This PET radiopharmaceutical has been tested in several pre-clinical and clinical studies, as reviewed here, showing an important correlation with other risk factors.Atherosclerosis is responsible for the majority of heart attacks and is characterized by several modifications of the arterial wall including an inflammatory reaction. The silent course of atherosclerosis has made it necessary to develop predictors of disease complications before symptomatic lesions occur. Vulnerable to rupture atherosclerotic plaques are the target for molecular imaging. To this aim, different radiopharmaceuticals for PET/CT have emerged for the identification of high-risk plaques, with high specificity for the identification of the cellular components and pathophysiological status of plaques. By targeting specific receptors on activated macrophages in high-risk plaques, radiolabelled somatostatin analogues such as  Cardiovascular atherosclerotic disease is a systemic condition that affects major arteries of the body, being the most common cause of death in the western world and expected to reach 22.2 million people by 2030 . Sudden Considering the dynamic nature of coronary atherosclerotic lesions, it is not easy to identify which plaques will become unstable based on just a single imaging modality. An approach that allows for the identification of cap thickness, necrotic core areas, and the degree of inflammation of the plaque is of paramount clinical relevance. Given the involved costs, the time required, and the potential adverse effects, invasive techniques are not suitable for screening asymptomatic patients. CT angiography can be used to identify the presence of lesions with high-risk characteristics, i.e., those with areas of low attenuation and expansive remodeling. Serial CT could also determine dynamic changes in the plaque as an indicator of instability, warning the clinician to adopt prophylactic measures . In thisNon-invasive imaging of atherosclerotic plaques includes optical imaging, computed tomography (CT), magnetic resonance imaging (MRI), ultrasound (US), single-photon emission computed tomography (SPECT), and positron emission tomography (PET). Furthermore, hybrid PET systems coupled with CT or MRI are available, providing both anatomical and molecular information about the disease ,8. PET i18F-fluoro-deoxyglucose (18F-FDG) is the most widely used and validated PET radiopharmaceutical for atherosclerosis imaging, since plaque uptake is an indicator of increased macrophage activity. Indeed, ex vivo studies have shown a strong correlation between 18F-FDG uptake and both macrophage density and the increased gene expression of inflammation markers [18F-FDG PET in patients with transient ischemic attacks, observing significant radiopharmaceutical uptake in symptomatic carotid plaques. In addition to findings supporting the use of 18F-FDG as a marker of arterial inflammation, preliminary studies have pointed out the value of this tracer to predict plaque rupture and clinical events [18F-FDG in cardiovascular disease, the non-specific uptake of the radiopharmaceutical is the most important drawback for imaging atherosclerosis [18F-methylcholine (FMCH), also uptaken by activated macrophages in vulnerable plaques [68Ga-DOTA-NOC-TATE-TOC, uptaken by somatostatin receptors expressed in activated lymphocytes and macrophages of the inflammed vulnerable plaque [18F-sodium fluoride, whose uptake is associated with active calcification, apoptosis, and necrosis, with good specific characteristics for the detection of high-risk microcalcification and early unstable atherosclerotic disease [ markers . Rudd et markers  reportedclerosis . To over plaques ; 68Ga-DOe plaque ; and 18F disease .Nevertheless, differences in histology and natural history occur between coronary plaques and large peripheral vessel plaques, such as those affecting carotid arteries. In coronary plaques, the calcium content seems to be an early and prognostic factor, whereas in large peripheral vessel plaques, the lympho-monocytic infiltration probably plays a major role in predicting vulnerability ,22.In coronary plaques, rupture is independent of plaque size, therefore there is the imperative need to differentiate stable from potentially unstable lesions in clinical practice . The ratIn this review, we will concentrate on the events occurring in coronary plaques and the role of PET/CT in imaging vulnerable coronary plaques. In particular, this review describes state-of-the-art imaging, with radiolabelled somatostatin analogues, and the processes associated with plaque rupture.Atherosclerosis is liable for the majority of hard events and is characterized by the accumulation of lipids, inflammatory cells, and connective tissue within the arterial wall . AlthougToday, it is accepted that endothelial cell dysfunction is the earliest detectable physiologic manifestation of coronary atherosclerosis  and thatThe atherosclerotic plaque develops as a consequence of an endothelial lesion and the entrance of LDL cholesterol into the intimal layer of the vessel wall. The consequence of this lesion is monocytes recruitment into the vessel wall and their differentiation into macrophages, which take up modified LDL . The macMacrophages deserve special mention because the outcome of the plaque is mainly determined by the actions of these cells, which are found in high densities in unstable coronary lesions , and theLibby et al. proposed a broader hypothesis to explain the plaque rupture mechanism, which is an expansion of the original work described by Henney et al. . These aIn addition to the previously described events during plaque progression, calcium deposition also occurs by differentiation of mesenchymal cells into osteoclasts and osteoblasts. As a consequence of necrosis, inflammation and cell apoptosis, microcalcification appears as a repair mechanism .Since atherosclerosis is a systemic, mainly lipid-driven inflammatory disease leading to multifocal plaque development , metabolThese physiopathological events have been used as potential targets for molecular imaging agents labeled with radioisotopes. Radiopharmaceuticals available for targeting atherosclerotic plaques are listed in In vivo visualization of inflammation at the cellular and molecular level has been possible through the use of different peptide receptor ligands and monoclonal antibodies. . Overexp111In, with high affinities for type 2 and 5 SSTR, has been a popular radiotracer for imaging purposes. Today, new PET radiopharmaceuticals for somatostatin receptor scintigraphy are commercially available, showing better affinity for different receptors, such as 68Ga-DOTA-TATE and 64Cu-DOTA-TATE (affinity to type 2 and 5 receptors); 68Ga-DOTA-TOC (selective for 2 and 5-type receptors); and 68Ga-DOTA-NOC  [SST was first isolated from the ovine hypothalamus and characterized as a tetradecapeptide . SST-proceptors) ,96.68Ga-DOTA-TATE has the potential to become a surrogate marker of inflammation for the study of plaque biology [18F-FDG and 68Ga-DOTA-TATE uptake are known today. So far, evidence shows a tendency to confirm the utility of radiolabeled SSTR with 68Ga-DOTA-TOC to identify vulnerable plaques, with a good correlation with cardiovascular risk factors [18F-FDG, which gives only global information about the inflammatory burden [Monocytes and macrophages as triggers of vascular inflammation play an important role in the pathogenesis of atherosclerosis, contributing to necrotic core formation, fibrous cap thinning, and plaque vulnerability . These c biology . Complex factors . The addy burden .68Ga-DOTA-octreotide tracers have been positioned as novel molecules for PET imaging due to physical characteristics, good spatial resolution, high sensitivity, and possibility for the quantitative assessment. The most used 68Ga-DOTA peptides are 68Ga-DOTA-TOC, 68Ga-DOTA-NOC, and 68Ga-DOTA-TATE. Recently, a new generation of radioligands based on SSTR-2 antagonists was described in a preclinical study, showing more favorable pharmacokinetics than agonists [111In-DOTA-JR11, demonstrating favorable biodistribution and targeting efficiency for detecting atherosclerotic plaque, with a higher target-to-background ratio contrast than that observed with DOTA-TATE (agonistic radioligand). After incubating a human endarterectomy sample with 111In-DOTA-JR11, they demonstrated uptake of the radioligand in sites where immunohistochemistry showed SSTR- 2 receptors and noticed no radioligand uptake or SSTR 2 expression in areas of macrocalcifications which were visible in CT.agonists . In this68Ga-DOTA-TATE for targeting activated macrophages in atherosclerotic plaques by detecting sites of SSTR-2 accumulation in the human body. Xiang et al. [68Ga-DOTA-TATE to macrophages and the normal endothelium ex vivo, as well as the uptake in inflammatory plaques in a mouse model. They reported the presence of SSTR-2 in activated macrophages and showed a high specific affinity of 68Ga-DOTA-TATE to atherosclerotic lesions by autoradiography. The potential of this probe in clinical settings has been confirmed by different authors.Pre-clinical studies have supported the use of g et al.  assessed68Ga-DOTA-TATE could provide a theranostic opportunity to characterize and modulate atherosclerotic plaque biology; in 11 oncological patients the baseline 68Ga-DOTA-TATE scans showed abnormal focal uptake in carotid vessels, which, after peptide receptor-targeted radionuclide therapy (PRRT), showed significant reduction in follow-up scans. They also documented a correlation between cardiovascular risk factors and radiotracer uptake at baseline; reported variables were: patient age ; number of calcified plaques ; and presence of hypercholesterolemia . They also noticed that PRRT-related reduction of 68Ga-DOTA-TATE uptake was stronger in non-calcified vs. calcified active plaques. Although this observation was focused on carotid plaques, it could serve as a starting point for further exploration of radionuclide-based anti-atherosclerotic molecular interventions aimed to decrease the degree of inflammation in high-risk atherosclerosis. In a SR Anzola et al. [68Ga-DOTA-TATE as a vascular inflammation imaging agent (VISION STUDY); they confirmed that high-target SSTR2 gene expression occurs exclusively among activated proinflammatory M1 macrophages in atherosclerosis and demonstrated the presence of SSTR-2 receptors from patients with coronary vascular disease. They found that 68Ga-DOTA-TATE correctly identified culprit coronary and carotid arteries in patients with acute coronary syndromes. Intra-observer correlation showed a coronary artery intraclass coefficient of 0.90, with an inter-observer correlation of 0.96. In culprit acute coronary syndrome lesions, 68Ga-DOTA-TATE uptake was higher than in the non-culprit segment (p = 0.008). According to ROC analysis, coronary 68Ga-DOTA-TATE mTarget to background ratio maximum values (TBRmax) greater than 2.66 showed 87.5% and 78.4% sensitivity and specificity, respectively. 