Moreover, FL-M2 co-cultures displayed enhanced angiogenesis, dissemination, and immunosuppression. Analysis of the CSF-1/CSF-1R pathway uncovered that CSF-1 was significantly higher in serum from grade 3A FL patients, and that high CSF-1R expression in FL biopsies correlated with grade 3A, reduced overall survival and risk of transformation. Furthermore, CSF-1R inhibition with pexidartinib (PLX3397) preferentially affected M2-macrophage viability and polarization program disrupting FL-M2 positive crosstalk. In vivo CSF1-R inhibition caused M2 reduction and repolarization towards M1 macrophages and antitumor effect cooperating with anti-CD20 rituximab. In summary, these results support the role of macrophages in FL pathogenesis and indicate that CSF-1R may be a relevant prognostic factor and a novel therapeutic target cooperating with anti-CD20 immunotherapy.Microenvironment contributes to follicular lymphoma (FL) pathogenesis and impacts survival with macrophages playing a controversial role. In the present study, using FL primary samples and HK follicular dendritic cells (FDC) to mimic the germinal center, together with mouse models, we have analyzed the three-way crosstalk of FL-FDC-macrophages and derived therapeutic opportunities. Ex vivo primary FL-FDC co-cultures ( Follicular lymphoma (FL) is the most frequent indolent B-cell lymphoma, characterized by a variable clinical course. Several treatments lead to remissions, but the disease will eventually return, thus FL is still considered incurable . FL arisNext generation sequencing studies have deciphered the FL mutational landscape, highlighting the prominent relevance of genetic alterations affecting histone-modifying enzymes present in almost every FL patient . In addiBlockade of colony-stimulating factor-1 (CSF-1) or its receptor CSF-1R represents a selective approach to manipulate macrophages in cancer patients  and someHere, we have analyzed the dynamic interaction between FL and its myeloid microenvironment. Using primary FL-FDC co-cultures to mimic the GC ex vivo and in vivo, we have demonstrated that FL-FDC niche orchestrates the recruitment and differentiation of monocyte to macrophages and their polarization into M2-like tumor associated macrophages (TAMs), which support FL survival, angiogenesis, and dissemination. In addition, we have found a dual role for CSF-1R as a prognostic marker and therapeutic target with the potential to increase the therapeutic benefit of standard immunotherapy.FL-FDC co-cultures were established with HK cells, a non-immortalized FDC cell line generated from normal tonsils which was kindly provided by Dr. Yong Sung Choi  at a 1:2FL-macrophage co-cultures were established at a 1:4 ratio (Mϕ:FL). Macrophages were derived from peripheral blood mononuclear cells (PBMCs) of healthy donors . Blood samples were enriched in the monocyte population using RosetteSep (Human monocyte enrichment cocktail)  and then cultured for 7 days with 100 ng/mL CSF-1 . When specified, differentiated macrophages were polarized to M1  + 100 ng/mL LPS ) or M2 ) for 24 h.6 cells/well) with monocytes from healthy donors at a 1:4 ratio (Mϕ:FL) for 7 days in standard six-well plates. The percentage of attached macrophages (CD11b + cells) was analyzed by flow cytometry and related to the positive control of differentiation (monocytes + CSF-1). LIVE/DEAD™ Fixable Aqua (Thermo Fisher Scientific) was used as a viability probe. To evaluate macrophage polarization, the expression of CD86 (M1-marker) and CD163 (M2-marker) was then analyzed in attached live macrophages  by flow cytometry as described in supplemental methods. In another set of experiments, monocyte differentiation was analyzed by imaging and co-culturing FL primary cells with monocytes labeled with CellTrace™ Far Red (Thermo Fisher Scientific). The number of attached macrophages (FarRed+) was analyzed by cell counting using Cytation™ 1 Cell Imaging Multi-Mode Reader (BioTek).Monocyte differentiation induced by FL was assessed co-culturing FL cell lines or primary cells  and primary cells from FL patients (FL). Of note, a significant number of monocytes differentiated into macrophages in these co-cultures, and more prominently when primary FL cells were used Fig. . Using MWe next validated in vivo these in vitro results using an FL-HK co-xenograft generated with two different FL cell lines (RL and WSU-FSCCL). The histological evaluation of the tumors showed a subcutaneous mass comprised of medium and large lymphocytes, which a diffuse distribution pattern with some areas vaguely nodular and a variable presence of cells with macrophage and (follicular) dendritic shape. In some areas the tissue had undergone necrosis but most of the areas were viable. The lymphocytes were of medium and large size, with slightly irregular nuclear membrane and small nucleoli, and homogeneously positive for B-cell markers such as CD20 (RL) or the germinal center marker of CD10 (WSU-FSCCL). When comparing tumors with or without FDC cells, we observed that FDC HK cells support lymphomagenesis as indicated by the increase in the tumor size of FL-HK tumors compared to FL cell lines alone Figs.  and S1D,It is noteworthy that the FL-HK tumors were significantly more infiltrated with mouse macrophages than FL tumors, identified with the pan-macrophage marker F4/80 Fig. . In thesIn summary, FL-HK niche favors tumor growth and monocyte/macrophage recruitment and their polarization towards a clear M2-like protumoral phenotype.p = 0.0002) increased the viability of FL cells . These genes belong to fundamental biological cancer pathways as cell proliferation, movement, adhesion, cytokine signaling, and immune response ). Similarly, spleen size decreased in clodronate-treated mice  were intravenously inoculated Fig. . At the ice Fig. p < 0.001ice Fig. .Thus, macrophages contribute to FL tumor growth and dissemination, suggesting that these immune cells may constitute new targets to modulate FL microenvironment support.As we have shown above, CSF-1 increased in FL-FDC co-cultures Fig.  and may CSF1 and CSF1R expression were overrepresented in FL-LN compared to NTS . Both NTS Fig. . The speNTS Fig. . Next, wIn summary, we have identified a for the first time a correlation between CSF-1R macrophage expression with FL grade 3A, reduced OS and risk of transformation, justifying the possibility to modulate macrophage activity in FL, and highlighting CSF-1R pathway as a new target for therapy.We first determined the expression of CSF-1R by flow cytometry in M1 and M2 macrophages generated from PBMC of healthy donors. CSF-1R was detected both intracellular and on the surface of M1 and M2 macrophages, although it was more prominent in M2 macrophages, which also expressed a higher proportion of CSF-1R in their plasma membrane Fig. . We nextCSF-1R inhibition by PLX3397 totally impedes the differentiation of monocytes to macrophages Fig. . PLX3397Of note, CSF-1R inhibition hampered FL-M2 positive crosstalk whereas it did not affect the viability of lymphoma cells in monoculture Figs.  and S6E.In summary, CSF-1R inhibition preferentially affects M2-macrophage viability and polarization program and disrupts FL-M2 positive crosstalk.p < 0.001 control vs PLX3397; p < 0.001 control vs rituximab at endpoint; Fig. p < 0.01 control vs PLX3397; p < 0.01 control vs rituximab; Fig. p < 0.05 rituximab vs rituximab+PLX3397; p < 0.001 PLX3397 vs rituximab+PLX3397; Figs. We next sought to determine if anti-CSF-1R therapy could increase the efficacy of standard lymphoma immunotherapy (anti-CD20 rituximab). To achieve this aim, we set up an in vivo experiment using the RL cell line sensitive to rituximab  in the cIn summary, targeting the CSF-1R axis cooperates with rituximab in terms of inhibition of tumor growth, B-cell depletion, and M2 macrophage infiltration.high CD86low) and in vivo (CD206high iNOSlow), cooperating in providing long-term protumoral support. Moreover, and despite the known limitations of FL cell lines, we have been able to validate these in vitro findings with primary FL cells, in mouse co-xenografts of FL cell lines and HK cells.FL arises from developmentally blocked GC-B cells that retain phenotypic and functional features of normal counterparts and a follicular organization reminiscent of GC . MicroarMacrophages participate in tumor initiation as a part of anti-inflammatory response and are immune activated in this scenario . HoweverNevertheless, the prominent results obtained with clodronate, should be interpreted with caution, as macrophage depletion may not be the best approach. Macrophages have dual roles in cancer depending on the specific scenario, and show antitumor functions in some therapeutic settings, such as in antibody therapy via ADCC and ADCP  or in drn = 17), these results are meaningful because they are concordant with those reported in CLL, where CSF1 transcripts are significantly more abundant in progressive CLL patients [+ TAMs correlate with reduced OS, similar to results found in HL using the same CSF-1R antibody [We first acknowledged that the mRNA expression of both the ligand and the receptor were enriched in FL-LN compared to normal tonsils. Additionally, we found increased levels of CSF-1 in the serum of grade 3A FL patients. Although the number of patient serum available was low  from the initiation of treatment onwards, arguing for a stabilization of the disease.Regarding CSF-1R/CSF-1 axis as a target for therapy, we have demonstrated that CSF-1R inhibition by PLX3397 affected more M2 than M1 macrophages, and induces their repolarization towards an M1-like phenotype, in accord with published results in glioma , 57. MorIn conclusion, our results support the role of M2 macrophages in FL pathogenesis and suggest that therapies manipulating FL-M2 crosstalk may be a new strategy for those patients with high macrophage infiltration, especially in combination with anti-B-cell therapies.Valero JG.Supplemental material


