plasma dasatinib concentration was 32 ng/mL (CSF-TPR: 0.23–1.5%) . Followi®). Seventeen matched pairs of CSF and plasma samples were collected. The CSF-UPRs were calculated by taking a 98% protein binding rate into account. The calculated concentration ratios were 12%, 20%, 30%, and 68%, pointing to large individual differences in the availability of the drug [Four leukemia patients (three diagnosed with Ph + ALL and one with Ph + CML and a blast crisis), all with CNS relapse after allogeneic stem cell transplantation, received nilotinib (Tasignathe drug . In a gr®) through the blood–brain barrier was also poor; however, it corresponded to the in vitro IC50 of the drug in an adult, male chronic lymphocytic leukemia patient diagnosed with trisomy 12, IGHV unmutated (VH4L) chronic lymphoid leukemia and experiencing a CNS relapse. Time-matched pairs of plasma and CSF samples were assayed after their collection in steady state, after 2 h and 23 h of drug intake, with 0.23% and 2.89% concentration ratios obtained. The unbound fraction of venetoclax is smaller than 1% of the total circulating amount; therefore, the CSF concentrations corresponded to approximately 10–29% of the unbound plasma levels [The penetration of the selective BCL2-inhibitor venetoclax (Venclyxtoa levels . Venetoca levels .®), another very heavily ( > 99%) protein-bound drug, was observed in a 3-year old girl diagnosed with Ph + acute lymphoblastic leukemia which had been confirmed to have penetrated the CNS [An extremely low CSF concentration (0.1 ng/mL) of ponatinib (Iclusig the CNS .®). This was a case report presenting a Japanese male patient diagnosed with NSCLC and developing carcinomatous meningitis. Ten days after the initiation of gefitinib treatment, the drug was assayed in serum and in CSF before and 2 h after the intake of 250 mg drug. At both time points, the observed CSF concentrations were negligible, 0.9 nmol/L, while serum concentrations of 117 and 132 nmol/L were attained. Assuming a 97% protein binding rate, this corresponds to CSF/unbound serum concentration ratios of 22.7% and 25.6% [The first manuscript discussing the quantitation of OACDs in CSF for monitoring their efficacy regarding the treatment of the CNS metastases of NSCLC was published on the epidermal growth factor receptor inhibitor gefitinib , regardless of the cytological response Two years after the publication of the first measurement of gefitinib concentrations in CSF, erlotinib concentrations were evaluated in three lung adenocarcinoma patients developing leptomeningeal metastases during gefitinib therapy. Twenty-eight days after switching to erlotinib, clinical improvement was observed, accompanied by 2.5–13.3% CSF-TPRs, corresponding to CSF-UPRs of 36–190% ,97. FourIn a comparative study conducted to evaluate the penetration rate of standard  versus pulsatile high-dose erlotinib (1500 mg on day eight and fifteen) and gefitinib (2500 mg/die from day eight to fifteen) in NSCLC patients with brain metastases who progressed on standard doses, both drugs attained higher concentrations in the CSF as a result of high-dose administration, with a constant CSF-TPR of 2% in the case of erlotinib, and a saturable penetration rate of gefitinib with no increases in CSF levels predicted for doses of 839 mg or higher . In addi®), with negligible blood–brain barrier penetration rates observed. A CSF-TPR of 0.26%  was found in a 29-year old Caucasian male diagnosed with stage IV NSCLC and treated first with cisplatin plus pemetrexed, then with erlotinib, and finally with crizotinib. The attained CSF concentration was substantially lower than the established 50% inhibitory concentration (IC50) required to inhibit mutant cell lines against which crizotinib had been tested [The next drug assayed in the CSF was crizotinib  twice a day in the fixed dose phase, five matched alectinib CSF-plasma concentration pairs were obtained. The CSF concentrations not only showed positive correlation with the unbound plasma fraction of alectinib , but were also equivalent or higher. The extrapolated trough CSF concentration exceeded the reported in vitro IC50 of alectinib for ALK inhibition [In a single-arm, open-label, multicenter phase 1/2 study conducted with the involvement of adult subjects with histologically confirmed, locally advanced or metastatic NSCLC with crizotinib-resistant ALK-positive rearrangement and receiving 600 mg or 900 mg alectinib (Alecensahibition . In an ihibition .