68Ga-DOTA-TATE also identified high-risk stable lesions in low-attenuation plaques detected by CT, where mTBRmax > 2.12 showed a sensitivity and specificity of 83.3% and 71%, respectively. They also reported a significant correlation between clinical risk factors and 68Ga-DOTA-TATE uptake . In a multivariate linear regression analysis, they demonstrated that these clinical factors were significant predictors of 68Ga-DOTA-TATE uptake. For patients receiving statins, they observed lower uptake providing anecdotal evidence that this probe may be used for monitoring the anti-inflammatory effects of specific drugs. The authors also compared the results with those obtained using 18F-FDG, and found a superiority of 68Ga-DOTA-TATE to discriminate high-risk vs. low-risk coronary atherosclerotic lesions, which was explained by the non-specific measurement of glucose metabolism of 18F-FDG in plaque cells , suggesting a possible use of this radiotracer as a non-invasive biomarker of cardiovascular risk. Pedersen et al. [64Cu-DOTA-TATE/MRI in a population of 10 patients with clinical symptoms of stroke or transient ischemic attack, reported a high uptake of the radiotracer in the lesions in symptomatic patients with a high correlation, with the presence of CD163 in activated macrophages at histology. Xian Li et al. [18F-FDG and 68Ga-DOTA-TATE PET/CT and reported significantly increased uptake in fibrotic and vulnerable plaques in large arteries compared to normal arteries. The comparison between the two molecules showed few differences in the low-risk group; nonetheless, in the high-risk group, 68Ga-DOTA-TATE showed significantly higher values than 18F-FDG. They also reported a significant correlation between the mean uptake, the clinical risk scores, and the presence of calcification. Mojtahedi et al. [68Ga-DOTA-TATE PET/CT in a population of 44 patients with neuroendocrine tumors (NET), reported that the TBR value in the normal group was lower than in the group with atherosclerotic plaques (p < 0.0001) as well than in the group with fibrotic plaques (p = 0.0043) They found a significant correlation between 68Ga-DOTATATE uptake and the progression to the formation of atherosclerotic plaques based on the coronary CT calcium score (p = 0.0026). When they compared risk factors with 68Ga-DOTA-TATE TBR values, they found a direct correlation between the two variables (p = 0.0068). These findings support the potential of 68Ga-DOTA-TATE PET/CT for the molecular assessment of coronary artery disease. Rominger et al. [68Ga-DOTA-TATE uptake in the LAD coronary artery with the presence of calcified plaques and cardiovascular risk factors. Higher uptake allowed the distinction between patients with and without coronary calcifications (r = 0.34); ROC analysis showed that a TBR greater than or equal to 1.5 could differentiate between the two groups. The authors observed a significant correlation between the TBR values and prior vascular events , suggesting the potential of the molecule to detect the presence of high risk vulnerable plaques.Malmberg et al. , in an on et al. , by usini et al. , in a rei et al. , by usinr et al. , correla68Ga-DOTA-TATE could be used to identify unstable plaque in two ways: the detection of activated macrophages and the angiogenesis within the atherosclerotic lesion [A report from Adams et al.  showed ac lesion .68Ga-DOTA-TOC to detect high risk, vulnerable, atherosclerotic plaques, and confirms a good correlation with risk factors, with the added value of high specificity for targeting inflammation and providing in vivo information on plaque vulnerability. Non-nuclear imaging modalities can give superb anatomic detail but are not able to look at early functional events and plaque vulnerability. The most important contribution of these radiotracers is their ability to detect inflammatory activity of the plaque, improving risk stratification and the prediction of hard clinical events. Furthermore, the information obtained could be potentially used for therapeutic decision-making and monitoring of anti-inflammatory treatment with statins. Nevertheless, SSTRs for PET imaging of coronary vulnerable plaques share the same technical limitations observed with other radiotracers used for these clinical indications, such as the limited spatial resolution, the size of the lesions with a low signal from the plaque and the partial-volume effect from adjacent structures. Other limitations are cardiac and respiratory motion artifacts entailing the need of specialized software to control these effects. Finally, while quantification is an important advantage of PET imaging, being the standardized uptake value (SUV) the most utilized parameter, an appropriate quantitative index for 68Ga-DOTA in atherosclerosis has not been established, which is important for an objective evaluation of the findings and to follow up in a clinical setting.Available evidence shows a tendency to support the utility of radiolabeled SSRS with 18F-aluminium, have been proposed for labelling biomarkers of vascular calcification such as matrix G1a protein (MGP), which is expressed by endothelial cells, fibroblasts, and chondrocytes. MGP binds to calcium crystals and may be suitable as a molecular imaging probe for the investigation of early vascular calcium deposits [Regarding future perspectives, hybrid molecular images using advanced systems such as PET/MRI together with more precise quantitative analysis will enable the simultaneous characterization of morphologic and biologic features of the vulnerable plaque, providing a greater potential for tissue characterization and risk assessment. New PET radiotracers , such asdeposits . Anotherdeposits . Antibod64Cu-labelled monoclonal antibodies for P-selectin and 18F-labelled peptide compounds for VCAM-1 imaging [111In-Danbirt and 99mTc-Demotate to determine atherosclerotic plaque inflammation and phenotypes. These authors reported that 111In-Danbirt discriminates the presence of different leukocyte subsets and differentiates the plaque phenotype of fibrous cap atheroma from both pathological intimal thickening and fibrocalcific plaque segments. Detecting the presence of leukocyte subsets of proinflammatory macrophages may improve the understanding of inflammation involvement in atherosclerotic progression.Since inflammation starts with endothelial activation, the expression of selectins and vascular cell adhesion molecule-1 (VCAM-1) constitute potential imaging targets on the activated endothelium  as has b imaging . Recentl imaging  reported68Ga-DOTA-TATE/TOC/NOC to provide prognostic information and evidence of treatment efficacy has been demonstrated; however, large-scale trials are required to determine the incremental value of plaque imaging in risk assessment and its influence on clinical outcome.Atherosclerosis is a systemic and multifocal disease of inflammatory origin with a silent course that makes it necessary to develop predictors of disease before symptomatic lesions develop. To date, new imaging tools have been studied in different preclinical and clinical scenarios which have provided knowledge about the presence, extension, and progression of the disease, and about the vulnerability of atherosclerotic lesions. To have the possibility to identify the atherosclerotic plaque in terms of molecular and cellular processes, focusing the aim of molecular imaging is crucial, which is the development of a tailored imaging radiotracers to characterize vulnerable plaque, leading to a better outcome through risk stratification, with the perspective to customize pharmacological treatment and optimize results. The ability of


Neurological examination revealed hypotonia, areflexia, paralysis of bilateral lower limbs (power = 0/5), and sensory loss below the level of T12. Magnetic resonance imaging (MRI) showed spinal meningeal enhancement surrounding the conus medullaris from the upper T12 level with enhancement of the cauda equina nerve roots (2O) and colorless cerebrospinal fluid (CSF) with normal glucose and protein levels. Cryptococcal antigen was present in high titer in the CSF (1:1024), and CSF culture revealed Cryptococcus neoformans. The nerve conduction study demonstrated mixed sensory and motor axonal neuropathy of the bilateral lower limbs, sparing the upper limbs. The clinical, electrophysiological, and radiological findings were suggestive of cryptococcal meningoradiculitis. Intravenous amphotericin B and flucytosine were started. However, the patient did not achieve neurologic improvement and eventually succumbed to nosocomial infection. A 38-year-old man with newly diagnosed advanced acquired immunodeficiency syndrome (AIDS) (CD4 count = 17 cells/mmve roots . Lumbar C. neoformansCryptococcal meningitis is an important opportunistic infection caused by the encapsulated yeast


Misfolded alpha-synuclein (α-Syn) is a lead candidate based on its crucial role in disease pathophysiology. Real-time quaking-induced conversion (RT-QuIC) analysis of CSF has recently shown high sensitivity and specificity for the detection of misfolded α-Syn in patients with Parkinson's disease (PD) and dementia with Lewy bodies (DLB). In this study we performed the CSF RT-QuIC assay in 236 PD and 49 DLB patients enriched for different genetic forms with mutations in GBA, parkin, PINK1, DJ1, and LRRK2. A subgroup of 100 PD patients was also analysed longitudinally. We correlated kinetic seeding parameters of RT-QuIC with genetic status and CSF protein levels of molecular pathways linked to α-Syn proteostasis. Overall, 85% of PD and 86% of DLB patients showed positive RT-QuIC α-Syn seeding activity. Seeding profiles were significantly associated with mutation status across the spectrum of genetic LBD. In PD patients, we detected positive α-Syn seeding in 93% of patients carrying severe GBA mutations, in 78% with LRRK2 mutations, in 59% carrying heterozygous mutations in recessive genes, and in none of those with bi-allelic mutations in recessive genes. Among PD patients, those with severe GBA mutations showed the highest seeding activity based on RT-QuIC kinetic parameters and the highest proportion of samples with 4 out of 4 positive replicates. In DLB patients, 100% with GBA mutations showed positive α-Syn seeding compared to 79% of wildtype DLB. Moreover, we found an association between α-Syn seeding activity and reduced CSF levels of proteins linked to α-Syn proteostasis, specifically lysosome-associated membrane glycoprotein 2 and neurosecretory protein VGF.The clinicopathological heterogeneity in Lewy-body diseases (LBD) highlights the need for pathology-driven biomarkers in-vivo marker of Lewy-body pathology and support its use for patient stratification in clinical trials targeting α-Syn.These findings highlight the value of α-Syn seeding activity as an The online version contains supplementary material available at 10.1186/s40478-021-01276-6. GBA gene (PDGBA) show extensive LB pathology, most PD patients with bi-allelic