EGFR was highly enriched in CSF (87% of samples). Compared with the other primary lung tissues, copy number gain of EGFR (27%), CDK4 (11%), PMS2 (11%), MET (10%), IL7R (8%), RICTOR (7%), FLT4 (5%), and FGFR4 (4%), and copy number loss of CDKN2A (28%) and CDKN2B (18%) were remarkably more frequent in CSF samples. CSF had significantly lower tumor mutation burden (TMB) level but more abundant copy number variant. It was also found that the relationships among co-occurrent and mutually exclusive genes were dynamically changing with LUAD development. Additionally, CSF (97% of samples) harbored more abundant targeted drugs related driver and fusion genes. The signature 15 associated with defective DNA mismatch repair (dMMR) was only identified in the CSF group. Cancer associated pathway analysis further revealed that ErbB (95%) and cell cycle (84%) were unique pathways in CSF samples. The tumor evolution analysis showed that CSF carried significantly fewer clusters, but subclonal proportion of EGFR was remarkably increased with tumor progression. Collectively, CSF sequencing showed unique genomic characteristics and the intense copy number instability associated with cell cycle disorder and dMMR might be the crucial genetic factors in BM of LUAD.Lung adenocarcinoma (LUAD) is most common pathological type of lung cancer. LUAD with brain metastases (BMs) usually have poor prognosis. To identify the potential genetic factors associated with BM, a genomic comparison for BM cerebrospinal fluid (CSF) and primary lung tumor samples obtained from 1082 early- and late-stage LUAD patients was performed. We found that single nucleotide variation (SNV) of  Lung adenocarcinoma (LUAD) is the most common histologic subtype in non-small cell lung cancers (NSCLC) and accounts for more than 38.5% of all lung cancers . Brain mEGFR genotyping of LUAD patients with BM [EGFR mutations  in patients with leptomeningeal metastasis  than brain parenchymal metastasis  [et al. discovered that the mutation ratio of EGFR in LM (81.8%) was higher than BPM (30%) in NSCLC patients with BM, and the status of EGFR mutation was consistent between CSF ctDNA and brain lesion tissue in five patients after surgical resection [Cerebrospinal fluid (CSF), containing cell-free DNA (cfDNA), had been considered as a vital liquid biopsy medium for lung cancer, which provides a less-invasive and routinely accessible method to dynamically acquire genomic information of BM patients in lung cancer . Previou with BM , and revPM, 10%) . Similaresection . Additioesection  and Osimesection . These sAlthough amounts of genes in CSF had been identified in previous researches, these studies have a limited the number of samples, and mainly focus on exploring the difference between BM lesions and primary lung tumor in advanced NSCLC, lacking of early-stage genomic comparison. Therefore, a systematic analysis of a large sample size was needed to further explore genetic alterations in the development of lung cancer. In this study, a 1021 cancer-related panel was used to detect gene mutations of 1082 unmatched samples from Chinese LUAD patients, including 135 CSF with BM (CSF group), 363 early-stage lung tumors (ESLT group), 396 late-stage lung tumors without BM (LSLT-noBM group), and 188 late-stage lung tumors with BM (LSLT-BM group) samples. To seek risk factors associated with metastasis by comparing genetic profiles between CSF and different stage primary lung tumors, thereby providing potential prognostic markers and therapeutic targets for LUAD with BMs, finally explaining the development of LUAD from a genetic perspective.A 1021 panel sequencing was performed on CSF, ESLT, LSLT-noBM, and LSLT-BM groups with the average depth of 1583, 1230, 1271, and 1254×, respectively. Tumor mutation burden (TMB) and somatic copy number variation (CNV) (including amplification and deletion) count were assessed. The median TMB of CSF was 4 mutations/Mb, which was remarkably lower than the other groups  Fig. . The medEGFR was the most prevalent gene in all groups, accounting for 87% in CSF, 51% in ESLT, 64% in LSLT-noBM, and 67% in the LSLT-BM. Thus, the variation characteristics of EGFR mutant patients were further analyzed. The median TMB of EGFR mutant CSF (EGFR-CSF) and ESLT (EGFR-ESLT) samples was 5 mutations/Mb, which was lower than EGFR-LSLT-noBM (6 mutations/Mb) and EGFR-LSLT-BM samples (6 mutations/Mb)  Fig. . CNV anaups Fig. . These aEGFR, TP53, and CTNNB1, and fewer RBM10, KRAS, SMARCA4, KEAP1, FAT1, and STK11 than ESLT group. The significantly differential genes in CSF and LSLT-noBM groups were EGFR, CTNNB1, RBM10, KRAS, and FAT1. Gene EGFR, KRAS, KEAP1, FAT1, and STK11 were significantly different between CSF and LSLT-BM group. Moreover, EGFR was enriched and FAT1 was rare in CSF compared to the other three groups , LSLT-noBM , and LSLT-BM group , suggesting that the co-mutation of EGFR and CTNNB1 might be associated with BM event.Genomic landscape of single nucleotide variation (SNV) for each group was shown in Fig. ups Fig. . AdditioEGFR-mutant patients , PIK3CA-MLL (1.5%), APC-NF1 (2.2%), APC-BRCA2 (2.2%), BRCA2-NF1 (1.5%), RB1-MLL3 (1.5%), CDKN2A-KRAS (0.7%), and PTCH1-KEAP1 (0.7%), reminding their possible relation with BM event. Genes that were specifically mutually exclusive in the ESLT group included EGFR and PTCH1/BRAC2/MLL2/CDKN2A, TP53 and CTNNB1/KRAS/RBM10, and KRAS and ERBB2. The mutual exclusion between TP53 and STK11 only appeared in the LSLT-noBM group. The mutually exclusive mutations that only exist in the LSLT-BM group are EGFR and ARID1A. The above results indicated that the relationships among genes were dynamically changing with LUAD development.Co-occurrence and mutually exclusivity analyses among mutant genes showed that les Fig. . NeverthA somatic mutational signature analysis was performed to describe which internal boundary or external environmental factors in the development of LUAD BM represents the most important carcinogenic factors. Signature 1 (correlates with the age of cancer diagnosis), signature 3 (associated with failure of DNA double-strand break repair by homologous recombination), and signature 13 (attributed to the activity of AID/APOBEC family cytidine deaminases) were mutual signatures in both early and late stages LUAD. Notably, signatures 5 and 15 were only identified in the CSF group Fig. . AlthougFANCF (6%) and FLT4 (5%) and deletion of CD274 (4%). Besides, the deletion of CDKN2A and CDKN2B, and the amplification of EGFR, CDK4, PMS2, MET, IL7R, RICTOR, FGFR4 were the most frequently observed CNV events in CSF compared with the other groups. Except for the high-frequency genes in CSF mentioned above, the significantly differential genes in CSF and ESLT group also included SDHA, FOXA1, NKX2-1, IFNG, RB1, and AXIN1. The significantly differential genes in CSF and LSLT-noBM group also included SDHA, IFNG, RB1, and AXIN1, while the significantly differential genes between CSF and LSLT-BM group were same as high-frequency genes in CSF.Next, we analyzed CNV features Fig. . Fifty  were enriched in CFS, including PI3K-Akt, Rap1, FoxO, Ras, ErbB, cell cycle, JAK-STAT, p53, mTOR, and AMPK pathways. Among them, only ErbB, cell cycle, JAK-STAT, mTOR, and AMPK pathways were remarkably enrolled in the CSF  and LSLT-noBM group (P = 0.007), excluding LSLT-BM (P = 0.091) group. Furthermore, EGFR L858R, T790M, exon 19 deletion (19del), C797S, exon 20 insertion, and L861Q were identified in all groups. Besides, other rare EGFR mutations L62R, L718X, and V834L also were identified in CSF and lung tissue samples, but only L792H, G873E, and H850Y mutated in CSF. PIK3CA, ERBB2, and KRAS were similar in all groups. These results showed that CSF could be used to discover actionable drug-targets in driver genes.We examined the driver genes including P = 0.0002), LSLT-noBM (P < 0.0001), and LSLT-BM (P < 0.0001) group , 2952 mutation clusters in the ESLT group with a median of 6 per sample , 3091 mutation clusters in the LSLT-noBM group with a median of 6 per sample , and 1615 mutation clusters in LSLT-BM group with a median of 6 per sample . CSF carried significantly fewer clusters than ESLT  resistance in NSCLC with BMs [FANCF and FLT4 and the deletion of CD274 only were identified in CSF samples. Studies had shown that the overexpression of FANCF and FLT4 resulted in proliferation, migration, and invasion of cancer cells [CD274 was most prevalent and frequent in NSCLC, which influenced its expression levels, was associated with dismal prognosis [Somatic mutations and copy number alterations possessed intratumor heterogeneity, and genome doubling and continuous dynamic chromosomal instability were related to intratumor heterogeneity, leading to parallel evolution of driver somatic copy-number alterations . In our ype EGFR . Also, tl in CSF , 25. PMSigenesis , and METwith BMs . Moreoveer cells , 29. Delrognosis . In geneERCC2 mutations in urothelial tumors [Somatic mutations found that cancer genomes may be the consequence of the intrinsic slight infidelity of the DNA replication machinery, exogenous or endogenous mutagen exposures, enzymatic modification of DNA, or defective DNA repair . Furtherl tumors , which ml tumors . CollectEGFR targeted therapy dramatically improved the prognosis of patients with NSCLC BMs [CDKN2A copy number loss) [EGFR subclones [EGFR mutant NSCLC patients under TKIs treatment. In our study, CDKN2A loss and EGFR subclone number were frequently increased in EGFR-CSF patients, which might be one of the reasons for the poor prognosis of LUAD patients with BM. In immune checkpoint inhibitors (ICIs) monotherapy, EGFR mutation patients did not respond better than chemotherapy [EGFR aberrations had been considered as a risk factor for hyper-progress in immunotherapy [MET amplification seemed close to that observed in unselected patients [EGFR and MET generally occurred in CSF samples, suggesting patients with BMs might be more not suitable for immune monotherapy.At present, several studies had confirmed that SCLC BMs , 37. Hower loss)  and the ubclones  are assootherapy , 41, andotherapy . ICI effpatients . Here, ECDK4 was remarkably amplified in CSF, supporting that the CDK4 inhibitors combined with radiation might be a promising therapeutic option for LUAD patients with BM. ICIs against programmed death-1 (PD-1) and cytotoxic T lymphocyte antigen-4 (CTLA-4) proteins were highly effective in advanced dMMR metastatic colorectal cancer, especially combination therapy provided improved efficacy relative to anti-PD-1 monotherapy [Genomic comparison of CSF and primary lung lesions at different stages also revealed several other possible therapeutical approaches for LUAD with BM. CDK4/CDK6 inhibitor abemaciclib had been verified to enhance radiosensitivity of NSCLC in vitro and in vivo . CDK4 waotherapy , 46. We otherapy , and divotherapy . Thus, eTo conclude, a multiple perspective analysis about molecular characteristics between CSF and different stages primary tumor tissues in LUAD patients revealed that CNV might be greater than the contribution of SNV to BM. ErbB pathway alterations, and the intense copy number instability associated with cell cycle disorder and dMMR might be the crucial genetic factors in BM of LUAD.EGFR prevalence of patients were summarized in Table Genomic and clinical information of 1082 LUAD patients with or without BM were analyzed, including 135 (12%) IV stage CSF with BM patients (CSF group), 363 (34%) I–III stage lung tissue patients (ESLT group), 396 (37%) IV stage lung tissue without BM patients (LSLT-noBM group), and 188 (17%) IV stage lung tissue with BM patients (LSLT-BM group). The clinical characteristics and mutated TM Cell-Free DNA Isolation Kit . Lung tumor tissue DNA was extracted using Maxwell® 16 FFPE Plus LEV DNA Purification Kit . PBL DNA was extracted using the DNeasy Blood & Tissue Kit . DNA concentration was measured using a Qubit fluorometer and the Qubit dsDNA HS (High Sensitivity) Assay Kit .CSF cfDNA was isolated using MagMAXMethods for sequencing libraries was previously described . DNA extSequence analysis used BWA   was used to identify mutational signatures within a single tumor sample based on a negative matrix factorization (NMF) algorithm , which rhttps://david.ncifcrf.gov/tools.jsp) was performed to identify significant gene clusters from SNVs and CNVs via annotating KEGG pathways, and 1021 cancer-related genes was considered as background gene set. The mutational frequency of pathways was obtained by computing the fraction of samples with at least one alteration in the corresponding pathway [An online analysis tool DAVID . The Fisher’s exact test was used to compare proportions between two groups. Mann–Whitney test was used for CNV count of per sample and TMB comparison among different groups. All statistical tests were two-sided, and the result with FigS1FigS2Supplementary figure legends