®) CSF levels was first described in a woman diagnosed with stage IV adenocarcinoma of the lung with an underlying mutation of the EGFR gene. Two CSF samples were assayed, and afatinib was found to attain a penetration rate lower than 1%, with a calculated CSF-UPR of 13.9% when 95% protein binding rate of the drug is assumed [The evaluation of afatinib , an OACD currently approved in China, into CSF was first evaluated in a phase 2 clinical study involving ten patients following the administration of 125 mg in a three-times-per-day regime. Meanwhile, WBRT was delivered in 3-Gy fractions once per day, five days per week, to a total dose of 30 Gy. The mean total plasma concentrations were 940.6±503.8 ng/mL (corresponding to 47.0±25.2 ng/mL unbound concentrations), while the mean CSF concentrations were 11.6±9.1 ng/mL in samples collected two hours after drug intake. The CSF-TPR was 1.4±1.1%, and the mean CSF-UPR can be calculated as 24.7% [The penetration of icotinib  was observed . In an o50 [Tepotinib plasma and CSF concentrations were evaluated in a male adult patient diagnosed with stage IIIA lung adenocarcinoma. EGFR mutation and ALK fusion gene were not detected. Following right lung pneumonectomy, a brain metastasis was identified in the left cerebrum which later progressed to leptomeningeal metastasis and hydrocephalus in spite of treatment with cisplatin and pemetrexed. A tepotinib regimen (500 mg/die) was started. On day 20 of therapy, the tepotinib CSF-TPR achieved 1.83% in the matched samples collected four hours post-dose. The attained concentration was judged to have exceeded the IC50 . In a fe50 .®) was monitored in the CSF in an ongoing, open-arm, multicenter phase 1/2 trial with the aim to further investigate the penetration of the drug into the CNS. Five patients with suspected or confirmed leptomeningeal carcinomatosis not visualized on magnetic resonance imaging, or carcinomatous meningitis, were included. Samples were collected at baseline and a later yet undefined point of the study. The CSF/plasma unbound lorlatinib concentration ratios were 61–96%, and showed very strong correlation (adjusted r2 = 0.96). The CSF/total plasma lorlatinib concentration ratios were 21–33%. The results indicated that lorlatinib concentrations exceeded the minimum efficacy concentrations in all of the patients regarding wild-type anaplastic lymphoma kinase (ALK) and the L1196M ALK resistance mutation. The authors concluded that this supported the broad coverage of these mutations, and, in approximately one-third of patients, the coverage of the G1202R ALK resistance mutation [Lorlatinib (Lorviquamutation .®) and osimertinib were investigated in an adult patient with an EGFR-mutant NSCLC with acquired RET fusions and meningeal metastasis after four months of co-treatment with pralsetinib and osimertinib. Pralsetinib attained concentrations of 91.3 µmol/L and 0.705 µmol/L in plasma and CSF, respectively . Osimertinib concentrations were 2.149 µmol/l and 0.0237 µmol/L, respectively . Despite the lower CSF/unbound concentration ratios, pralsetinib levels were judged to be sufficiently high both in plasma and in CSF to inhibit the CCDC6-RET-mutated protein, indicating that pralsetinib is more efficient than osimertinib to treat this mutation [The CSF concentrations of pralsetinib  ductal carcinoma yielding CNS metastases. The CSF-UPRs can be calculated as 8.6–12.9% in these two patients ,105. Ner®), then with sunitinib, concentrations of the administered OACD were monitored in plasma and in ascitic fluid. The concentrations measured in the ascitic fluid were equivalent to or higher than those assayed in the systemic circulation, and, following an early phase with sufficient plasma levels, systemic concentrations became subtherapeutic [Malignant ascites is a rare condition secondary to abdominal malignancies . In an erapeutic . The ascCML occurs very rarely during pregnancy, at an estimated rate of 1:750 000. Imatinib is employed for treating Ph + cases developing during pregnancy, an approach which may cause harm to the fetus and the newborn. Assaying the drug in breast milk is valuable for characterizing the exposure of the infant. The first appearance of the measurement of imatinib in breast milk was the description of a case with the imatinib concentrations being approximately 60% of the lower limit of the currently accepted blood reference range (1000–3000 ng/mL). Its