mutations in the recessive gene parkin  show nigral degeneration without LBs [LRRK2 mutations (PDLRRK2) is also remarkably variable, including typical LB pathology, misfolded tau deposition or nigral degeneration without LBs [in-vivo. With disease-modifying treatment options targeting α-Syn underway, patient stratification according to α-Syn-specific enrichment strategies is a much-needed prerequisite to introduce patients to clinical trials.The current gold-standard diagnosis for Parkinson’s disease (PD) is based on clinicopathological criteria that include neuronal loss in the substantia nigra pars compacta and Lewy-body (LB) pathology related to alpha-synuclein (α-Syn) aggregation –3. Howevhout LBs . The hishout LBs –7. This in-vivo, research has focused on CSF. Yet, it is unclear whether CSF profiles of α-Syn species reflect α-Syn brain pathology. Analyses in sporadic PD and PDGBA demonstrated decreased CSF levels of total α-Syn compared to controls with the highest decrease in PDGBA patients carrying severe mutations [As we have no reliable imaging marker to assess the cerebral load of α-Syn utations –10. Howeutations , 12. Botutations , 14. Howutations .GBA mutations as a proxy for pronounced α-Syn pathology, and PD patients with parkin, PINK1 and DJ1 mutations as representatives for nigral degeneration with sparse or no α-Syn aggregation. Moreover, PD patients with LRRK2 mutations were assessed. As no study has yet analyzed α-Syn seeding capacities longitudinally, we also evaluated α-Syn seeding profiles throughout the disease course in repeatedly collected CSF samples.To further asses the value of α-Syn RT‐QuIC, as pathology-driven biomarker for LBD, we assessed CSF α-Syn seeding activity in two large cohorts with LBD enriched for genetic forms. We included PD and DLB patients with Besides, as several molecular pathways associated with α-Syn proteostasis have been identified over the last decade , we evalwildtype), 99 PD patients with GBA mutation (PDGBA), 9 PD patients with LRRK2 mutation (PDLRRK2), 20 PD patients with mutations in parkin, PINK1, or DJ1 , 33 DLB patients without GBA mutation (DLBsporadic) and 16 DLB patients with GBA mutation (DLBGBA) was available. See Additional file Between 2005 and 2020, 236 PD patients, 49 DLB patients, 14 asymptomatic mutation carriers, and 26 healthy controls have been recruited at the University Hospital of Tuebingen. Specifically, CSF of 108 sporadic PD patients . See Additional file Repeated lumbar punctures allowing longitudinal CSF measurements were performed in 100 PD patients  were excluded.. In addition to the negative control, we ran the same positive sample throughout all experiments to optimize the comparison between fluorescent responses in different plates. To overcome batch-to-batch variations and intrinsic plate-to-plate variability, we normalized the relative fluorescent units (RFU) for every time point to the maximum intensity reached by the positive control within each plate and expressed it as percentage. Intra-assay and inter-assays CVs before and after normalization are reported in Additional file We performed purification of recombinant wildtype human α-Syn and the various steps of the RT-QuIC assay as described . In addiWe calculated the threshold as the average normalized fluorescence value of negative control repeats during the first 10 h of recording, plus 30 standard deviations. The cut-off was set at 30 h. When only one of the four replicates crossed the threshold, the analysis was considered "unclear" and repeated up to three times. In those participants who showed a positive RT-QuIC α-Syn seeding profile (at least 2 out of 4 runs), we measured the area under the curve (AUC), the peak of the fluorescence response (Imax), and the lag phase (LAG) (time required to reach the threshold).RT-QuIC experiments were performed at the Institute of Neurological Science of Bologna (ISNB). Results were reported blinded of the clinical diagnosis and genetic status. The assay showed a high specificity (98.7%) for LB pathology in a series of 121 CSF samples from individuals referred to the laboratory of neuropathology at ISNB for dementia of various etiologies in which the presence of LBs and abnormal α-Syn deposits was excluded by neuropathological examination .CSF levels of total α-Syn were assessed using an ELISA kit for human α-Syn . Intra-assay imprecision of 4.4% was calculated from duplicate analyses and expressed as median of the range to average of the duplicates. Inter-assay imprecision of < 10% was determined using two quality control CSF pool samples.Neurotransmitter secretion: chromogranin-A (CHGA), secretogranin-2 (SCG2), neurosecretory protein VGF (VGF).Synapse plasticity: neuronal pentraxin-1 (NPTX1).Autophagy, including endocytosis, lysosomal function, and ubiquitin–proteasome system: AP-2 complex subunit beta (AP2B1), cathepsin F (CTSF), ganglioside GM2 activator (GM2A), lysosome-associated membrane glycoprotein 2 (LAMP2), ubiquitin.Measurements of the following protein concentrations were performed using parallel reaction monitoring mass spectrometry (PRM-MS) as previously described , 27.Neur50μL CSF was mixed with 50μL of an internal standard mixture containing stable isotope-labelled peptides , 13C-labelled ubiquitin  and bovine serum albumin , diluted in 50 mM NH4HCO3. Reduction and alkylation was performed by addition of 50μL 15 mM 1,4-dithiothreitol in 50 mM NH4HCO3, shaking for 30 min at 60 °C, cooling down at room temperature for 30 min, and addition of 25μL 70 mM iodoacetamide in 50 mM NH4HCO3 followed by shaking at room temperature in the dark for 30 min. Samples were digested by the addition of 25μL 0.08 μg/μL sequencing grade modified trypsin  diluted in 50 mM NH4HCO3 and incubated at 37 °C shaking for 18 h. Digestion was ended by addition of 25μL 10% trifluoroacetic acid. Solid-phase extraction was performed using Oasis® HLB 96-well μElution Plates  by conditioning (2 × 300μL methanol), equilibration (2 × 300μL H2O), loading of samples, washing (2 × 300μL H2O), and elution (2 × 100μL methanol). Samples were dried by vacuum centrifugation and stored at − 80 °C.Samples were dissolved by addition of 50μL 50 mM NH4HCO3 and shaken at room temperature for 1 h. Forty μL of sample were injected and separated using a Dionex™ UltiMate™ 3000 standard-LC system  and a Kinetex® EVO C18 column  with a SecurityGuard™ ULTRA cartridge prefilter (Phenomenex Inc.). On a 60 min method, with solvents A (0.1% formic acid in H2O (v/v)) and B (84% acetonitrile and 0.1% formic acid in H2O (v/v)), using a flow rate of 300μL/min, the gradient went from 3 to 5% B over one minute followed by 5–26% B over 48 min. The column temperature was set to 50 °C. Separation by high-performance liquid chromatography was performed in online mode coupled to a Q Exactive™ Hybrid Quadrupole-Orbitrap™ mass spectrometer (Thermo Fisher Scientific Inc.). Using a HESI-II ionization probe (Thermo Fisher Scientific Inc.) electrospray ionization was performed in positive ion mode with the following settings: spray voltage + 4.1 kV, heater temperature 400 °C, capillary transfer tube temperature 380 °C, sheath gas flow rate 25, auxiliary gas flow rate 10, and S-Lens RF level 60. Acquisition of data was performed using single microscan in PRM mode with an isolation window of m/z 2 centred on the second isotope of the precursor ion. The resolution setting was 70 k with an AGC target of 1 × 106 and a 256 ms injection time. Fragmentation was performed using higher energy collision-induced dissociation.Skyline v.19.1 was used to calculate and export fragment ion peak areas and to monitor fragment ion traces and ratios. The ratio between tryptic peptide and isotope-labelled peptide peak area was used for quantification.1-42, t-Tau and p-Tau were measured using ELISA kits from INNOTEST, Fujirebio GmbH, Germany. CSF levels of NFL were measured using the UmanDiagnostics NF-light®assay. Intra-assay coefficients of variation for each CSF parameter were below 15%.CSF levels of Aβgroup (seeders vs. non-seeders) and time (time of follow-up in years), their interaction and the random variable subject, modelled by random intercepts. Hypothesis testing was 2-sided and p values ≤ 0.05 were considered statistically significant.Statistical analysis was performed using IBM SPSS 26.0 software. Group comparisons of dichotomous data were analyzed using the likelihood-ratio chi-square test as it is suited for unequally distributed samples sizes. Inter-group comparisons  of demographics, clinical markers, and CSF parameters were calculated using ANOVA/ANCOVA including sex, age, age at onset, and disease duration as co-variates as appropriate. Pearson’s correlation was used to evaluate associations between RT-QuIC seeding profiles and clinical markers and CSF parameters. Longitudinal analysis of RT-QuIC α-Syn seeding was done using linear mixed model with fixed factors Demographic and clinical characteristics of all groups and subgroups are given in Table GBA, 1 LRRK2) were α-Syn seeding positive. A single PD patient showing 1 out of 4 positive replicates by RT-QuIC in three runs was excluded from analyses given the inconclusive result.Eighty-five percent of PD and 86% of DLB patients showed a positive RT-QuIC α-Syn seeding activity compared to 8% of controls Table . Of the p > 0.05).There were no significant differences in RT-QuIC seeding kinetics  between PD and DLB patients with positive α-Syn seeding (p = 0.019) and higher numbers of positive RT-QuIC replicates .Male sex and higher age were associated with positive α-Syn seeding  showed positive α-Syn seeding activity, 78% of PDLRRK2 and only 59% of PDrecessive _heterozygous gave a positive reaction. None of the PDrecessive_bi-allelic showed positive α-Syn seeding   and lowest in PDrecessive _heterozygous . Accordingly, PDGBA_severe had the shortest LAG phase  .PDGBA showed positive α-Syn seeding activity compared to 79% of DLBwildtype (p = 0.014)   and VGF .Positive α-Syn seeding and higher numbers of positive replicates were associated with lower CSF levels of LAMP2 , CHGA , CTSF , SCG2 , ubiquitin , and VGF    and VGF  compared to non-seeders . No associations were found with disease duration.Higher age and female sex were associated with higher CSF levels of AP2B1, CHGA, GM2A, LAMP2, SCG2, ubiquitin, and VGF  and lower MoCA scores .Positive α-Syn seeding and higher numbers of positive RT-QuIC replicates were associated with higher UPDRS-III  and lower MoCA scores  .Despite a shorter disease duration (7 years), PD patients with positive α-Syn seeding activity had a higher UPDRS-III (26) and lower MoCA score (25) compared to non-seeders  whereas sex, age, age at onset, disease duration, prevalence of parkinsonism and the interval between onset of parkinsonism and onset of dementia were not statistically significant when compared to non-seeders. DLB RT-QuIC non-seeders had higher mean CSF levels of t-Tau  and NFL  compared to RT-QuIC seeders   . Similarly, α-Syn seeding positives