Treponema IgG by enzyme-linked immunosorbent assay (ELISA) for NS diagnosis. We conducted a retroprospective monocentric study including adults with neurological symptoms who had serum and cerebral spinal fluid (CSF) samples collected between 2006 and 2021. Two NS definitions were used. NS1 included patients with neurological symptoms, positive Treponema pallidum particle agglutination (TPPA) serology, and CSF-TPPA of ≥320, as well as CSF-leukocytes of >5 cells/mm3 and/or CSF-protein of >0.45 g/L and/or a reactive CSF-VDRL/RPR test. NS2 included patients with acute ocular and/or otologic symptoms, positive TPPA serology, and a response to NS treatment. Controls were patients with central nervous system disorders other than neurosyphilis. Anti-Treponema pallidum IgG were measured simultaneously in serum and CSF, and AI was calculated according to Reiber diagram. We assessed the AI test area under the curve (AUC), sensitivity/specificity, and estimated positive and negative predictive values. In total, 16 NS1 patients, 11 NS2 patients, and 71 controls were included. With an AI of ≥1.7 as a positive test for NS diagnostic, specificity was 98.6%  and sensitivity was 81.3% (CI 95% of 54.4 to 96.0) for NS1 and 98.6% (CI 95% 92.4 to 100.0) and 27.3% (CI 95% 6.0 to 61.0), respectively, for NS2. Positive and negative predictive values were >95% for NS1 and >85% for NS2, for prevalence above and below 20%. Measuring an AI for intrathecal synthesis of specific anti-Treponema pallidum IgG is a new promising tool highly specific for NS diagnosis.Neurosyphilis (NS) diagnosis is challenging because clinical signs are diverse and unspecific, and a sensitive and specific laboratory test is lacking. We tested the performance of an antibody index (AI) for intrathecal synthesis of specific anti-IMPORTANCE In the context of a lack of a gold standard for the diagnosis of neurosyphilis due to either nonspecific or nonsensitive tests, we present in this article a new promising tool highly specific for NS diagnosis. This new test involves measuring an intrathecal synthesis index of specific anti-Treponema IgG by ELISA. Treponema pallidum, occurs worldwide with a steadily increasing incidence. Neurosyphilis (NS) rate is thus expected to rise  in CSF are indicative of an inflammatory reaction in the CSF, but they are not specific markers for NS (Treponema test (VDRL/rapid plasma reagin [RPR]) in CSF associated with neurological symptoms is commonly used as diagnostic for NS, it is only 30% sensitive /Treponema pallidum particle agglutination (TPPA) in CSF are sensitive but suffer a poor specificity owing to the passive transfer of immunoglobulins across the blood-CSF barrier  by demonstrating a production of local pathogen-specific antibodies. Normally, blood-brain barriers (BBB) restrict leakage of systemic antibodies into CSF, but their function changes in the presence of inflammation. Therefore, to discriminate between locally produced antibodies and systemic antibodies, a correction is needed to take the blood-CSF barrier function into account and correct for polyspecific antibody production in the brain.In the current diagnostic study, we assessed the diagnostic performance and the clinical utility of measuring an antibody index (AI) for intrathecal synthesis of specific anti-P < 0.001). Neurological, ocular, or hearing symptoms or a combination of symptoms were found, respectively, in 44.4%  and the type III (n = 6) oligoclonal bands (OCB) but was restricted to NS1  of 0.96 (CI 95% of 0.91 to 1.00) . AI specFor NS2, AUC was 0.88 (CI 95% of 0.81 to 0.96). AI specificity was still high at 98.6% (CI 95% of 92.4 to 100.0), but sensitivity was reduced to 27.3% (CI 95% of 6.0 to 61.0). Positive and negative predictive values were >95% for a prevalence above 50% and below 6%, respectively.In NS1 patients, 13/16 had a positive intrathecal synthesis (type II or III OCB). All positive CSF-OCB correlated with a positive specific AI, except in one case with positive CSF-OCB and a negative AI (<1.7), but this intrathecal immune reaction may be triggered by its HIV coinfection.Among controls, 34/71 (47.9%) had a positive syphilitic serology (TPHA/TPPA of ≥80), but they did not meet the criteria for NS. All patients with other neurological pathologies and a nonreactive treponemal antibody test in the serum had a negative AI (<1.7), except one.Treponema pallidum test and CSF abnormalities. In this study, we investigated the diagnostic performance of CSF-Treponema pallidum-specific antibody production by measuring an AI in NS patients classified according to two “gold” standard definitions combining clinical and biological manifestations.The diagnosis of NS is still an issue and relies on a combination of neurological manifestations in patients with a reactive serological anti-In NS1, we found that AI had a high sensitivity and specificity as well as good positive and negative predictive values in the studied population. Although the number of cases included is limited, we believe that this test performs better than others and can be used to diagnose NS alone. Indeed, it reflects a specific intrathecal synthesis by taking into account a possible dysfunction of the BBB, which is not the case when using treponemal tests in the CSF alone, as they can be positive when their blood titer is high or when there is a rupture of the BBB . FurtherTreponema pallidum tests suffer from low sensitivity in ocular and otologic NS . Among the 11 cases analyzed, 3 had no CSF abnormalities, 5 had only high CSF-WBC, and 3 had a CSF-WBC and elevated CSF-protein, but none had a CSF-TPPA of ≥320. For those patients, our AI appears to be helpful to confirm NS diagnosis if positive, but it cannot be used to exclude the diagnosis. This is consistent with a previous study reporting that CSF-logic NS . It is lstrategy , we raisThis study has several limitations. First, our initial NS1 definition including a CSF-TPPA above or equal to 320 can lead to underdiagnosis of NS. Indeed, it is known that a CSF-TPPA of ≥320 has a low sensitivity , and thTreponema pallidum IgG. However, in otologic or ocular syphilis, with few or no CSF abnormalities, AI is not sufficient to rule out the diagnosis of NS, and further investigations should focus on those cases.Our data suggest that the lack of specificity of the current NS diagnostic criteria could be improved by intrathecal synthesis index of specific anti-We conducted a monocentric diagnostic study with retrospective and prospective recruitment between July 2007 and May 2021 at Geneva University Hospitals, Switzerland.The study protocol was approved by the Ethics Committee of the Swiss Cantonal Research Ethics Commission (study number 2019-00232).Eligible patients were all adults presenting neurological and neurosensory signs , with serum and CSF sampled less than 3 days apart. CSF samples with red blood cells (RBC) of >200 M/L and albumin quotient of >20 were considered contaminated by blood and were thus further excluded.Cases were patients with NS defined on the basis of composite criteria both clinical  and biological according to European and American guidelines (reference definitions) , 7, 8.3 and/or CSF-protein of >0.45 g/L and/or a reactive CSF-VDRL/RPR test.Our first composite definition (NS1) includes the following: (i) neurological symptoms or signs consistent with NS, (ii) a positive TPHA/TPPA serology (TPHA/PPA ≥80) and a positive CSF-TPHA/TPPA test of ≥320 , 22, 27,CSF examination being normal in 90% of otosyphilis cases and in 30 to 40% of ocular syphilis cases , 28, we Controls were patients diagnosed with any other CNS pathologies: infections (neuroborreliosis), inflammatory diseases , dementia, stroke, acute psychiatric disorders, etc. They could have a positive TPHA/TPPA serology (≥80).Patient charts were reviewed and the following data were collected in an online case report form using the Research Electronic Data Capture (REDCap) software: age, gender, neurological symptoms , blood parameters , and CSF results .Albumin and total IgG determination was performed using Atellica Neph630 . TPPA, TPHA, RPR, and VDRL titrations were performed manually using reagents from Fujirebio, Siemens, Human diagnostic, and Bio-Rad, respectively.3) and evaluating the proportion of different cell types after CSF cytocentrifugation and May-Gründwald Giemsa staining. The BBB integrity was evaluated according to Reiber method . Serial dilutions of CSF (1/6 and 1/36) and serum  were performed and analyzed with DSX Dynex automate to obtain an optical density (OD) value within the linear range of the test. Only OD values within the linear range of the test were chosen to quantify anti-Treponema IgG. Assays were performed by batch between 2018 and 2021.All matched CSF/serum samples were tested in the same series for anti-LimIgG, with QLimIgG = 0.93 × −3, Q-trep IgG = 1,000 × (CSF-trep IgGRLU/mL/serum-trep IgGRLU/mL), Q-total IgG = CSF-IgGmg/L/serum-IgGg/L. Reiber’s diagrams for NS1 and NS2 can be found in Fig. S1 in the supplemental material. According to Reiber, a cutoff value of 1.4 should be used, whereas others suggested a cutoff value of 2 or 3 to demonstrate local pathogen-specific antibody production in CSF  and controls: continuous variables were presented by mean (± standard deviation [SD]) and median (interquartile range), and categorical variables were represented by frequencies and relative percentages. We compared data (age) between NS1 and NS2 and then across the three groups  using Mann-Whitney nonparametric test. We compared HIV status and sex between NS1 and NS2 and then across the three groups  using either chi-squared or Fisher’s exact test, depending on the application conditions.−1 ratios were both above 10, moderately useful when they were between 5 and 10, and poorly useful when they were between 2 and 5.We estimated the area under the curve (AUC) of the AI test with its 95% confidence interval (95% CI) for the diagnosis of NS according to definitions 1 (NS1) and 2 (NS2). We reported the optimal threshold of the AI test that maximized the number of well-classified subjects; we reported the sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratios, and their 95% CIs using this optimal cutoff. The interpretation of the AI test utility was judged to be highly useful when positive and (negative likelihood)Raw data can be shared upon request.