pharmacologically active metabolite, however, displayed accumulation in breast milk . AnotherEverolimus is primarily administered as an immunosuppressant based on its ability to inhibit the mammalian target of rapamycin (mTOR) functional complex mTORC1. In a heart-transplanted patient, everolimus therapy was continued during pregnancy and following delivery. At 48 h postpartum, the drug was not detectable in the colostrum, indicating that the evaluation of the immunosuppression of the newborn had to be based on its prepartum administration .Oral anticancer medications have serious adverse effects, including low blood cell counts, resulting in an increased susceptibility to infections and, potentially, bleeding, as well as dermal and gastrointestinal symptoms. Several of these may prompt the discontinuation of therapy. Efforts have therefore been made to identify the relationships between the presentation of the drugs in non-targeted organs and fluid compartments, and the development of adverse symptoms.Pleural effusion may be induced by tyrosine kinase inhibitors. In a young male adult patient who had developed pleural effusion from dasatinib earlier, nilotinib therapy again led to the formation of the effusate. The measured nilotinib concentrations were 927 ng/mL and 2092 ng/mL in plasma and in the pleural effusate, respectively, clearly indicating the accumulation of nilotinib in the latter medium. Other possible causes, including malignancy, were excluded. The severity of this adverse effect is shown by the fact that, eventually, performing endotracheal intubation and left thoracic drainage was required .®) levels in saliva was investigated in 11 cancer patients receiving everolimus in a once-daily (10 mg) or twice-daily (2 × 5 mg) regime. Both the plasma and saliva concentrations of the drug were higher in patients with stomatitis than in those who did not develop this condition. While the statistical significance of this difference was low, this result may indicate the utility of everolimus saliva assays concerning the prevention of the occurrence of stomatitis. Of note, the rate of the penetration of everolimus into saliva was extremely low (0.8%) with high interindividual variability (67.7%) [The relationship between the occurrence of stomatitis and everolimus (Afinitor (67.7%) .Imatinib has been demonstrated to cross the brain–testis barrier and to reach equilibrium. Imatinib concentrations reached concentrations of 1471 ± 570 ng/mL and 1397±425 ng/mL in the plasma and the semen of eleven male CML patients, respectively. The clinical relevance of the assay was confirmed by the finding that the number, the survival rate, and the activity of sperms were reduced in these patients. Reproductive hormone structures and sex hormone concentrations were unaffected .Panobinostat was not detected in the CSF of patients diagnosed with human immunodeficiency virus (HIV) infection . In addiIt is increasingly acknowledged that certain OACDs may be effective against neurodegenerative and autoimmune diseases ,108. NilIn an open label pilot study conducted to investigate the safety and tolerability of two doses of nilotinib, the drug penetrated readily into the CSF, and remained detectable there for five hours, when administered to stage 3–5 Parkinson’s disease patients in low doses (150–300 mg/die). This was accompanied by a steady increase in plasma concentrations. The CSF-TPR was higher when the lower dose was administered, with a comparable level of Abl inhibition . FurtherThe most recent evaluation of nilotinib delivered results which contradicted some key findings of the above works, although the safety and tolerability of low-dose nilotinib was still acceptable. The measured CSF penetration was in concert with previous findings. This was a six-month, multicenter, randomized, parallel-group, double-blind, placebo-controlled trial conducted with the involvement of 76 participants, 51 of whom received nilotinib (150 mg or 300 mg pro die). Based on the evaluation of the geometric means of measured drug concentrations, the administration of 150 mg or 300 mg resulted in 0.61–1.10 ng/mL and 1.10–1.90 ng/mL peak CSF nilotinib concentrations along with 343.3–524.4 ng/mL and 485.8–621.2 ng/mL peak serum concentrations, respectively, after three months of treatment. This corresponded to 0.16–0.23% and 0.20–0.32% CSF penetration rates, respectively. In contrast to the favorable outcomes of the earlier