showed stable results of AUC (p = 0.576), Imax (p = 0.558) and LAG phase (p = 0.324) over time  Fig. A nor in e p = 0.34 over tiGBA, especially those classified as severe, showed the highest percentage of positive α-Syn seeding activity in both PD (93%) and DLB (100%). In contrast, PD patients with bi-allelic mutations in recessively inherited genes like parkin or PINK1 did not show CSF α-Syn seeding at all, whereas those carrying heterozygous mutations in these genes showed less α-Syn seeding than wild-type cases with a reduced positivity rate of 59%. Also, PD patients with LRRK2 mutations showed a reduced rate of α-Syn seeding (78%), although not to the extent previously reported in a Spanish cohort carrying the p.G2019S mutation [LRRK2 on α-Syn pathology.The present results show a significant association between RT-QuIC α-Syn seeding activity and mutation status across the LBD spectrum. Remarkably, mutations in mutation . The broGBA given that they were not characterized genetically. Interestingly, patients with DLB that were RT-QuIC seeding negative had fewer clinical core features related to α-Syn pathology such as RBD [GBA mutations, who were all positive by α-Syn RT-QuIC, showed normal mean CSF profile of t-Tau, p-Tau and Aβ1–42, whereas the group of sporadic DLB patients without GBA mutations presented higher mean CSF levels of t-Tau, p-Tau and NfL as well as reduced Aβ1–42 levels indicating a typical Alzheimer’s disease profile. The heterogeneity in α-Syn seeding activity among the different genetic forms of PD mirrors histopathological findings in these cases. While PD patients with GBA mutations show extensive α-Syn-positive LB pathology, those with bi-allelic mutations in parkin or PINK1 show nigral degeneration without LB, whereas those carrying LRRK2 mutations show LB pathology in most cases, but also misfolded tau deposition or nigral degeneration without LB pathology [Overall, 85% of PD and 86% of probable DLB patients showed α-Syn seeding activity, which is slightly lower than the percentage obtained in previous studies –13. The h as RBD . These rh as RBD . Consistathology .1–42 [in-vivo the mechanistic link between proteins linked to α-Syn proteostasis and seeding, which was previously shown only in post-mortem studies and cell models [Additionally, we demonstrated an association between RT-QuIC parameters expressing higher α-Syn seeding activity  with lower CSF levels of proteins related to α-Syn proteostasis, including autophagy, endocytosis, lysosomal function, ubiquitin–proteasome system and neurosecretion. Accordingly, PD and DLB patients with positive RT-QuIC seeding showed reduced CSF levels of lysosomal-associated membrane glycoprotein 2 and neurosecretory protein VGF compared to patients with negative seeding. Recent studies reported lower CSF protein levels of AP-2-complex subunit beta, cathepsin F, ganglioside GM2 activator, lysosomal-associated membrane glycoprotein 1 and 2, and ubiquitin in PD compared to patients with Alzheimer’s disease and controls, and decreased CSF levels of chromogranin-A and B, neurosecretory protein VGF, and secretogranin species in PD and DLB compared to controls , 31. Int1–42 , 33. Thel models –39.in-vivo, it will be essential to determine whether α-Syn RT-QuIC may have a role in predicting clinical trajectories. Increased α-Syn seeding capacities might predispose to accelerated/widespread α-Syn aggregation, thereby promoting faster progression to clinical disease milestones such as cognitive impairment. Our finding of an association between RT-QuIC kinetics parameters  with more severe motor impairment and cognitive dysfunction suggests that this might be the case. However, additional studies in prospective longitudinal cohorts where CSF and clinical data are available in de-novo PD patients with several years of follow-up, combined with further refinement of the quantitative capabilities of the assay are needed to fully address the value of the α-Syn RT-QuIC as a prognostic marker in LBD patients.Besides the capacity to accurately discriminate patients with LB pathology GBA, 1 LRRK2) were older and had lower MoCA scores than the negative ones. The observation is in line with a recent study in LRRK2 mutation carriers where only 19% showed a positive seeding compared to 20% of the control group. Those with positive seeding activity had higher likelihood ratios for probable prodromal PD [Most asymptomatic mutation carriers did not show RT-QuIC α-Syn seeding. The two RT-QuIC α-Syn positive mutation carriers , 99 PD patients with GBA mutation (PDGBA), 9 PD patients with LRRK2 mutation (PDLRRK2), 20 PD patients with mutations in parkin, PINK1, or DJ1 , 33 DLB patients without GBA mutation (DLBsporadic) and 16 DLB patients with GBA mutation (DLBGBA) was available. Repeated lumbar punctures allowing longitudinal CSF measurements were performed in 100 PD patients .Aditional file 3: Table S2. Intra-batch and inter-batch  coefficients of variation (%) of quantitative RT-QuIC parameters Imax and AUC of the positive control, before (raw) and after normalization. The intra-batch coefficients of variation (CV) of the maximum intensity of fluorescence (I max) and area under the curve (AUC) are expressed as percentage of the ratio between standard deviation and average.Additional file 4: Table S3. RT-QuIC seeding profiles in DLB stratified by GBA mutation status. The table shows the α-syn seeding profiles of the analysed DLB group  together with the quantification of CSF Aβ1-42, t-Tau, p-Tau and NFL .3.Additional file 3: Table S3 (.txt). RT-QuIC seeding profiles in DLB stratified by GBA mutation status. The table shows the α-syn seeding profiles of the analysed DLB group  together with the quantification of CSF Aβ1-42, t-Tau, p-Tau and NFL .Aditional file 5: Table S4. Correlations between CSF RT-QuIC alpha-synuclein seeding parameters with clinical measures and with CSF protein levels related to alpha-synuclein proteostasis in PD and DLB. RT-QuIC alpha-synuclein seeding activity is associated with higher motor impairment as measured by the Unified Parkinson Disease Rating Scale III (UPDRS III) and lower cognitive function measured by Montreal Cognitive Assessment (MoCA). Moreover, higher RT-QuIC alpha-synuclein seeding capacity is associated with lower CSF levels of proteins that are linked to lysosomal dysfunction and neurotransmitter secretion.Additional file 6: Table S5. Clinical and CSF characteristics of RT-QuIC seeders vs. non-seeders. p-Value significantly different between RT-QuIC alpha-synuclein seeders vs. non-seeders in the respective diagnostic groups.


To characterize the evolution of central nervous system (CNS) inflammation in HIV-1 infection applying a panel of cerebrospinal fluid (CSF) inflammatory biomarkers to grouped subjects representing a broad spectrum of systemic HIV-1 immune suppression, CNS injury and viral control.This is a cross-sectional analysis of archived CSF and blood samples, assessing concentrations of 10 functionally diverse soluble inflammatory biomarkers by immunoassays in 143 HIV-1-infected subjects divided into 8 groups: untreated primary HIV-1 infection (PHI); four untreated groups defined by their blood CD4+ T lymphocyte counts; untreated patients presenting with subacute HIV-associated dementia (HAD); antiretroviral-treated subjects with ≥1 years of plasma viral suppression; and untreated elite controllers. Twenty HIV-1-uninfected controls were included for comparison. Background biomarkers included blood CD4+ and CD8+ T lymphocytes, CSF and blood HIV-1 RNA, CSF white blood cell (WBC) count, CSF/blood albumin ratio, CSF neurofilament light chain (NfL), and CSF t-tau.HIV-1 infection was associated with a broad compartmentalized CSF inflammatory response that developed early in its course and changed with systemic disease progression, development of neurological injury, and viral suppression. CSF inflammation in untreated individuals without overt HAD exhibited at least two overall patterns of inflammation as blood CD4+ T lymphocytes decreased: one that peaked at 200–350 blood CD4+ T cells/μL and associated with lymphocytic CSF inflammation and HIV-1 RNA concentrations; and a second that steadily increased through the full range of CD4+ T cell decline and associated with macrophage responses and increasing CNS injury. Subacute HAD was distinguished by a third inflammatory profile with increased blood-brain barrier permeability and robust combined lymphocytic and macrophage CSF inflammation. Suppression of CSF and blood HIV-1 infections by antiretroviral treatment and elite viral control were associated with reduced CSF inflammation, though not fully to levels found in HIV-1 seronegative controls. Infection of the central nervous system (CNS) is a nearly universal facet of untreated human immunodeficiency virus type 1 (HIV-1) infection and is accompanied by increased CNS immune activation and inflammation with frequent cerebrospinal fluid (CSF) lymphocytic pleocytosis and increased concentrations of a variety of inflammatory biomarkers . Though CNS HIV-1 infection develops very early in systemic infection –15 and ccompartmentalized, and also may more frequently favor replication in macrophages and related myeloid cells (M-tropic) [Systemic disease progression is also accompanied by evolution in the genetic and phenotypic characteristics of CNS infection with both shared and divergent HIV-1 populations when compared to those in plasma –25. Duri-tropic) , 31. Fin-tropic)  and AIDS-tropic) , 34, and-tropic) . ThroughWhile primary systemic infection and the early phases of chronic CNS infection are most often asymptomatic, they may not always be neurologically innocent. Neurological dysfunction and evidence of CNS injury have been documented during the first year of infection , while mactive CNS disease, and complements quantitative neuropsychological test documentation of cognitive-motor impairment that may reflect the cumulative effects of both active and inactive (legacy) HIV-related and non-HIV-related CNS injury [Measurement of CSF neurofilament light chain protein (NfL) provides a sensitive objective biomarker of ongoing neuronal injury, indicating S injury , 38. CSFS injury , 40, butS injury .CSF HIV-1 escape in which CSF HIV-1 RNA levels exceed those of plasma, either with clinical impact (neurosymptomatic CSF escape) [asymptomatic CSF escape) [secondary CSF escape) [As with its systemic counterpart, combination ART has a major impact on CNS HIV-1 infection, so that individuals with systemic viral suppression with reduction of plasma HIV-1 RNA levels to below standard clinical detection limits usually also exhibit effective CSF HIV-1 RNA suppression –45. Inde escape) , 48 or n escape) , or in t escape) . While t escape) .Like systemic HIV-1 infection , 53, CNSin vivo injury remain largely inferential [While CNS immune and inflammatory reactions may participate in local viral control, they also may contribute to brain injury , 59. In erential –62. Furterential . While