A classification tree was used to analyze background factors for granulocyte colony-stimulating factor (G-CSF) preparation selection for febrile neutropenia (FN) prophylaxis in Japanese patients with non-Hodgkin B-cell lymphoma receiving the first R-CHOP cycle.This was a subanalysis of the retrospective observational study STOP FN in NHL 2 (UMIN000029534). Patient characteristics, changes in neutrophil count, incidence and severity of neutropenia, and risk factors for dose reduction/delay of R-CHOP were assessed by G-CSF formulation.Among 234 patients in cycle 1, 25.6% received no G-CSF preparation, 52.1% received daily G-CSF, and 22.2% received pegfilgrastim. Pegfilgrastim use was most frequent among patients aged ≥ 80 years, while that of daily G-CSF was most frequent in patients with lymphocyte count (LC) < 1000 cells/μL. Changes in neutrophil count were more marked with pegfilgrastim compared with daily G-CSF and no G-CSF. Relevant factors for G-CSF preparation selection in the first R-CHOP cycle were age ≥ 80 years and LC < 1000 cells/μL; for chemotherapy dose reduction were FN onset in cycle 1 and female sex; and for dose delay was hemoglobin (< 12 g/dL). After cycle 2 and onward, pegfilgrastim use increased markedly (72.6%) compared with cycle 1 (22.2%), with significantly greater proportions continuing pegfilgrastim use and switching from daily G-CSF.Relevant factors for G-CSF preparation selection were age ≥ 80 years and LC < 1000 cells/μL. The use of pegfilgrastim increased markedly after cycle 2. These results may be useful for selecting appropriate G-CSF preparations in the first R-CHOP cycle.https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000033733.UMIN000029534; registered on 13 October 2017, The online version contains supplementary material available at 10.1186/s12885-021-08068-0. Febrile neutropenia (FN) is the most common and severe complication of patients receiving chemotherapy for cancer . DevelopIn the past decade, the prophylactic use of granulocyte colony-stimulating factor (G-CSF) to mitigate the development of FN has become more frequent and has led to a reduced incidence of FN and a corresponding decrease in the number of patients experiencing chemotherapy dose reductions , 9.We reported that Japanese patients treated with two types of G-CSF preparations, daily G-CSF and pegfilgrastim, had incidences of FN of 7.3 and 3.7%, respectively, while patients who did not receive any G-CSF preparation had an incidence of FN of 23.0% . ClinicaStudies have shown that prophylaxis with daily G-CSF or pegfilgrastim can reduce the incidence of FN in patients receiving R-CHOP therapy , 17, 18.Furthermore, although it is known that the onset of FN in cycle 1 brings about dose reduction and delay of chemotherapy in subsequent cycles, no previous studies in Japan have reported on risk factors that may cause R-CHOP dose modification. In this study, a classification tree was used to analyze background factors for drug selection by G-CSF preparation in the first R-CHOP cycle. Additionally, we analyzed FN incidence, patterns of G-CSF preparation and identified possible predictors of dose reduction and delay of R-CHOP therapy among Japanese patients with B-NHL receiving chemotherapy in a real-world setting.This was a subanalysis of a retrospective observational study conducted between January 2015 and June 2017 at the Cancer Institute Hospital of the Japanese Foundation for Cancer Research, Tokyo, Japan.Full details of the STOP FN in NHL 2 study (UMIN000029534) have been published . In brieThe study was approved by the institutional Ethical Review Board. The analysis was conducted in accordance with the Declaration of Helsinki and Ethical Guidelines for Medical Research on Individuals, the Law Concerning the Protection of Personal Information, and all other applicable laws and regulations concerning the handling of personal information. As this was a retrospective observational study on data from medical records, the need for informed consent from patients was waived.Patients diagnosed with B-NHL who had received and completed R-CHOP regimens, or patients who had discontinued the R-CHOP treatment after receiving three or more scheduled cycles were eligible for inclusion in this study. The main exclusion criterion was a diagnosis of human immunodeficiency virus-related B-NHL.For this subanalysis, patients were divided into three groups according to the type of FN prophylaxis received in cycle 1: those who did not receive any prophylactic G-CSF preparation (no G-CSF administration); those who received daily G-CSF; and those who received FN prophylaxis with 3.6 mg pegfilgrastim by subcutaneous administration.Characteristics of patients prior to the initial R-CHOP regimen were collected for evaluation, including age, sex, performance status (PS), body mass index (BMI), disease characteristics , presence or absence of complications , presence or absence of previous history (last [< 1 month prior to initiation of (R) CHOP regimens] infection or FN), and hematologic parameters .Data related to each chemotherapy cycle, including details related to the prescribed R-CHOP regimen , were collated for analysis. Dose reduction was defined as more than a 20% reduction in the dose of cyclophosphamide or doxorubicin in each cycle after cycle 2 compared with that in the first cycle. Dose delay was defined as more than 7 days behind schedule in each cycle after cycle 2.Information related to the development of FN after completion of the R-CHOP regimen was also analyzed, including body temperature, neutrophil counts, preventive and therapeutic measures , and hospitalizations for the development of FN. For hospitalized patients, temperature measurements were taken daily and prior to initiation of the R-CHOP regimen and blood samples were collected every 2 days, wherever possible. For outpatients, temperature measurement was self-reported by the patient.The specific outcomes assessed during the study were: identification of patient background factors that may aid in the selection of G-CSF preparation (pegfilgrastim or daily G-CSF); changes in neutrophil count in cycle 1 by G-CSF preparation; duration (number of days) of neutropenia (< 500 cells/μL), the incidence of grade 4 neutropenia (< 500 cells/μL), lowest neutrophil count (neutrophil nadir), and day of onset of the neutrophil nadir, all by type of G-CSF preparation; identification of risk factors that may cause dose reduction or delay of R-CHOP therapy after cycle 2; and actual status of treatment with pegfilgrastim, daily G-CSF, or no G-CSF after cycle 2.A target sample size for this analysis was not statistically calculated, and all patients identified in the database who met the study eligibility criteria during the study period were included. A classification tree was used to analyze 15 background factors for drug selection by G-CSF preparation in the first R-CHOP cycle . SupplemThe STOP FN in NHL 2 study  enrolledTable ). The classification path of the tree analysis was as follows: age , lymphocyte count , age (< 65 and 65–79 years), and neutrophil count (< 4235 and ≥ 4235 cells/μL), with those who had neutrophil count < 4235 cells/μL being further categorized based on BMI (< 23 and ≥ 23 kg/m2). The most frequent use of pegfilgrastim was in patients aged ≥ 80 years. Conversely, the frequency of daily G-CSF use was greatest in patients aged ≤ 79 years and with lymphocyte counts of < 1000 cells/μL, and these patients were also found to have the highest FN rate. This classification tree analysis showed that the most relevant factors for selecting a G-CSF preparation in the first R-CHOP cycle were age ≥ 80 years and lymphocyte count < 1000 cells/μL, followed by neutrophil count < 4235 cells/μL and BMI < 23 kg/m2.As one of the specific objectives of this subanalysis was to identify the patient background factors that may aid in the selection of G-CSF preparations (pegfilgrastim or daily G-CSF), we developed a classification tree using the background factors of patients undergoing the first R-CHOP cycle as explanatory variables to predict the FN incidence rates at that time Fig. . The claWe evaluated whether receiving no G-CSF preparation or receiving daily G-CSF or pegfilgrastim during cycle 1 would have any effect on neutrophil count Fig. . Over ti). Of note, the duration of neutropenia and incidence of grade 4 neutropenia were significantly higher in the no G-CSF administration group  and the daily G-CSF group (p < 0.0001 for both) compared with the pegfilgrastim group. The neutrophil nadir was significantly lower in the no G-CSF group and the daily G-CSF group (p < 0.0001 for both) compared with the pegfilgrastim group. The day of onset of the minimum neutrophil count was significantly later for patients in the no G-CSF and daily G-CSF groups (p < 0.0001 for both) compared with the pegfilgrastim group. Although patients receiving pegfilgrastim had a significantly earlier onset of neutrophil nadir, they had a significantly shorter neutropenia duration, a lower incidence of grade 4 neutropenia, and a significantly higher neutrophil count nadir compared with patients treated with daily G-CSF and those who did not receive any G-CSF preparation.We also compared the duration of neutropenia (< 500 cells/μL), the incidence of grade 4 neutropenia (< 500 cells/μL), lowest neutrophil count , and day of onset of the neutrophil nadir by type of G-CSF preparation Fig. . Of noteWe compared the dose reductions and dose delays after cycle 2 by no administration of G-CSF preparation, daily G-CSF, and pegfilgrastim groups . Among patients receiving daily G-CSF during cycle 1, 81.9% switched to pegfilgrastim (p < 0.0001), while 14.8% remained on daily G-CSF. Those who received no treatment had the highest incidence of FN (23.3%) during cycle 1, and slightly more than half switched to pegfilgrastim   after cycle 2 and onward Fig. . OverallThis subanalysis of the retrospective STOP FN in NHL 2 study clarified the patient background factors that may aid in selecting a G-CSF preparation to prevent FN during chemotherapy. Additionally, we clarified possible predictors of dose reductions or delays among patients receiving R-CHOP and analyzed the current treatment patterns of G-CSF preparations in this population. These findings are relevant as these aspects of G-CSF prophylaxis have not been evaluated in Japan thus far.In this analysis, daily G-CSF and pegfilgrastim were administered to patients primarily based on age and lymphocyte count. The more frequent use of pegfilgrastim in patients aged ≥ 80 years was likely due to clinicians adhering to the Japanese treatment guidelines , 16, andOf note, older Japanese patients seemed to be receiving G-CSF preparations as recommended in the local guidelines. In contrast, studies in patients in other countries indicate that G-CSF prophylaxis was generally suboptimal –22, withIn this study, the lymphocyte count (< 1000 cells/μL at baseline before treatment) was more relevant than neutrophil count, according to the classification tree analysis. The lymphocyte count may be an important detail in future decision-making as, generally, the