studies, this trial ended with the conclusion that the low penetration rates were associated with no treatment-related alterations of dopamine metabolites in the CSF. Therefore, the changes in the protein biomarkers  alone provided weak evidence of the clinical efficacy of nilotinib treatment .In animal models of neurodegeneration, nilotinib promoted the degradation of proteins Aβ/amyloid protein and the microtubule-associated protein tau . This reThe above works included the detailed evaluation of quantitative changes in the pharmacodynamic variables, such as microtubule-associated protein tau or amyloid proteins. Although the penetration rate of nilotinib into the CSF was very low, these were associated with statistically significant pharmacodynamic improvement and measurable clinical efficacy.All of the described bioanalytical methods relied on chromatographic separation using high-performance or ultra-high performance liquid chromatography. Mass spectrometry was chosen for detection by most authors, but multiple examples of applying ultraviolet–visible (UV–VIS) light absorbance detection for the assessment of afatinib, erlotinib, gefitinib, imatinib, nilotinib, and vemurafenib were found.In the majority of cases, sample pretreatment consisted of deproteinization. The removal of proteins was performed using organic solvents  , dasatinDetailed information on the methods employed for monitoring OACDs in peripheral fluid spaces is provided in The rapid growth of the number of related publications reflects the increasing clinical interest in monitoring OACDs in therapeutically relevant extravascular fluids. Nevertheless, the range of substances that have been monitored in these compartments with the aim of supporting clinical decision making comprises the minor segment of marketed OACDs. Currently, imatinib is the most extensively studied drug, followed by erlotinib, gefitinib, and nilotinib. Interest in studying recently approved entities, such as dasatinib, osimertinib, panobinostat, and ribociclib, is also rising.To date, frequently monitored peripheral fluid spaces have included cerebrospinal fluid, and, to a lesser extent, breast milk. Sporadic examples of monitoring OACDs in pleural effusion fluid, ascitic fluid, the intracellular space of peripheral blood mononuclear cells, semen, and saliva have been encountered. Collecting, handling, and processing samples originating from these fluid spaces requires expertise and, regarding CSF, pleural effusate, and ascitic fluid, specialized clinical infrastructure. Due to this limitation, as well as to the need to use specialized and resource-intensive analytical technology, it is likely that OACD monitoring in peripheral fluid spaces remains a competence of centers of excellence in oncology.The attainment of very low OACD concentrations in CSF seems to have been unexpected by several authors. One explanation could be the poor permeability of the blood–brain barrier to these drugs, but this assumption has been contradicted by results showing that WBRT, an adjuvant intervention undertaken to increase this permeability, had not always led to increased penetration rates ,72,91. TAnother interpretation is that the CSF concentrations of OACD substances could be associated with the unbound plasma fractions. Several examples of a correlation observed between unbound serum/plasma concentrations and CSF levels confirm this assumption . Since tThe extent of plasma protein binding may not be the only factor of the penetration of OACDs through the blood–brain barrier. Guntner et al. have shown with seven OACD substances that molecule size and the affinity of the molecule to p-glycoprotein  are also key determinants. In accordance, the permeability of the blood–brain barrier to dasatinib, imatinib, regorafenib, ribociclib, and vorinostat was higher than to nintedanib or panobinostat. The comparison of experimental results to those obtained using computer models nevertheless indicated that further variables, currently unidentified, are likely to play an important role in this process .50 established for the given malignant cell line, and based on this relationship, by creating a mathematical link between the pharmacokinetic and pharmacodynamic properties of the administered drug. In the future, this approach may prove useful in developing precision dosing schemes with pharmacokinetic–pharmacodynamic targets, in an analogy to those already employed for guiding