terential –67.While a number of the components of local CNS inflammation have been documented in different cohorts or case series, the overall character of evolving CNS inflammation has not yet been clearly delineated in relation to the stages of systemic infection, CNS injury and antiretroviral treatment (ART) , 54, 57.To begin to address this deficiency, we undertook this cross-sectional exploration of a set of subject groups encompassing the main stages of HIV-1 systemic infection and CNS injury using a panel of 10 functionally diverse, soluble inflammatory biomarkers measured in CSF and blood with major attention to the former. With these measurements we examined questions related to the compartmentalization of CSF inflammation and the evolving course of inflammation from early (primary) infection, through declining CD4+ T-lymphocyte counts and the development of HIV-associated dementia (HAD), and after treatment or during endogenous viral suppression.This cross-sectional study was an extension of an earlier published study using a set of archived CSF and matching blood samples derived from two cohorts: one in San Francisco, California, USA and the other in Gothenburg, Sweden . The samCSF was obtained according to standard protocols as previously described , 68, 69.All subjects underwent routine clinical bedside screening for symptoms or signs of CNS opportunistic infections or other conditions that might impact CSF biomarker concentrations; individuals with CNS opportunistic infections or other conditions confounding these analyses were omitted. Most of these subjects were studied before publication of the more formal Frascati criteria  and were® ELISA kit, UmanDiagnostics AB, Umeå, Sweden) and analyzed in a single run in the Laboratory of Neurochemistry at the University of Gothenburg by board-certified laboratory technicians blind to clinical data using a single batch of reagents for each assay; intra-assay coefficients of variation were below 10% for all analyses. To compare NfL values across all groups, we calculated age-adjusted NfL values for 42 years of age as outlined by Yilmaz and colleagues, and compared to the 42 year-old upper limit of normal of 757.8 ng/L [HIV-1 RNA levels were measured in cell-free CSF and plasma at each site using the ultrasensitive Amplicor HIV Monitor assay , Cobas TaqMan RealTime HIV-1  or the Abbott RealTime HIV-1 assay . All recorded viral loads that were below 40 copies per mL were standardized to an assigned ‘floor’ value of 39 copies per mL for descriptive purposes. Each study visit included assessments by local clinical laboratories using routine methods to measure CSF white blood cell (WBC) count, CSF and blood albumin in order to assess blood-brain barrier integrity, and blood CD4+ and CD8+ T lymphocyte counts by flow cytometry. CSF neurofilament light chain protein (NfL) concentration was measured using a sensitive sandwich method . Linear and quadratic orthogonal (“appropriate”) contrasts across the four NA groups explored trends across using ANOVA were performed using SAS version 9.4 .Biomarker associations were analyzed across the entire sample set using Spearman’s rank correlation, graphically presented as a heat map. Comparison of biomarker concentrations between subject groups to address selected In this combined section of Results and Discussion, we will first outline the background characteristics of the cross-sectional sample, follow with a description of the overall results of biomarker measurements and analysis of CSF biomarker associations, and finally explore comparisons of selected subject group differences and their implications for understanding the evolution of CNS inflammation and its response to, and potential role in, CNS HIV-1 infection and CNS injury over the course of disease. Our main emphasis will be on the CSF inflammatory biomarker findings, while their blood counterparts will be considered mainly among the features of the background context to CNS inflammation.Fig 1A depicting the concentrations of each of the 10 biomarkers in CSF and plasma across the 9 subject groups, HIV-1 infection induced an enhanced inflammatory state in CSF (and blood) across its full spectrum. The complete data set for the study results in S1 Table (deposited in Dryad: https://doi.org/10.7272/Q61R6NRZ).As shown in 1. CSF biomarker concentrations varied in relation to their blood counterparts in absolute terms at ‘baseline’ (HIV negative) and also in relative terms over the spectrum of HIV-1 disease; 2. the CSF inflammatory biomarkers exhibited higher concentrations in the untreated HIV-1-infected groups than in the uninfected controls in CSF (and in blood with the exception of MMP9), though the patterns of change across the groups differed among the biomarkers; 3. high CSF but not blood levels of all the markers were found in the HAD group; and 4. viral suppression by ART or by elite control reduced the CSF biomarker elevations, though not always to levels found in the HIV-negative controls. 5. Similarities in the patterns of some of the CSF biomarker changes allowed them to be grouped into hypothetical pathogenetic vectors representing more general inflammatory processes during evolution of untreated infection.Among the major features of the broad and robust CSF inflammatory response were: In order to dissect the elements of the CSF inflammatory features of HIV-1 infection, we will first examine the broad correlations among the 10 biomarkers across the entire sample. We follow this with more focused attention on selected subject groups and group comparisons to understand inflammatory transitions during disease progression and their associations with the characteristics of CNS infection and neurological injury.Fig 2 presents the Spearman’s correlation coefficients and a visual overview of the biomarker associations.To explore the relationships among the 10 CSF inflammatory biomarkers and their associations with both these same biomarkers in blood and the salient background biomarkers, we examined biomarker inter-correlations across the entire sample irrespective of group designation. The inclusion of the broad range of subject groups in this study provided a wide sampling of the HIV-1 systemic and CNS disease spectrum for this overall analysis. The heat map in aBox  drawn in the upper left corner of Panel A of Fig 2), suggesting relationships in their biological activity across HIV-1 progression. Similarly, CSF sCD14, sCD163 and sVCAM1 exhibited very high inter-correlations ; this indicates a second group of biomarkers with related responses across the full spectrum of disease. Moreover, these two drawn boxes of correlating biomarkers exhibited additional overlapping correlations, though excluding MMP9 (outlined by the third cBox  to the right of a and above b). These correlations highlight associations or interactions among 7 of the CSF biomarkers that form two overlapping clusters.In general, the inflammatory biomarker concentrations were extensively inter-correlated, consistent with the broad CNS and systemic immune activation and inflammation associated with HIV-1 infection. Notably, some of the biomarkers aggregated into discernible group patterns. For example, CSF MMP9, TNFα, CXCL10 and neopterin exhibited high levels of inter-correlations, with CXCL10 showing the highest correlations with the other three of these markers . Blood concentrations of both these biomarkers correlated relatively strongly with their CSF counterparts and almost as strongly with CSF sCD14 and sCD163 suggesting that their blood concentrations—and the systemic inflammatory state that they indicate—were associated with CNS inflammation. These two blood markers correlated strongly with CD4 count, plasma HIV-1 RNA and somewhat less strongly to CSF RNA . Additionally, among the blood markers, sCD14 and neopterin showed strong correlations with CSF NfL , at a level similar to that of the CSF markers correlating with injury, suggesting a systemic inflammatory profile predisposing to CNS injury. While these CSF and blood inflammatory patterns do not establish cause or effect, they suggest pathobiological links between components of CNS and systemic inflammation with axonal injury.Individual blood biomarker concentrations generally showed only relatively weak correlations with their CSF counterparts, with the notable exceptions of CXCL10 and neopterin  to a nine-fold higher CSF than blood concentration (CCL2). Seven of these concentration differences were highly significant, the exceptions being TNFα, with high variability in the sample, and both neopterin and IL-6 with CSF concentrations that were close to those in blood.Before comparing the changes in inflammatory biomarkers during infection, it is pertinent to examine CSF concentrations in the HIV-uninfected controls which varied considerably among the 10 biomarkers both in absolute concentrations and in relation to their blood counterparts. Comparison of mean CSF and blood inflammatory biomarker concentrations are shown in This analysis indicates that even in the absence of infection the CSF biomarkers are compartmentalized with a range of concentration relationships to blood. Thus, even in the absence of HIV-1 infection each of these inflammatory factors is ‘regulated’ in an individual manner, presumably by different levels of local production but perhaps also by variable kinetics of exchange between blood and CSF and cell uptake. They indicate that the ‘physiological’ role of these and other inflammatory molecules in the ‘baseline’ state is a worthy subject of further study , 84.Fig 1B). In an earlier study of a larger sample with PHI, we found that the albumin quotient was elevated in about 20% of subjects [Like CNS infection, CSF inflammation is clearly established during the first year of infection , 15, 85.subjects ; in the Fig 3.In order to examine the CSF (and blood) inflammatory reactions during this first year in more detail, including how they compared to uninfected individuals, on the one hand, and the early phase of chronic infection, on the other, we isolated comparisons of the biomarkers results in three subject groups: 1. uninfected HIV negative controls, 2. PHI and 3. HIV-1-infected NA group with highest CD4+ T-cell counts, >350 cells/μL, applying the Kruskal-Wallis test and Dunn’s multiple comparison test as shown in Table 3). The median change was greater than a twofold increase, with the change reaching significance in this analysis for 4 of the 10 biomarkers: TNFα, CXCL10, sCD14 and neopterin  with a less notable significance for sVCAM (P <0.05).The concentrations of all the inflammatory biomarkers were higher in CSF (and blood) during PHI than in the HIV-negative controls. These increases varied from 33% (TIMP-1) to 500% (TNFα and MMP9) of the HIV-negative level  exhibit lower mean levels in the CD4 >350 group, emphasizing that the first year of infection exhibited a particularly enhanced inflammatory state that subsided somewhat after transition to