neutrophil count is the standard measure to assess G-CSF administration in current clinical practice. Other studies have also reported that the lymphocyte count was relevant in determining FN risk and was required for G-CSF administration , 25. A pIn this study, patients with low BMI had a higher daily G-CSF administration rate. Lean patients and those with worse nutritional status are considered to be at a high risk of developing FN , 27 and Regarding changes in neutrophil count in cycle 1, there was no significant difference in the progression of neutrophil counts between patients who did not receive G-CSF and those who received daily G-CSF. This finding may be attributed to the administration timing. We reported that, in cycle 1, only a small percentage of patients (2.4%) started daily G-CSF treatment on days 0 to 3, with a mean of approximately 10 days for starting daily G-CSF treatment. In contrast, most patients (90.7%) treated with pegfilgrastim started treatment between days 0 and 3, with a mean of approximately 3 days for starting pegfilgrastim treatment. Further, the mean dosing period of daily G-CSF was approximately 3 days. Thus, it is possible that daily G-CSF was administered late during the cycle, or the dosing period was too short [Although it is well known that the onset of FN during a treatment cycle can lead to chemotherapy dose delays or reductions, few studies have reported on the factors that may contribute to these dose modifications among patients with B-NHL undergoing FN prophylaxis with G-CSF. In our study, FN onset in cycle 1 and female sex were identified as risk factors for chemotherapy dose reduction, and hemoglobin (< 12 g/dL) as a risk factor for subsequent treatment delay. Taken together, current findings detailing the risk factors for FN, G-CSF treatment outcomes, and chemotherapy modification may help to inform future therapeutic decisions and improve patient health status and survival.p < 0.0001, each). Consequently, the proportions of patients receiving daily G-CSF and no treatment decreased considerably. In Japan, while the first cycle of chemotherapy is administered on an inpatient basis, the subsequent cycles are handled in an outpatient manner. Pegfilgrastim is administered subcutaneously using a prefilled syringe, and only needs to be administered once per cycle. Thus, the prescription of pegfilgrastim may have increased from cycle 2 because of its convenience for administration.Additional analyses were performed to evaluate the status of G-CSF preparation use after cycle 2. Overall, the proportions of patients receiving pegfilgrastim during cycle 2 and onward increased by more than 3-fold. Among patients who developed FN in cycle 1, pegfilgrastim use increased from 8.0 to 92.0% in cycle 2 and onward. A significantly greater proportion of patients continued to use pegfilgrastim or switched from daily G-CSF to pegfilgrastim . However, this analysis is based on the data from the first chemotherapy cycle, which is administered on an inpatient basis in Japan. This particularity could signify accurate assessments and patient evaluations in a real-world setting.In this analysis of Japanese patients diagnosed with B-NHL, who received at least three cycles of R-CHOP treatment, a classification tree analysis was performed to assess the use and type of prophylactic G-CSF preparation. This analysis showed that a lymphocyte count < 1000 cells/μL at baseline was associated with more frequent administration of daily G-CSF compared with pegfilgrastim, and that these patients had a high FN rate despite daily G-CSF treatment.For the first time, we have clarified the characteristics of patients based on the actual use of G-CSF preparations. The main determinants of G-CSF preparations seemed to be age and lymphocyte count. Patients aged ≥ 80 years were generally prescribed pegfilgrastim. Patients aged ≤ 79 years and with a lymphocyte count of < 1000 cells/μL were most likely to be prescribed daily G-CSF, and they tended to have higher rates of FN. Additionally, we report the predictors of R-CHOP therapy dose reductions or delays among Japanese patients with B-NHL, which were female sex, FN onset during cycle 1, and hemoglobin level. Finally, we confirmed that changes in neutrophil counts were observed when daily G-CSF or pegfilgrastim were administered and that these changes were significantly more marked with pegfilgrastim compared with daily G-CSF and without G-CSF administration. We consider that these findings have important implications on clinical decision-making, particularly when selecting the most beneficial G-CSF preparation for patients with B-NHL who are planned to undergo R-CHOP.Additional file 1 : Supplementary Table 1. Analysis settings and analysis usage variables. BMI body mass index, G-CSF granulocyte colony-stimulating factor, PS performance status. Supplementary Table 2 Univariate logistic regression analyses with and without dose reduction and delay after cycle 2. ALC absolute lymphocyte count, ANC absolute neutrophil count, BMI body mass index, DLBCL diffuse large B-cell lymphoma, CI confidence interval, FL follicular lymphoma, FN febrile neutropenia, G-CSF granulocyte colony-stimulating factor, OR odds ratio, PS performance status. Supplementary Figure 1. Treatment received by cycles in patients who developed FN. FN febrile neutropenia, G-CSF granulocyte colony-stimulating factor.


Congenital hydrocephalus occurs in one in 500–1000 babies born in the United States and acquired hydrocephalus may occur as the consequence of stroke, intraventricular and subarachnoid hemorrhage, traumatic brain injuries, brain tumors, craniectomy or may be idiopathic, as in the case of normal pressure hydrocephalus. Irrespective of its prevalence and significant impact on quality of life, neurosurgeons still rely on invasive cerebrospinal fluid shunt systems for the treatment of hydrocephalus that are exceptionally prone to failure and/or infection. Further understanding of this process at a molecular level, therefore, may have profound implications for improving treatment and quality of life for millions of individuals worldwide. The purpose of this article is to review the current research landscape on hydrocephalus with a focus on recent advances in our understanding of cerebrospinal fluid pathways from an evolutionary, genetics and molecular perspective. Historically, hydrocephalus has been divided into communicating versus non-communicating, and congenital versus acquired. Acquired forms can result from a plethora of environmental insults such as traumatic brain injuries, tumors, cysts, hemorrhagic and ischemic stroke. The term hydrocephalus often implies increased intracranial pressure (ICP) as dictated by the Monroe-Kellie doctrine, but is not necessarily synonymous with elevated ICP, as in conditions like normal pressure hydrocephalus (NPH) and low-pressure hydrocephalus. While the earliest use of the term hydrocephalus has been attributed to Hippocrates (466-377BC),spiritus animalus in Latin.Hydrocephalus, or “Diamectylus viridescens by Gage in 1893, Blake in 1900 reported the presence of the metapore only in humans and three Old World monkey species and absent in all other species he examined.Although there has been much speculation as to why human brains contain CSF reservoirs, the exact purpose is still unclear. The existence of a clear fluid around brain tissue was first scientifically demonstrated by Cotugno in the 18th century and Francois Magendie from 1825–1842, for which the midline foramen in the fourth ventricle was named, also termed the metapore. Luschka then derived the name of the lateral recesses of the fourth ventricle in 1854 and Key and Retzius expounded on the main structures of the pia, arachnoid and dura mater in 1875. Interestingly, although a metapore was noted in the amphibian newt Cushing later helped to further develop the canonical view of CSF flow from the choroid plexus to the subarachnoid space and venous sinuses; however, this theory is notably inconsistent with other species lacking a metapore. Further examination of CSF morphology across species reveals that a connection between the hindbrain linking the ventricles to an external CSF compartment is not consistent across species; some groups of fish including elasmobranchs, teleosts, cyclostomes and dipnoans do not contain any external CSF compartment . These CCSF may also play a role in the normal development of the central nervous system (CNS). First, increases in embryonic CSF volume exerts a positive pressure against the neuroepithelial walls during development and generates an expansive force, contributing to brain expansion and neurogenesis.Embryonic CSF also contains a number of growth factors and cytokines which contribute to the regulation of basic neural progenitor cell functions including survival, proliferation and differentiation.Aside from the role of CSF in normal brain development, it is also speculated to act as a cushioning system and buoyant force which reduces the overall weight of the brain from 1500 to 25–50 g.,,42/ Aβ40 has been used to predict cortical amyloid deposition.42 levels compared with age-matched controls.The composition of CSF is similar to blood serum except chloride concentrations are higher and the potassium and calcium concentrations significantly lower,The choroid plexus is thought to be responsible for 70–80% of CSF production. The term originally derived from the Latin for “chorion,” or delicate, with “plexus” based on its vascular architecture. Indeed, all vertebrate species share a choroid plexus, and in human embryos, the choroid plexus accounts for over 60% of the total ventricular surface, signifying an important and evolutionarily conserved function. The choroid plexus receives more blood per gram of tissue than any other part of the brain. It is composed of an interstitial stromal layer, a fenestrated vascular core, and a single layer of polarized cuboidal epithelium or modified ependymal cells joined by tight junctions . These cBoth CPECs and ependymal cells contain cilia, although they differ in structure. CPECs have between one to two dozen primary cilia, whereas ependymal cells have hundreds of motile cilia equipped with axonemes containing a central pair of microtubules in the 9+2 configuration (versus 9+0 for primary cilia). Although primary cilia are typically non-motile, they still contain receptors with the capacity to respond to mechanical and chemical stimuli.,The remainder of CSF production is believed to come from ependymal cells lining the ventricles and in the subarachnoid space as well as fluid transport across the blood brain barrier (BBB), formed by the tight junctions of endothelial cells along cerebral capillaries. Fluid that is transported across the BBB becomes interstitial fluid (ISF) which includes water-soluble metabolites making up the extracellular space between neurons and glia in the parenchyma. CSF and ISF then communicate in the pericapillary spaces (Virchow robin spaces) across the pia as well as through the ependymal liningAs previously mentioned, Cushing helped to develop the canonical