antibiotic therapy. In sum, more research is needed to find dosages and monitoring approaches that result in the attainment of clinically sufficient CSF concentrations in all patients. Aggressive dosing, the artificial facilitation of the penetration of drugs through the blood–brain barrier, or the administration of drug combinations containing a component which inhibits p-glycoprotein or other drug-eliminating proteins relevant to a specific OACD are potential strategies for the more efficient therapy of CNS malignancies. A methodology is also emerging to predict OACD treatment efficacy by comparing the drug concentrations measured in the target peripheral fluid to the in vitro ICIn sharp contrast to the observations made in the CSF, high penetration rates or even the accumulation of OACDs were consistently described in exudates formed by pleural effusion and malignant ascites, and in excreta such as breast milk and semen. Imatinib showed considerable accumulation in buffy coat cells and in peripheral blood mononuclear cells. These findings indicate that the consideration of third spaces as pharmacokinetic compartments may be rational in patients treated with lung or breast cancer, as well as in leukemia patients.There has been a solid consensus in relying on liquid chromatography-based analytical approaches for the therapeutic monitoring of OACDs in peripheral fluid spaces. Several early methods relied on the use of UV–VIS detectors, but LC–MS/MS has by now emerged as the primary analytical technique as a result of ensuring sufficient selectivity and sensitivity, requiring small sample volumes for the analysis, and allowing the high-throughput processing of peripheral fluid space samples. When applied for the clinical analysis of OACDs in these compartments, the main steps of these methodologies were reversed phase chromatographic separation followed by positive electrospray ionization and multiple reaction monitoring. The simple and rapid process of deproteinization was in most cases sufficient for the pretreatment of samples. A common weakness of the analytical methodologies employed in the reviewed records is that they had not undergone comprehensive validation, lowering the credibility of the presented results.The application of equilibrium dialysis to retrieve direct clinical pharmacological information fits into a series of related emerging approaches, such as the rapid assessment of protein binding, or the partitioning between plasma and red blood cells. Such technologies are expected to facilitate the reporting of truly individualized, and, in a clinical sense, substantially more relevant information on the pharmacokinetic properties of drugs including OACDs in the future ,147.An important limitation of the performed evaluation is that only a minority of the retrieved publications described the outcomes of registered clinical trials. The majority of the works reported small-scale, researcher-initiated, unicentric studies, case series, or case reports. In addition, the methodologies employed for sample collection and analysis were uniquely developed by most investigators, limiting the comparability of results. Only a fraction of the subjects involved in the studies had given their consent for collecting CSF samples; consequently, the number of available CSF concentrations was small in several publications. Indeed, peripheral space drug monitoring was conducted as a collateral tool of diagnosis or patient status monitoring in several cases.Malignancies are the leading causes of premature death worldwide, with breast and lung cancers underlying the largest number of new cases . The impThis review has revealed that the therapeutic monitoring of OACDs in peripheral fluid spaces is an important diagnostic tool for the assessment of the penetration of these substances into CSF and third space fluids, which is imperative for the optimization of drug administration, and of their appearance in excreta, which may convey important information on adverse effects and other forms of toxicity. LC–MS/MS is an established analytical technology for performing these measurements, with little effort required to transfer conventional, blood-based TDM methods. Nevertheless, dedicated centers of excellence are needed to perform such measurements routinely.A range of indications has been identified for which the TDM of OACDs in peripheral fluid spaces can provide clinically powerful information. More systematic