chronic infection. Notably, this robust CSF inflammation during the first year of infection involved a group of biomarkers that also exhibited intercorrelations over the entire samples as discussed earlier and outlined in Fig 4 extracted from Fig 1 with statistical comparisons added. This isolation emphasizes the varying patterns of biomarker change across the four CD4+ strata. Some first rose before falling, exhibiting an inverted ‘U’ pattern, while others gradually increased or remained relatively static across the four groups.To focus on the changes in CSF inflammatory biomarkers in the face of chronic untreated infection and progressive immunosuppression in the absence of overt HAD, we separately examined the four viremic NA groups defined by blood CD4+ counts; these are shown in isolation in Table 4). These analyses confirmed statistical differences in the patterns of changes among the CSF biomarker concentrations. Notably, the four biomarkers with the early rise in PHI—TNFα, CXCL10, sCD14 and neopterin—along with MMP9, exhibited significant inverted ‘U’ patterns with a peak in the midrange CD4 groups followed by decline as blood CD4 cells fell to <50 cells/μL (Table 4). Two other markers, sCD163 and CCL2, showed significant linear increases with declining CD4 cells. VCAM, IL6 and TIMP1 did not show either type of trend with CD4 decline. CSF MMP9, in addition to the inverted ‘U’ quadratic trend, exhibited a significant linear decrease, with the concentration in the CD4 <50 group lower than the CD4 >350 group.To more formally test for significant inverted ‘U’ shaped or linear relationships observed visually with descending blood CD4+ counts, we applied linear and quadratic trends analysis to CSF and blood inflammatory biomarkers and to other salient background variables in these four groups (pathogenetic vectors in the evolution of CNS/CSF infection and CNS pathology (see below). These links are supported by associations of different biomarkers with CSF HIV-1 RNA and WBC counts, on the one hand, and with neurological injury indicated by CSF NfL, on the other. Thus, both CSF HIV-1 RNA and CSF WBC counts shared the inverted ‘U’ pattern of change with the TNFα, MMP9, CXCL10, sCD14 and neopterin group of biomarkers, while CSF NfL and QNPZ-4 testing followed a linear trend of worsening with CD4+ T-cell loss over these four non-HAD groups like CSF sCD163 and CCL2. We should also note that CSF WBCs also showed some linear trend while NfL exhibited a minor quadratic component, suggesting more complexity and elements of mixed associations of these trends. In contrast to the different trends among the CSF biomarkers, in general the blood inflammatory biomarkers exhibited linear increase , linear decrease in the case of MMP9, or non-significant  trends with CD4 decline across these four subject groups.These patterns of change allow definition of two different inflammatory group patterns which we refer to in the ensuing discussion as Fig 1A), and all 10 were higher in the HAD group than the untreated groups with comparable blood CD4+ T cell counts (the <50 and 50–199 CD4 groups). This contrasted with the blood inflammatory biomarkers that did not appreciably increase in HAD compared to the comparable blood CD4+ groups.The CSF inflammatory biomarker concentrations in the HAD group stood out conspicuously from the other subject groups. Concentrations for several were the highest of any group  or comparable to the highest levels in one of the CD4-defined groups (MMP9 and CXCL10) (1) subjects with blood CD4 >200 cells per μL and normal age-adjusted NFL , total subject N = 36; 2) those from the two groups with <200 CD4 counts (<50 and 50–199 cells/μL) with CSF NfL concentrations below the age-adjusted laboratory cutoff ; 3) those from the same two lower CD4+ groups with elevated CSF NfL ; 4) the subacute HAD group as initially defined by their clinical presentation . The results of this re-sorting are shown in Fig 5.To provide further perspective on the associations of the 10 biomarkers with neural injury, we focused not only on the HAD group defined by their clinical neurological presentation, but also on a second ‘type’ of injury identified by CSF NfL concentrations above the age-corrected norm  within tThis regrouping helps to highlight some of the salient differences between the more highly inflammatory CSF profile in HAD compared to less-inflammatory injury in the NFL+ group.Fig 5A). Thus, the high level and breadth of the inflammation in the HAD group add to its pathobiological distinction from other types of CNS injury and enforce the clinical diagnostic differentiation of this condition, joining with abnormal t-tau and CSF/blood albumin ratios , again emphasizing the broad character and high level of inflammation associated with clinically overt HAD. Concentrations of some of the CSF inflammatory biomarkers, including TNFα, CXCL10 and sCD14, were higher in the HAD group than in all three of the other groups which did not differ among themselves .Fig 5A). The QNPZ-4 performance was also not as severely impaired (not shown), and the CSF t-tau concentrations and BBB permeability were normal in this group . The CSF inflammatory profile in the NFL+ group was also very different from HAD. CSF levels of TNFα, CXCL10, sCD14, TIMP1 and neopterin were distinctly lower than those of HAD and, importantly, not significantly different from those of the NFL- group and, indeed, also the >200 group, suggesting that these biomarkers do not associate with the neural injury in this NFL+ group. By contrast, CSF sCD163, CCL2, and IL6 were intermediate between HAD and the NFL- group supporting a possible association with this type of injury . Additionally, blood concentrations of sCD14, CCL2, TIMP-1 and neopterin also were intermediate or nearer to those of HAD than the groups without elevated NfL, further raising the question of an association of systemic inflammatory responses with this type of injury. These distinctions in both CSF and blood further define a profile of this second type of low-inflammatory injury and its distinction from the high-inflammation character of subacute HAD.The regrouping segregated individuals with the second type of HIV-related CNS injury (the NFL+ group) from the HAD group. In general, the NFL levels in this group were lower than in HAD but overlapped with its lower quartile  that are active or predominate during different phases of progressive infection and CNS injury.The analysis of the 10 CSF biomarkers and the differences among the subject groups provides the basis for proposing a tentative ‘model’ of CNS inflammatory factors in relation to other features of CNS infection and injury. Table 4). These biomarkers included CSF TNFα, MMP9, CXCL10, sCD14 and neopterin. Notably, they also prominently correlated with CSF HIV-1 RNA, which along with the CSF WBC, exhibited a similar quadratic pattern of change with systemic disease progression . CSF CXCL10 and MMP9 showed the highest overall correlation with the CSF WBC count. These observations, along with the known biological functions of these markers, support the hypothesis that a lymphocytic vector of CSF inflammation associates with local meningeal HIV-1 replication that develops early in infection, and in the absence of subacute HAD, reaches its peak when blood CD4+ counts are between 200 and 350 cells per μL. This is also associated with the pattern of CSF HIV-1 RNA levels, though to what degree these mediators drive lymphocyte entry and local HIV-1 replication or are a response to infection is uncertain. Likewise, it is uncertain why their concentrations were attenuated after further depression of the blood CD4+ T cell count below 50 cells per μL While systemic lymphopenia may provide a partial explanation, it is likely an incomplete one, since robust inflammation (including increase in these CSF biomarkers and pleocytosis) was generated in HAD despite similar low blood CD4+ T cell counts. By itself, this early CSF lymphocytic inflammation was associated with limited neuronal injury , and, hence, appeared to have relatively low direct neuropathic potential. Using traditional terminology, this may most commonly accompany a clinically silent aseptic meningitis that is a frequent feature of untreated HIV-1 infection.This phase begins during PHI but is most clearly defined in the four CD4+ groups in which lymphocytic CSF biomarkers exhibited an inverted U-shaped pattern conforming to a quadratic solution in the trends analysis (Table 2). CSF NfL also exhibited this strong linear trend, and these two macrophage biomarkers were among those that correlated well with CSF NfL, along with CSF sCD14 and sVCAM-1. These associations are consistent with the prevalent hypothesis that HIV-1-related neuronal injury is associated with macrophage recruitment into and activation within the CNS [This second inflammatory component increases progressively with falling CD4+ T cell counts and, in this series, predominated in more advanced systemic disease as blood CD4 cells fell to 200 cells per μL and below. The CSF biomarker elevations associated with this component most notably included two myeloid CSF biomarkers, sCD163 and CCL2, both exhibiting significant linear trends through the range of falling blood CD4+ T cells  by its robust CSF inflammatory profile with the components of the lymphatic and myeloid vectors boosted above the levels in the non-HAD groups with comparable blood CD4 counts. Indeed, all the CSF inflammatory biomarkers were either frankly higher in HAD than the other groups or nearly so. Among the markers, only the median CSF MMP9 was higher in one or more of the NA groups. Thus, HAD, defined by its subacute clinical presentation, was a broadly and distinctly inflammatory CNS disorder. It was also coupled with other unique features, including common disruption of the blood-brain barrier to albumin as previously reported in a larger case series  and elevWhile we studied blood biomarkers mainly for comparison with CSF and did not include them in this simple model, we also found that some of the blood biomarker changes correlated with CSF NfL, most notably blood neopterin and sCD14. While this may simply be related to CD4+ T-cell loss without direct effect on CNS events, it might also suggest that the character of systemic inflammation predisposes to development of neuronal injury by influencing the character of CNS inflammation or, more directly, by mechanisms that are not yet apparent. Whatever the reasons for the association, elevations of some of these blood inflammatory biomarkers in the NfL+ and HAD subjects may help to distinguish individuals at higher risk of CNS injury.Fig 7).Suppressive ART clearly attenuated the HIV-1-related CNS inflammatory response, reducing all the measured CSF inflammatory biomarkers, while elite control similarly was accompanied by reduced inflammation compared to patients with viremia Click here for additional data file.S1 File(PDF)Click here for additional data file.