view of CSF flow from the choroid plexus to the subarachnoid space through the foramen of Luschka and Magendie, and then to the cerebral venous sinuses, through which it is absorbed via arachnoid granulations. Arachnoid granulations or arachnoid villi (on a microscopic level) are outpouchings of arachnoid mater located along the venous sinuses and intercavernous sinuses in the skull and have classically been considered the cells responsible for the absorption of CSF in a one-way valve-like type flow mechanism. At birth, however, arachnoid granulations are not fully present, and the absorption of CSF is believed to occur through the venous plexus of the inner surface of the dura, which is more robust in infants.Historically, the brain was believed to be an immune privileged environment due to the lack of a canonical lymphatic system. However, recent evidence has shown that the brain has an alternative to a traditional lymphatic system which involves multiple pathways of CSF circulation and egress for the maintenance of fluid homeostasis as well as immune surveillance. First, CSF may flow along olfactory and spinal nerves to the cervical and spinal lymphatics, and experiments whereby drainage from the cribriform plate was blocked after isolation of cranial from spinal lymphatics, led to increases in resting ICP in a sheep model.Theoretically, hydrocephalus may be caused by any mechanism that disrupts the equilibrium between CSF absorption and production. This could include, for instance, the hypersecretion of CSF at the level of the choroid plexus or the blockage of CSF absorption at the level of the arachnoid villi. The explanation in these situations is direct and mechanical. In fact, the presence of blood within the ventricular system after intraventricular hemorrhage (IVH), one of the leading causes of secondary, acquired hydrocephalus, has been thought of as a direct form of outlet obstruction, causing blood clots to obstruct the drainage of CSF through the arachnoid villi. Additionally, inflammation from blood products in the intraventricular space, meningitis, or any other type of pro-inflammatory signal can cause fibrosis and scarring, serving as another mechanical barrier to normal CSF flow. Although hydrocephalus may form in the acute phase, due to direct mechanical obstruction, a delayed reaction to inflammatory blood breakdown products is also the means by which hydrocephalus may occur in a delayed fashion and become chronic.This concept of hydrocephalus as a simple disequilibrium in production and absorption is based on a bulk flow model of CSF flow proposed by Harvey Cushing in 1925.Transport proteins are critical in maintaining homeostasis in the brain through the transport of ions between intracranial compartments. Three of the most well recognized and studied proteins implicated in hydrocephalus are NKCC1, Aquaporin-1 (AQP-1), and Aquaporin-4 (AQP-4). NKCC1 is found in the apical membrane of CPECs, where it can transport sodium, potassium and chloride ions and help drive CSF secretion . NKCC1 iAQP-4, unlike AQP-1, is predominantly located on the basolateral membrane of ependymal cells as well as astrocytic perivascular end feet where it is involved in water transport from the interstitial to intracellular space. AQP-4 null mice have smaller ventricles, decreased CSF production and higher brain water content.Ependymal cells and CPECs play a critical function in normal CSF dynamics. Directional flow of CSF is generated through the synchronous beating of the cilia of ependymal cells and during development, a steady ependymal flow is required to maintain the patency of the cerebral aqueduct. Cilia not only modulate the directional flow of CSF but may also act as chemoreceptors to regulate the production of CSF.Not surprisingly then, some of the genes implicated in congenital hydrocephalus in humans function in ciliated ependymal cells and CPECs. In humans with syndromic hydrocephalus, at least 20 genes encoding proteins that are required for either the biosynthesis or proper function of the cilium and/or choroid plexus epithelium have been identified.49Foxj1, and, not surprisingly, de novo heterozygous mutations in FOXJ1 cause a motile ciliopathy characterized by hydrocephalus, airway disease, and deficits in right/left asymmetry.Many other genes with direct functions in ependymal cells and cilia have also been discovered in animal models of hydrocephalus. In eight of twelve novel autosomal recessive hydrocephalic mutants identified in a study using 4650 knockout mouse lines, the mutation was associated with motile ciliogenesis and ciliary function.de novo mutations in TRIM71, SMARCC1, PTCH1 and SHH. The authors conclude that genes associated with congenital hydrocephalus typically fall into one of two categories; those involved in CSF circulation, or those involved in normal cortical development.Although ependymal cells and CPECS are modified epithelial cells, they are actually derived from the neuroepithelium or neuroectoderm, which gives rise to cortical neurons and glia.As a whole, therefore, many of the genes associated with both congenital and syndromic hydrocephalus in both animal models and humans play a role in the development, maintenance, and/or function of ciliated ependymal cells and/or CPECs. Furthermore, many of these genes may overlap between CPECs, ependyma and neural progenitors, with abnormalities of the ependyma and choroid plexus epithelium correlating with changes in the adjacent subventricular zone, and vice versa.Although the examples discussed above are associated with forms of congenital hydrocephalus, perturbations of normal CSF ependymal and CPEC ciliary function also occur in models of secondary, acquired hydrocephalus. For example, there is evidence that post hemorrhagic and post infectious hydrocephalus may cause an inflammatory reaction which impairs normal ependymal and choroid plexus function. The presence of foreign inhabitants of CSF  often sets off a non-specific inflammatory response, including the release of cytokines such as Il-1β, IL-6 and TNF-α, as well as local immunoglobulin production by plasma cells.Intraventricular injection of autologous whole blood also causes a buildup of iron in the CSF. Iron has also been shown to play a distinct role in hydrocephalus and may cause direct damage to ependymal cilia. Normal ependymal cells take up iron from the CSF and prevent its diffusion to the rest of the brain; consequently, levels of iron can accumulate in brain parenchyma if ependymal cells are damaged. In turn, systemic deferoxamine treatment has been found to partially reverse this iron accumulation and alleviate hydrocephalus in a rat model of IVH.Altogether, hydrocephalus is one of the most common yet least well understood clinical entities in neurosurgical practice. Our methodology for the treatment of hydrocephalus has also not changed significantly since the 1950s, when the first silicone ventriculoperitoneal shunt (VPS) was placed by Richard Ames.Although clinical focus has been on treating hydrocephalus once it occurs, understanding the mechanisms by which this entity develops may help us to identify novel therapeutic strategies. Moreover, the model of a unidirectional CSF flow system from the ventricles to the subarachnoid space and uptake via arachnoid granulations is overly simplistic. CSF can permeate all brain compartments, and perivascular networks may play a much more important role than previously recognized. In fact, “glymphatic dysfunction” is being recognized as a hallmark of many different pathological processes, including sleep disturbances and psychiatric conditions such as major depressive disorder.Ependymal cells and CPECs also play a variety of key roles in both congenital and acquired hydrocephalus. In addition to maintaining directional CSF flow through motile cilia, ependymal cells direct transport of ions and solutes in the surrounding intraventricular and parenchymal compartments, the production of growth factors that may be important for neurogenic proliferation and survival, and the choroid plexus epithelium acts as a barrier at the CSF-brain interface, maintaining cell junctions at the choroid plexus. Both ependyma and CPECs can contribute to both genetic causes of hydrocephalus  and to acquired causes, as they are subject to functional impairment after any secondary injury that causes associated inflammatory responses and damage.Given the limitations and complications associated with shunt systems, a therapeutic strategy targeting the genes and/or proteins involved in the development of hydrocephalus is a promising alternative solution. Choroid plexus and ependyma are especially suitable targets for therapeutic interventions including viral, RNA or protein based therapies given their location as the cells directly lining CSF spaces in the brain and spinal cord. For example, AAV vectors have been shown to produce strong long-term expression within the choroid plexus and ependyma in mouse models,In order to identify these novel therapeutic strategies, however, it is first essential to understand the molecular mechanisms underlying the pathogenesis of hydrocephalus. In deciphering these key regulators of hydrocephalus at a genetic and molecular level, the ultimate goal is to develop novel therapies to modify the function of ependymal and CPECs and ultimately abrogate the need for ventricular shunting. It will be especially exciting to see how our understanding of CSF dynamics and hydrocephalus changes over the coming years, and how this will come to influence future neurosurgical practice.Many questions about CSF physiology and hydrocephalus remain to be addressed. First and foremost, what is the purpose of CSF reservoirs? What are the key molecular pathways that regulate the brain's response to changes in intracranial pressure? How does the choroid plexus respond to these changes and what are the molecular cues that govern the regulation of CSF production? How can we manipulate the functions of choroid plexus and ependymal cells to prevent and/or treat patients with hydrocephalus? Once we learn the answers to some of these fundamental questions, we may begin to tackle the problem of hydrocephalus and its association to the most common cerebral pathologies such as trauma, brain hemorrhages, stroke, neurodegenerative disease and even abnormal ageing.References for this review were identified by searches on both Pubmed and Google Scholar from 1960 to June 2021 and references from relevant articles. The search terms “hydrocephalus”, “glymphatic system”, “cerebrospinal fluid dynamics”, “cerebrospinal fluid physiology”, cerebrospinal fluid evolution”, and “hydrocephalus mechanisms” were used. There were no language restrictions. The final reference list was based on relevance to the topics covered in the review.AB conceived of the topic of the review, performed the literature searches and wrote the manuscript. PF, KK, and EA contributed to intellectual development and helped to edit the final manuscript. All authors read and approved the final version of the manuscriptThere are no conflicts of interest to declare.