studies with rigorous quality control are needed, however, for elucidating the pharmacokinetic properties of OACDs, for setting quantitative therapeutic targets, and for establishing standard analytical methodology. Related research in pediatric populations still remains an unmet need.After more than 20 years of using OACDs, an alarmingly small number of these substances has ever been investigated in a clinically important peripheral fluid space. In several malignancies, the administration of these medications cannot be optimized without knowledge regarding their quantities in these fluid spaces, especially in CSF; therefore, research should be focused on gathering information on all OACDs in this respect.


Accumulating evidence supports the role of monocytes and neutrophils in radiographic axSpA (r-axSpA). Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a growth factor for both leukocyte lineages and a pro-inflammatory cytokine activating myeloid cells and promoting osteoclastogenesis. It acts through the JAK-STAT pathway. We measured serum GM-CSF and markers of bone metabolism in patients with r-axSpA before and after anti-TNF treatment.Patients with active r-axSpA despite treatment with NSAIDs, all eligible for treatment with a biologic agent, were recruited. Healthy donors were sampled as controls. Serum was collected before (baseline) and after 4–6 months (follow-up) of anti-TNF treatment and the following molecules were measured with ELISA: GM-CSF, sclerostin (SOST), and dickkopf-1 (Dkk-1).p=0.049), significantly lower Dkk-1 , but similar levels of SOST  compared to controls. Anti-TNF treatment did not affect GM-CSF, Dkk-1, or SOST levels. Spearman correlation analysis showed that GM-CSF correlated positively with ASDAS at baseline , while no correlations were identified between bone markers  on one hand and GM-CSF or disease activity indices on the other.Twelve r-axSpA patients  with a median disease duration of 1 year and 16 age- and sex-matched controls were included. At baseline, patients had mean BASDAI 6.3±2 and ASDAS 3.2±0.7, which decreased to 4.1±1.7 and 2.2±0.6 at follow-up, respectively. At baseline, r-axSpA patients had significantly higher mean serum levels of GM-CSF (150 vs 62pg/ml, GM-CSF is increased in patients with active AS and strongly correlates with disease activity. TNF inhibition does not affect GM-SCF levels, despite improving disease activity. GM-CSF may represent an important pathway responsible for residual inflammation during TNF blockade, but also a potential target of JAK inhibitors, explaining their efficacy in r-axSpA.The online version contains supplementary material available at 10.1186/s13075-022-02888-6. Radiographic axial spondyloarthritis (r-axSpA), also known as ankylosing spondylitis, is the main representative of the axSpA family and a chronic inflammatory disease characterized by inflammation of spinal structures and peripheral entheses, which gradually results in aberrant bone formation creating syndesmophytes and enthesophytes . In receNevertheless, there is now increasing evidence that myeloid cells, such as neutrophils and macrophages, are also involved in both the inflammatory and osteoproliferative processes of axSpA. Disease activity in r-axSpA has been positively correlated with the proportion of neutrophils in the peripheral blood , 4, as wGranulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor for both neutrophils and monocytes, acts as a pro-inflammatory cytokine inducing myeloid mobilization, activation, and cytokine production, and it is also involved in osteoclast differentiation .In addition, molecules inhibiting the bone anabolic process, such as the Wnt pathway inhibitors sclerostin (SOST) and dickkopf-1 (Dkk-1) display altered expression and/or function in patients suffering from r-axSpA. Indeed, SOST expression is impaired in patients with r-axSpA, whereas low levels of sclerostin are linked to increased structural damage and progression . MoreoveWe therefore sought to conduct a pilot study to measure serum GM-CSF levels in patients with r-axSpA before and after treatment with a tumor necrosis factor (TNF) blocker and explore for correlations between GM-CSF levels, disease activity, and serum markers of inflammation and bone remodeling.U test, and Spearman’s rank order correlation test on the Statistica software. The level of significance was set at p<0.05.The study included biologic-naïve patients diagnosed