Meningiomas have been classified as the most commonly occurring primary brain tumors. Although the majority of meningiomas are benign and slow-progressing, the tumors that grow to a larger size are associated with various risks during surgical procedures. Early detection of meningiomas is crucial to the treatment as those detected early can be treated through non-invasive methods. Due to their benign nature, meningiomas contain homogeneous protein biomarkers that can be easily identified. Cerebrospinal fluid (CSF) has a high protein composition which can be used to diagnose various brain tumors. Because CSF comes into direct contact with the brain during its functioning, it is one of the factors that makes it an important source of different biomarkers. An analysis of biochemical changes occurring in the CSF can be useful in assessing the condition of the periventricular white matter and the parenchyma.In this review, PubMed, Medline, PubMed Central, and Google Scholar were used to identify studies discussing meningiomas regarding their assessment, types, diagnosis, and treatment, with more attention directed towards the application of CSF proteome analysis in diagnosis. Priority was given to studies published within the last 15 years. The following keywords were used in the literature search: “cerebrospinal fluid,” “meningiomas,” “brain tumors,” “primary brain tumors,” “protein biomarkers,” “proteome analysis,” and “diagnosis.” Subsequently, the 15 most relevant studies were selected for inclusion in the review. We excluded studies discussing different types of non-brain tumors as well as older articles. The selected studies also underwent a quality appraisal process using corresponding assessment tools. The selected articles were highly informative about meningiomas and the processes of diagnosis and treatment that are currently in use as well as those that are being developed or implemented. The use of CSF proteins in the diagnostic process is also discussed in this review. The studies also describe proteomics as a less invasive procedure that allows for the analysis of entire proteins and the projection of diagnostic images with higher resolutions that aid in the diagnosis. According to the World Health Organization (WHO), meningiomas account for roughly 37.6% of all diagnosed primary brain tumors, and 50% of these are benign, implying that they are not harmful to the individual . MeningiStatistically, meningiomas are the most frequently occurring primary brain tumors, with incidence rates of 10.82 per 100,000 people annually and prevalence rates of 97.5 per 100,000 people annually  OR Clear Cell Meningioma OR Xanthomatous Meningioma OR Meningiomas Xanthomatous OR Fibrous Meningioma OR Fibrous Meningiomas OR Intracranial Meningioma OR “Benign Meningioma” [Majr] OR Malignant Meningioma AND Cerebrospinal Fluid OR “cerebrospinal fluid proteins” [Mesh] AND “Tumor Biomarkers” [Mesh] OR Carcinogen Markers OR Biological Tumor Markers OR “Tumor Markers” [Mesh] OR Cancer Biomarkers.Inclusion and Exclusion CriteriaThe articles identified were filtered using the following inclusion criteria: priority was given to English-language articles published within the last 15 years. The papers were also prioritized based on the type of research carried out, with human brain cell experimental and observational studies given preference. Studies were also preferred based on the type of brain tumor investigated, with a priority given to those on meningiomas in line with the research objectives.The exclusion criteria used to filter studies included in the review included studies involving other specific types of metastasized brain tumors and those that were descriptive in nature.Quality AppraisalThe selected articles underwent quality appraisal to assess their quality using various existing tools. The Cochrane Risk of Bias assessment tool was employed to assess any bias in the studies transcending biases that could affect the overall quality of the clinical trials. The Newcastle Ottawa Scale was used to assess any bias in the observational studies used in the research. The Assessment of Multiple Systematic Reviews 2 tool was employed to assess the quality of systematic reviews. The Critical Appraisal Skills Randomized Controlled Trials Checklist was used to assess the quality of the results reported in the various studies used in the research.ResultsDuring the initial search for the articles related to meningioma and meningioma diagnosis, 188 articles were identified, which were then further filtered to 100 articles related to meningioma diagnosis through the use of CSF biomarkers, with 30 articles reporting the use of protein biomarkers. The selected articles underwent further screening to assess if they conformed to the inclusion and exclusion criteria, and a total of 15 articles were finally selected for inclusion in the review. The quality assessment did not reveal any quality concerns.Figure DiscussionCurrent Methods and DiagnosisThe current methods employed for the diagnosis of meningiomas and other primary brain tumors are based on patients’ family history, physical examination, and as well as the use of radiological methods. Radiological methods include head contrast computed tomography (CT) scans and MRI. The concentration of water within living tissues is depicted by MRI, which is speculated to predict parts of their biomechanical activity. MRI signal intensity has been used to predict the consistency of a tumor and its histological subtype in meningiomas. Currently, MRI is the preferred gold standard of diagnosis .Although effective, these methods have been reported to have various concerns, such as the invasive techniques currently in use, and this research has been conducted to find better methods, which has included numerous studies on various components that come into contact with the neural systems such as the CSF .The protRecently, research has been directed towards profiling Ki-67, an immunohistochemical marker that can correlate with severity and outcomes. The study by Liu et al. focused on pooled hazard ratios (HRs) for overall survival (OS) and disease/progression/recurrence-free survival (D/P/RFS) that were calculated using a fixed or random-effects model. The study comprised 43 trials with a total of 5,012 patients. In meningiomas, higher Ki-67 expression levels were linked to poor overall survival  and D/P/RFS . They concluded that an increase in Ki-67 in patients with meningioma correlates with poor outcomes and an indefinite need of follow-up .Most studies have emphasized that the early detection of brain tumors can lead to a better prognosis and it is important to implement ways that aid in early diagnosis .Proteomic Analysis of Cerebrospinal FluidProteomics technologies have become more sensitive and dependable because of recent improvements in mass spectrometry. Targeted proteomics, a relatively new field of mass spectrometry-based proteomics, has shown enormous promise in resolving the limitations of traditional molecular biology-based techniques such as Western blotting and immunohistochemistry .Ghantasala et al. emphasized the necessity of focused proteomics in detecting and validating proteins involved in brain tumor pathophysiology. They demonstrated that APOE can be a potential tumor progression marker in meningioma based on the multiple reaction monitoring (MRM) tests utilizing CSF .Figure Several factors are related to the effectiveness of proteome analysis as a method of diagnosing brain tumors, including the availability of the CSF proteins in large amounts, which increases the chance of interaction with the molecules produced by various brain tumors; the direct interaction of the CSF with the brain, as well as the nature of the proteins in terms of patterns. The proteome analysis methods are also less invasive as the sampling is done through a lumbar puncture .CSF analysis can help with brain cancer detection and clinical prognosis. MicroRNA (miRNA) technology has rapidly enabled sensitive recognition of distinct miRNAs that are molecularly stable in the CSF and can distinguish between different kinds of CNS cancers. Analysis of CSF miRNAs can be a useful addition to clinical care due to the ease with which a CSF specimen can be acquired .Because changes in CSF are believed to sensitively reflect pathological processes in the CNS, such as neoplastic disorders, it has been recognized as a valuable source for prospective biomarker screening in this era of proteomics . BecauseMeningioma DiagnosisIn one study, meningioma-specific proteins were identified through analysis of CSF samples using 2-DE and mass spectrometry. The results are then validated through a Western Blot analysis that analyzes the patterns and compares them to those of patients already diagnosed with meningiomas. The proteins identified include APOE, alpha-1-antitrypsin (AAT), and PTGDS proteins, as discussed above. The patterns on the proteins are also essential in grading meningiomas according to the grades discussed above .Figure Another study focuses on the use of miRNAs, which are proteins found in the CSF because it is also a readily available protein in the CSF that can aid in the early detection of brain tumors . The PTGThe study by Kopkova et al. showed how miRNA detection in CSF is a multi-step procedure that includes CSF management and storage, miRNA isolation and expression analysis, and final data standardization, analysis, and interpretation. The research emphasized that there is a clinical need for improved brain tumor biomarkers that would allow for more precise diagnosis, prognosis, and therapy response prediction. Brain cancers linked to miRNAs produced and identified in CSF appear to be promising proteins from this perspective. Unfortunately, several factors can influence the entire procedure, which are yet to be standardized .Another study has defined CSF proteome analysis-based methods as those that are effective in the early detection of brain tumors. The effectiveness of early diagnosis leads to a better prognosis, which implies that the outcomes of most brain tumors can be improved after treatment before cancer progressively grows and metastasizes . As alreMiRNAs are single-stranded, non-coding RNAs that are 18-25 nucleotides long and influence gene expression post-transcriptionally. These molecules are tissue-specific and play a role in the development of various neurological illnesses. Circulating miRNAs have been discovered in nearly all human bodily fluids, including CSF, and appear to be extremely stable and resistant to harsh circumstances. Furthermore, unregulated levels of CSF miRNAs have been linked to CNS tumors including meningiomas. Thus, miRNAs in CSF of brain cancer patients may potentially aid in the development of a novel diagnostic approach that allows for more accurate diagnostic techniques, including in patients with meningiomas .The contribution of miRNAs to the clinical spectrum of benign meningiomas MiRNAs provide a second line of amplification of growth-promoting cellular signals by deregulating the translation of genes involved in signaling pathways known to be critical for meningioma genesis and progression. MiRNAs as indicators for meningioma diagnosis could be valuable and should be investigated further in the future .The importance of miRNA as an important biomarker for meningioma is further solidified in the study by Slavik et al. It aimed to identify molecular genetic markers for more accurate prediction of meningioma recurrence and improved targeted therapy. The research included microarrays that revealed the presence of miRNA expression in primary and recurring meningiomas of all WHO grades. In a group of 172 patients, those reported to be unregulated were further verified using quantitative real-time polymerase chain reaction. The resulting dataset was statistically analyzed, and predictors of meningioma recurrence were discovered. The study concluded that MiR-15a-5p, miR-146a-5p, and miR-331-3p were found to be the most significant prognosticators in both adjusted and non-adjusted models of time to relapse. In several models, the final validation phase demonstrated the critical importance of miR-146a-5p and miR-331-3p, as well as clinical parameters such as the type of resection  and WHO grade. Following stepwise selection in a multivariate model on an enlarged cohort, the model with decreased miR-331-3p expression  and partial tumor resection  was found to be the best predictive. Furthermore, in univariate models adjusted for clinical variables, both miRNAs remained prognosticators