In this proposed work, direct green 6 (DG6) decorated carbon paste electrode (CPE) was fabricated for the efficient simultaneous and individual sensing of catechol (CA) and hydroquinone (HY). Electrochemical deeds of the CA and HY were carried out by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) at poly-DG6-modfied carbon paste electrode (Po-DG6-MCPE). Using scanning electron microscopy (SEM) studied the surface property of unmodified CPE (UCPE) and Po-DG6-MCPE. The decorated sensor displayed admirable electrocatalytic performance with fine stability, reproducibility, selectivity, low limit of detection (LLOD) for HY (0.11 μM) and CC (0.09 μM) and sensor process was originated to be adsorption-controlled phenomena. The Po-DG6-MCPE sensor exhibits well separated two peaks for HY and CA in CV and DPV analysis with potential difference of 0.098 V. Subsequently, the sensor was practically applied for the analysis in tap water and it consistent in-between for CA 93.25–100.16% and for HY 97.25–99.87% respectively. Nevertheless, DHB is venomous and hard to degrade, menacing the environment and crippling animal and human health, it leads to incurring the harm to kidney and liver function affected by CA and HY have also been reported12. In addition, simultaneously sensing of CA and HY is tough because they mutual interfere with each other during their identification because of their similar structures, properties and coexistence14. Consequently, the fabrication of simple, selective, speedy, accurate and sensitive analytical method was needed for the detection and quantifying of CA and HY16. Up to now, numerous techniques have been testified for sensing of CA and HY, such as spectrophotometry, capillary electrochromatography (CEC), high-performance liquid chromatography (HPLC), gas chromatography/mass spectrometry (GC/MS), fluorescence  and electroanalytical method24. Amongst, the electrochemical analysis has owing prominent in current years to its advantages like simplicity, portability, low cost, time saving, convenient operation, speedy response, as well as high efficiency, sensitivity and selectivity30.Dihydroxybenzene (DHB) isomers like catechol (CA) and hydroquinone (HY) are extensively utilized in the agronomic practices and chemical industry, such as tanning, plastic, pesticides, dyes, rubber, pharmaceuticals and so forth34. Direct green 6 (DG6) mainly utilized in fibers, soap, leather, regenerated cellulose fibers film shading, colouring of paper and also employed in the production of colour pigment sediment36  have accomplished more attention due to their homogeneity, reproducibility, good constancy and strong adherence to electrode surface6 Scheme . The fabVoltammetric analysis was tested by CV and DPV performances expending a traditional three-electrode cell of CH Instrument-660 (CHI-660c) electrochemical workstation. UCPE and Po-DG6-MCPE were employed as working, saturated calomel electrode (SCE) as a reference and a platinum wire as a counter electrode individually.–4 M) was prepared in double distilled water. DG6, sodium dihydrogen phosphate (NaH2PO4), disodium hydrogen phosphate (Na2HPO4) were gained from Merck chemicals of AR grade and all aqueous solution were prepared with double distilled water. All the reagents are utilized in this study with analytical grade and used as received.HY and CA procured from Sigma-Aldrich and standard solutions (25 × 10–1 at 10 multiple cycle.The UCPE was fabricated by blending of graphite powder and silicone oil in the ratio 70:30 (w/w) for about 30 min and get homogeneous mixture. The gotten blend was then filled with homemade Teflon cavity consuming 3 mm internal diameter and cooper wire was utilized for the electrical contact. The pre-treated carbon paste electrode (PCPE) was constructed by electrochemical oxidised by cycling the potential between − 0.6 to 1.0 V in 0.1 M NaOH with speed rate of 0.05 V s–1 for 10 polymerization cycles as elucidates in Fig. 37. The deposition of DG6 on UCPE was carried by changing the polymer series 5 to 25 cycles  was carried on the surface of UCPE using cyclic voltammetry in the existence of 0.1 M NaOH (supporting media) and cyclic the potential scanned between − 0.6 to 1.0 V with speed rate 0.05 V s.8 mM was4[Fe(CN)6] (25 × 10–3 M) as redox probe in 1 M KCl (supporting media). Figure –1. At UCPE, it implies small peak current and in the similar form Po-DG6-MCPE reveals superior boost in peak current than UCPE. Therefore, this development in peak current donated substantial increase in electroactive surface area. This active surface area of the electrode was identified using Randles–Sevick’s Eq. (38. Compared to Po-DG6-MCPE (0.061 cm2) the UCPE (0.030 cm2) was consumed less electroactive surface area. The approximate adhered modifier thickness or surface average concentration on UCPE was calculated by utilising Eq. (39 and got at 0.271 × 10−10 M cm−2.The characteristic property of Po-DG6-MCPE was verified using K)6] 25 × 0–3 M as 41.A is the area of working electrode (cm2), C0 is the concentration of the electroactive species (mol/cm3), n is the electrons transformed, ν is the sweep rate (V/s), D is the diffusion coefficient (cm2 s−1), Ip is the peak current, Γ (M/cm2) is the surface average concentration and R, F, T are the physical constants.The surface changes of before and after fabrication of UCPE was characterized by SEM using ZEISS Ultra-55. The Fig. −1. The UCPE provides CVs with reduced and broad response and at PCPE it gave voltammetric response with poor response. Compared UCPE and PCPE the designed electrode signifies ultimate enrichment in peak current with very precise sensitivity with sharp peaks. The peak potential difference (ΔEp = Epa-Epc) was acquired at 0.153 V (BCPE), 0.095 V (PCPE) and 0.017 V (Po-DG6-MCPE) individually. At tailored electrode the electron transfer was easier than UCPE and PCPE, because where ΔEp value is inferior and transfer of electron rate will be greater. Therefore, the Po-DG6-MCPE accomplished as a good sensor and prominent for the analysis of CA. The redox response of CA was portrayed in Scheme The sensing proficiency of Po-DG6-MCPE towards the CA was scanned by CV technique as most sensitive and precise voltammetric technique. In Fig. −1) and minute move of their peak potential to positive and negative side. The linear correlation between anodic peak current (Ipa) versus speed rate (ν) and Ipa versus square root of the speed rate was drawn in Fig. 2) was initiate at 0.9991 and 0.9937 correspondingly. Consequently, by spotting the overhead practical result the kinetic property of Po-DG6-MCPE was originated at adsorption controlled process43. Heterogeneous rate constant (k0 in s−1) were computed by Eq. 3 and attained results are tabulated in Table 0, ΔEp, ν, is the heterogeneous rate constant in s-1, difference in peak potential, speed rate respectively.The Po-DG6-MCPE process was assessed by changing the speed rate. Figure −1. These figures evidently portray that the peak current was boosted noticeably when the analyte concentration was rises. Inset Fig. 2: 0.9992 and 0.9993. Utilizing the slope value (M) and standard deviation (S) of the Ipa . The acquired slope value 49 mV pH−1 was very close to the Nernstian theoretical value (59 mV), so it clearly advises the equal number of electron and proton was involved in the electrode reaction45. Figure The choice of supporting electrolytes plays major part in the electrochemical study. Figure −1 at Po-DG6-MCPE. At BCPE (scattered row), it portrays low peak current with poor response because of sluggish electron transfer. Similarly, at established Po-DG6-MCPE (hard row) it provided superior enlargement in redox peak current with very precise sensitivity with sharp than UCPE. The ΔEp value for Po-DG6-MCPE and UCPE was gotten at 0.018 and 0.07 V respectively. Where ΔEp is lower than electron transfer rate will be greater, hence at the constructed electrode electron transfer is easier than UCPE. Thus, the Po-DG6-MCPE achieved as good sensor and prominent for the investigation of HY and redox mechanism was signifying in Scheme The electrocatalytic sensing of HY was traced by cyclic voltammetry and Fig. −1). By witnessing the Fig. 2 value was originate at 0.9997 and 0.9984 respectively and electrode process was governed by adsorption controlled process.The speed rate study offered the essential confirmation about the electrode process. Figure −1. As noticed in Fig. 2 value 0.9984 and 0.9979. The LLOD and LOQ was establish at 0.11 and 0.36 μM correspondingly. The Po-DG6-MCPE offered low LLOD for CA and HY than other fabricated sensor and displayed in Table The LLOD and LOQ was calculated utilizing Eqs.  and 5)  5) for H46. Thus, to validate the potentiality of designed electrode for the simultaneous exploration of CA and HY. Figure −1. The UCPE (scattered line) was failed to depicts the two oxidation peaks but gave only one oxidation peaks located at 0.258 V. Moreover, the fabricated Po-DG6-MCPE (solid streak) clearly verified the two separated oxidation peaks for CA and HY (0.15 and 0.03 V) individually with magnificent development in peak current compared to UCPE. Figure The simultaneous revealing of CA and HY was very challenging in mixed solution at UCPE. Because in the mixed solution they are overlapped each other due to their approximately same oxidation potentialThe efficiency and selectivity detection of CA and HY was testified at Po-DG6-MCPE by applying the DPV technique. Figure −1 by utilizing CV system. As depicted in Fig. n/Ip148, where Ip1 and Ipn are the 1st and nth cycle Ipa respectively. The retained steadiness of the Po-DG6-MCPE was 95.43% and this confirms the established electrode have magnificent stability.The stability of the Po-DG6-MCPE was assessed for CA (10 µM) in presence of 0.2 M PBS for 10 cycles with speed rate of 50 mV s2. There was no considerable interference was perceived for the analysis. Thus, the proposed Po-DG6-MCPE sensor reveals the excellent anti-interference ability.The anti-interference capability of Po-DG6-MCPE were tested for CA and HY in presence of some coexisting compounds such as resorcinol, uric acid, serotonin, NaCl and KCl and CaCl50. This advises the recommended electrode exposes satisfactory recovery for each sample addition and attained results are recorded in Table Finally, the fashioned Po-DG6-MCPE was testified for the practical applicability of CA and HY in a local tap water sample using standard addition methodThe proposed work reports Poly-DG6-MCPE was accomplished as a sensor for the detection of CA and HY. The surface morphology of UCPE and Po-DG6-MCPE was characterized by SEM examination. This proposed electrode exposed the strong electrocatalytic activity, high sensitivity, stability and gave improved electron transfer response than UCPE with respect to the oxidation of CA and HY. The impact of pH study, concentration variation and speed rate study was tested at fabricated electrode. The Po-DG6-MCPE involves adsorption-controlled procedure and displayed low detection limit value compared to other reported electrodes. Simultaneous firmness of CA and HY was tracked by CV method. The constructed modified electrode results in good stability, selectivity and offers satisfactory recovery of analytes. Therefore, the mentioned Po-DG6-MCPE was substantial potentiality for the specific and simultaneous investigation and this constructed electrode was utilized for the further analysis of other electro-active biomolecules.