with r-axSpA with active disease despite treatment with non-steroidal anti-inflammatory drugs (NSAIDs), all being eligible for treatment with a biologic disease-modifying anti-rheumatic drug (bDMARD) and with the decision for treatment with a TNF inhibitor made by the treating rheumatologist independent of this analysis. Serum was collected before  and after 4-6 months  of TNF inhibitor treatment. Age and sex-matched healthy donors were sampled as controls. The following molecules were measured in sera using ELISA: GM-CSF, SOST, and Dkk-1, according to the manufacturer’s instructions (R&D Systems). All subjects gave their written informed consent and the study was approved by the Institutional Boards of the Ruhr-University Bochum, Germany, and University Hospital of Alexandroupolis, Greece. Statistical analysis was performed using Fisher’s exact test, Mann-Whitney Twelve patients with r-axSpA  with a median age of 37 years (range 22–52) and 16 controls  with a median age of 37 years (range 22–55) were included. Median duration of r-axSpA symptoms was 6.5 years (range 0.5–29), while the median time since r-axSpA diagnosis was 1 year (range 0.5–25). Eight (66.7%) patients were HLA B27 positive. At BL, r-axSpA patients had a mean Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) 6.3±2 and Ankylosing Spondylitis Disease Activity Score (ASDAS) 3.2±0.7. At FU, the mean BASDAI decreased to 4.1±1.7 and ASDAS decreased to 2.2±0.6 (Table p=0.049), the levels of Dkk-1 were significantly lower , whereas the levels of SOST were not different  compared to controls. At FU, the r-axSpA patient mean serum levels of GM-CSF were 152pg/ml, of Dkk-1 975pg/ml, and of SOST 379pg/ml, none of which showed significant change compared to BL , but not at FU . They were also negatively correlated with age , but not with disease duration since diagnosis  or symptom duration . Notably, a significant negative correlation between GM-CSF levels and age was found in healthy controls , as well. No correlations were identified between serum levels of Dkk-1 and SOST on one hand and GM-CSF levels, disease characteristics or activity indices on the other.At BL, serum levels of GM-CSF were significantly higher in r-axSpA patients , GM-CSF levels remained unaffected, while the correlation with ASDAS was abolished. This finding suggests that GM-CSF is increased in r-axSpA and persists despite the suppression of the TNF-mediated inflammatory pathways. As a consequence, increased GM-CSF keeps on enhancing myelopoiesis and the pro-inflammatory functions of myeloid cells, including neutrophils and monocytes. This may explain the residual disease activity despite anti-TNF treatment, since also at the same time both mean BASDAI and ASDAS values decreased but not to a status of ‘low’ disease activity (BASDAI <4 or ASDAS <2.1). On the other hand, while GM-CSF levels did not correlate with r-axSpA symptom duration or time since r-axSpA diagnosis, they inversely correlated with age. However, the same correlation was also observed in healthy controls, suggesting that this observation is not r-axSpA-specific, but may be a normal finding.Several studies reported a positive correlation between peripheral neutrophil-to-lymphocyte ratio and clinical disease activity , 4, whicThe role of myeloid cells in the pathogenesis of spondyloarthritis (SpA) is gradually unveiled. A recent analysis of the transcriptome of peripheral monocytes from two axSpA cohorts revealed a skewing of monopoiesis towards granulocyte monocyte progenitors (GMPs) rather than monocyte dendritic cell progenitors, particularly in subjects with established r-axSpA versus non-radiographic axial SpA . These oTo date, several anti-GM-CSF monoclonal antibodies are being developed and some of them have successfully been tried in rheumatoid arthritis . MoreoveThe results presented above derive from a small and preliminary study with a short observation period. Consequently, since both GM-CSF measurements occurred only a few months apart, it is uncertain whether GM-CSF levels remain relatively stable or vary over years possibly with relation to disease activity or in response to different treatments. However, our results suggest a potential pathogenetic role of GM-CSF in r-axSpA and corroborate previous preclinical and clinical observations that support further studies on the biology and clinical utility of GM-CSF in this disease.Additional file 1: Suppl. Table 1. Single patient data.