in the subgroup of total resections. Thus, the presented models may allow for a more accurate prediction of time to recurrence of meningioma and, consequently, optimum postoperative therapy. Furthermore, combining this model with current knowledge of the molecular pathways behind recurrence should allow for the identification of distinct meningioma subtypes and more precise therapy .Shalaby et al. reported that the use of CSF analysis is not being implemented clinically for various reasons, including the lack of collaboration between oncologists and other physicians involved in brain tumor diagnosis and the researchers involved in carrying out this research. Therefore, a gap exists in the sharing of knowledge, thus delaying the transfer of this technology to the field where it would be most valuable. Shalaby et al. also note that the transfer of this technology would simplify the work done by health professionals and, as discussed above, aid in the early and effective diagnosis of brain tumors .Application of Cerebrospinal Fluid biomarkers in Miscellaneous ConditionsCSF biomarkers play an important role in additional neurological condition in both the adult and pediatric populations.Moyamoya disease (MDD) is a rare cerebrovascular disorder with an unknown cause, described by stenosis or blockage of bilateral internal carotid arteries and an abnormal vascular network. Araki et al. analyzed CSF samples taken from 20 patients with MMD and 12 healthy controls to discover biomarkers. Surface-enhanced laser mass spectrometry (SELDI-TOF-MS) with an anion exchange chip was used to generate mass spectral data in three distinct buffer conditions. A comparison analysis was performed after expression difference mapping, utilizing the derived protein profiles. The study concluded that the statistically significant biomarkers found in patients with MDD have the ability to detect and classify the disorder, implying that the CSF proteins may be important in the diagnosis of various brain disorders .Multiple sclerosis (MS) is an autoimmune disease that affects the brain and spinal cord (CNS), leading to neurodegeneration. Biomarkers in body fluids may be able to predict and study neurological impairment in MS patients. CSF oligoclonal bands (OCB) are key indicators that can aid in MRI diagnosis and prevent false-positive diagnoses. New biomarkers such as aquaporin4 (AQP4) have already been established in clinical practice, while others such as anti-myelin oligodendrocyte glycoprotein (anti-MOG) and neurofilament light (NFL) are being investigated. When it is important to draw a distinction between a very active disease course and a mild progression, prognostic CSF proteome analysis may alter the treatment options for MS patients. Currently, the most promising prognostic CSF indicators include protein chitinase 3-like1 (CHI3L1) and NFL. Increased CSF concentrations of oligoclonal IgM bands (OCMB) and protein 14-3-3 have been linked to MS and a worsening of disease progression .Huang et al. demonstrated the importance of T-cell activation in MS disease progression. Several chemokine receptor CXCR3 ligands, such as CXCL9/10/11, were shown to be overexpressed in the CSF of MS patients. This receptor is only expressed by Th1 cells and possibly certain regulatory T-cells. Researchers noted a higher infiltration of CCR5+ and CXCR3+ T-cells in active lesions, as well as higher production of the ligands MIP-1a and CXCL10. MIP-1a and CXCL10 levels in the CSF of MS patients are elevated. Several newly found CSF biomarkers, including CD6, IL7, and IL12B, as well as its component IL12A, have previously been related to inherited risk factors for various diseases. Many other CSF molecules, such as CHI3L1, IL-6, and CXCL13, have the potential to be used as clinical indicators; however, more research is needed to confirm their significance. The relevance of these proteins in MS is further supported by overlapping findings from proteomics and genetics .Another important autoimmune neurological condition is autoimmune encephalitis (AE). It is an immune-mediated CNS disease that is not infectious. It causes immune-mediated brain injury which can manifest in various ways, ranging from mild cognitive impairment and behavioral abnormalities to life-threatening epileptic seizures.Levraut et al. analyzed the CSF concentration of interleukins (ILs). In 20 AE patients, the researchers evaluated the CSF concentrations of each cytokine and compared them with ILs. Overall, 13 patients with CNS demyelinating disorders and 20 non-inflammatory controls had their IL-6 and IL-17A levels tested. The concentration of IL-17A in the CSF of AE patients was higher than in either of the control groups. The findings suggested that CSF IL-17A could be useful in determining the severity of AE at the outset. Consequently, CSF IL-17A could be a promising therapeutic target and an effective tool for evaluating early immunosuppressive therapy .Neurodegenerative disorders are becoming more common, and new biomarkers for diagnosis, prognosis, and therapy efficacy are urgently needed. Alzheimer’s disease (AD) is a progressive neurodegenerative illness marked by cognitive deterioration and the presence of amyloid plaques and neurofibrillary tangles. It is the most prevalent cause of dementia, which is defined as a loss of cognitive  and behavioral abilities to the point where they interfere with everyday life and activities. The protein composition of the CSF reflects structural and functional brain changes. Kinney et al. highlighted different CSF proteins in the disease course; TNF, IL-6, and TGF are involved in inflammation, disease progression, and neurological decline . In retrPrimary brain tumors are the most prevalent solid tumors and the main cause of cancer-related death in children under the age of 18 . ImproveThe efficiency of brain tumor treatment is dependent on early tumor diagnosis, thus it is critical to identify and implement biomarkers that can help. One such biomarker is miRNAs, which have been studied but require further research to optimize their role as biomarkers .Figure Figure Therefore, protein biomarkers in the CSF can be useful in not only diagnosing but also treating and monitoring reoccurring tumors, which can help oncologists and other physicians clinically, both in the pediatric and adult populations .Bruschi et al. compared CSF samples obtained from the extraventricular drainage (EVD) of 17 non-tumor individuals with CSF samples sourced from the EVD of 29 consecutive patients treated for common brain tumor types, encompassing both malignant and benign histopathologies, and discovered some possible protein biomarkers. The goal of the study was to use the large volume of CSF produced by EVD to look for predictive protein biomarkers for assessing the likelihood of a cancerous condition versus any other requiring an EVD, despite the presence of abnormal CSF production (controls). The study concluded that potential biomarkers could distinguish between different types of brain tumors to some extent .Apart from the miscellaneous conditions discussed above, CSF protein analysis may play a fundamental role in pediatric neuropsychiatric diseases such as autism. Because brain-specific proteins are not found in other tissues, non-invasive blood testing can be used to detect structurally and functionally damaged brain areas, as well as the severity and prognosis of neuropsychiatric disorders . For exaTo conclude the application of CSF protein in miscellaneous conditions, CSF protein evaluation plays an important role in more current events such as the coronavirus disease 2019 (COVID-19). In the study by Tandon et al., CSF immunoglobulin M (IgM) and a combination of severe acute respiratory syndrome coronavirus 2 serum and CSF reverse transcriptase-polymerase chain reaction (CSF RT-PCR) testing were explored in patients with COVID-19 who had neurological symptoms. CSF RT-PCR, CSF (IgM) tests, and CNS-targeting antibodies can be used together to guide diagnosis, treatment, and outcomes. The most prevalent finding of CSF analysis was an increase in CSF protein, which was occasionally accompanied by an increase in mainly lymphocytic cell count. CSF protein levels were shown to be increased in all stages of neurological disease . Other sOverall, treatment for cancer is increasingly being tailored based on tumor-specific genetic changes in the modern era of medicine. Next-generation sequencing (NGS) of tumor-derived circulating nucleic acids in CSF can help with precision treatment in the field of brain cancer. Baumgarten et al. examined if NGS from the CSF of neuro-oncologic patients could reliably detect tumor-specific genetic alterations and if it could assist clinicians in better managing patients with CNS cancer. In 23 of 27 patients, NGS proved successful (85%). In 20 out of 27 patients, 74% had genomic changes, and 11 (40%) were potentially actionable. Therefore, the study found that NGS from CSF fluid is practical in clinical practice and delivers therapeutically significant changes in a wide sample of patients. This method could be integrated into a cancer medicine program to enable patients with brain tumors to have more therapeutic alternatives .Table The use of a highly sensitive modality in the diagnosis of brain tumors, particularly meningioma, has been an important topic that many researchers, including the aforementioned researchers, are aiming to answer. The use of CSF protein analysis to diagnose meningioma early is one that merits further attention because it allows the diagnosis of asymptomatic meningioma patients, potentially giving patients a better prognosis.The current work focuses on a sensitive technique for diagnosing brain meningioma that enables early detection of the disease. As a result, the management can be changed. Although it is not extensively used by clinicians, it might be of utmost significance in the future.LimitationsEach type of study covered has its own set of limitations. The number of samples collected for analysis in studies that use CSF directly may be less than what is required for accurate and dependable results. However, this might be solved by using replication to obtain more samples, as shown in the study by Kim et al. The variety of studies employed in research primarily focused on studying literature may be limited, resulting in a limited amount of knowledge about the topic. Further, the majority of the studies included in this study concentrated on meningiomas and other types of primary brain tumors, leaving secondary brain tumors, which are also common. Another limitation is the limitation to articles that have been published in the last 15 years. Because many of the studies were based on previous research, this could result in a limited knowledge base as there may be older researches with results that could contribute significantly to the study.In conclusion, brain tumors are rampant in the population and their prognosis depends on early diagnosis which influences the treatment. The current diagnostic methods for brain tumors include the use of CT scans and MRI, with the latter being considered more effective. However, these diagnostic methods have been described as invasive, either with the use of contrast dye or related to other invasive procedures. The use of circulatory fluids such as CSF that come into direct contact with the brain to detect brain tumors has been under investigation for years, with promising results for future easier, faster, and less invasive techniques of diagnosing brain tumors. Meningiomas are a type of brain tumor that are classified into typical, atypical, and anaplastic types. CSF has been recently identified as a reliable source of protein biomarkers for brain tumors because it has a high protein content and comes into direct contact with the brain.Despite the favorable recommendations, the implementation of these procedures in the clinical setting has not attained the expected popularity. This is due to a lack of communication between researchers and oncologists and clinicians who interact with patients directly. There is also ongoing research on the application of this type of analysis to other types of brain tumors. Consequently, CSF proteomic analysis can be used as a diagnostic tool for brain tumors, which would be a significant contribution to health care by improving current diagnostic methods, which are invasive and time-consuming, and by increasing the likelihood of early detection of brain tumors, which would improve the prognosis and the quality of life for brain tumor patients.