Sc in post-mortem tissues as indication of infection and disease. Since PrPSc detection is not considered a reliable method for in vivo diagnosis in most TSEs, it is of crucial importance to identify an alternative source of biomarkers to provide useful alternatives for current diagnostic methodology. Ovine scrapie is the prototype of TSEs and has been known for a long time. Using this natural model of TSE, we investigated the presence of PrPSc in exosomes derived from plasma and cerebrospinal fluid (CSF) by protein misfolding cyclic amplification (PMCA) and the levels of candidate microRNAs (miRNAs) by quantitative PCR (qPCR). Significant scrapie-associated increase was found for miR-21-5p in plasma-derived but not in CSF-derived exosomes. However, miR-342-3p, miR-146a-5p, miR-128-3p and miR-21-5p displayed higher levels in total CSF from scrapie-infected sheep. The analysis of overexpressed miRNAs in this biofluid, together with plasma exosomal miR-21-5p, could help in scrapie diagnosis once the presence of the disease is suspected. In addition, we found the presence of PrPSc in most CSF-derived exosomes from clinically affected sheep, which may facilitate in vivo diagnosis of prion diseases, at least during the clinical stage.Diagnosis of transmissible spongiform encephalopathies (TSEs), or prion diseases, is based on the detection of proteinase K (PK)-resistant PrP C) to an infectious and pathological conformation (PrPSc) in a self-propagating reaction [Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases that affect humans and animals . TSEs rereaction . Ovine areaction . As in oreaction . To dateSc in the brain tissue of diseased individuals is used as a biochemical marker for the presence of infection and disease [Sc is minimal in accessible tissues and body fluids such as blood or urine [Sc isoforms have been discovered that are not detectable by current diagnostic methods [Sc that can be detected from accessible tissues or fluids to provide useful alternatives for current diagnostic methodology.Post-mortem detection of PrP disease . Howeveror urine . In addi methods ,8. ThereExosomes are small vesicles surrounded by membrane with a variable size ranging from 30 to 150 nm . These pC and infectious PrPSc by different types of infected cells in association with exosomes [In vitro studies have demonstrated the release of PrPexosomes ,13,14,15exosomes . The relexosomes . Autophaexosomes , so thisC among the protein cargo of blood circulating exosomes has been demonstrated in humans [Sc was first detected by protein misfolding cyclic amplification (PMCA) in extracellular vesicles (EV) isolated from blood plasma of mice infected with mouse-adapted vCJD [Sc in plasma-derived exosomes have been carried out in natural scrapie sheep using Western blot [The presence of PrPn humans  and sheen humans . PrPSc wted vCJD  and fromted vCJD . Besidesern blot . More seC in CSF have been considered to be a potential biomarker for several neurodegenerative diseases [C [Sc in EV from CSF has not been previously described. Brain pathophysiology is reflected in CSF and the presence of EV in this biofluid represents a potential source of biomarkers for neurological diseases . The levdiseases , includidiseases , as its diseases ,27. It heases [C  but, to Sc, which could facilitate diagnosis and be crucial for prion transmission. Proteins and microRNAs (miRNAs) are molecular constituents of exosomes and promising biomarkers for many diseases, including neurodegenerative diseases [The interest of exosomes in TSEs is not limited to the determination of PrPdiseases ,30. Exosdiseases . In a prdiseases . In thisSc  by PMCA, which would facilitate in vivo diagnosis. Then, their infectivity or ability to spread disease in the CNS was evaluated by bioassay. Finally, as the miRNA profile in exosomes could change with the pathological status of the individual, the potential of exosomes as a source of biomarkers was evaluated analysing the levels of candidate miRNAs described to be altered in different prion diseases and their models. These included miR-342 [The relevance of exosomes in prion diseases has only been analysed in vitro and in murine models of TSE. The aim of this work was to analyse exosomes from blood and CSF in sheep naturally infected with scrapie, as an animal model of prion diseases. First, we evaluated the presence of PrP miR-342 ,32,33,34 miR-342 ,34, miRN miR-342 ,31,35, m miR-342 ,37, miR- miR-342 . TM Exosome Isolation Kit  and Total Exosome Isolation Kit ). Exosomes were visualised using transmission electron microscopy (TEM). We successfully isolated exosomes from plasma and CSF using two commercial kits  assays showed that vesicles isolated from plasma displayed a mean diameter ranging between 30 and 120 nm B. DLS in−7–10−8 dilution, suggesting that the presence of PrPres in plasma-derived sEVs, if any, is at least 8 log10-fold lower than that detected in sheep brains  in six out of eight samples from clinically affected sheep and three out of three samples from sheep at terminal stage  and two scrapie-infected sheep  were also tested for infectivity by bioassay in Tg338 transgenic mice. A total of 27 mice were intracerebrally inoculated: nine mice with CSF-derived sEVs obtained with Exiqon kit, nine mice with CSF-derived sEVs obtained with Invitrogen kit and nine mice with total CSF. In each group, three mice were inoculated with CSF or CSF-derived sEVs from the negative control sheep and six mice with CSF or CSF-derived sEVs from the aforementioned two positive scrapie sheep.Most animals were sacrificed by end point criteria (from 232 days post inoculation (dpi) of a mouse inoculated with scrapie CSF to 647 dpi of a mouse inoculated with negative CSF) or at the end of the study. Three of the mice inoculated with CSF-derived sEVs isolated with the Exiqon miRCURY kit died without showing symptoms compatible with scrapie or any other end point criteria at ages older than 381 dpi.Sc in any inoculated mice  in sEVs obtained from scrapie-infected sheep (p = 0.065). As a general observation, the dispersion between samples was greatly increased in scrapie sheep compared with controls.We recently described alterations in miRNA profile in plasma from scrapie-infected sheep . In the ed sheep . Additiop < 0.05) in CSF from scrapie-infected sheep compared to healthy control sheep  to separate plasma, divided in aliquots and stored at −80 °C until analysis. A volume of approximately 15 mL of CSF was obtained from each sheep immediately after sacrifice, using a 10 mL syringe inserted into the cisterna magna and avoiding blood contamination. CSF was also aliquoted and stored at −80 °C until analysis.Plasma was obtained from 5 mL of blood, which was collected by jugular venipuncture from each animal. Blood was centrifuged for 10 min at 1300× TM Exosome Isolation Kit—Serum and plasma (Exiqon). The initial amount of plasma used for sEV isolation was 250 µL for the first kit and 500 µL for the second one. We followed the manufacturer’s instructions, with a final resuspension of sEV pellets in 100 µL of PBS  or 270 µL of Resuspension Buffer (Exiqon). sEVs isolated with the first kit were subjected to PMCA for PrPSc detection (n = 24), whereas those obtained with miRCURYTM kit were used for further RNA extraction and miRNA profiling (n = 16).Two commercial kits were used for sEV isolation from plasma: Total Exosome Isolation Kit (from plasma)  and miRCURYTM Exosome Isolation Kit—Cells, Urine and CSF (Exiqon) was used to isolate sEVs for miRNA profiling (n = 12). Both kits used a starting sample of 1 mL CSF to precipitate sEVs, with a final resuspension step in 50–75 µL of PBS (Invitrogen) or 100 µL of Resuspension Buffer (Exiqon). In all cases, the purified sEV samples were stored at −20 °C until their use.Total Exosome Isolation Kit (from other body fluids) (Invitrogen) was used for sEV isolation from CSF for further PMCA analysis (n = 19), and miRCURYsEV morphology was analysed in two sEV samples obtained from plasma and CSF. The morphology was evaluated by transmission electron microscopy (TEM) using the methodology previously described ,76. A voThe size distribution of nanoparticles was evaluated using dynamic light scattering (DLS) assays as previously described . An aliqSc in sEVs isolated from plasma  and CSF  was analysed by PMCA.Presence of PrPC substrate was prepared from brain tissue (10% brain lysate) of uninfected transgenic Tg338 mice expressing ovine PrPC in cold PMCA buffer . One teflon bead  was added to each well. Each sample was run in duplicate using 1/5, 1/10 and 1/50 dilutions in PMCA buffer. Amplification was performed in a modified sonicator (QSonica Q700), using a water recirculation system (39.5 °C) [−3 to 10−9 diluted) of brain homogenate from ARQ/ARQ ovine scrapie sheep was included on each PMCA run to check the amplification performance. Brain homogenates were prepared in PBS (10% weight/volume) from medulla oblongata.PMCA was performed by mixing 5 µL of the seed with 45 µL of substrate per well in a 96-well PCR microplate . The PrP39.5 °C) . MicroplSc in each PMCA round. PMCA products (18 µL) were supplemented with 2 µL of 3% SDS and treated with PK  for 1 h at 37 °C. Digestion was stopped by adding an equal volume of Laemmli buffer and heating at 95 °C for 5 min. A 5-µL volume of sample was mixed with 25 µL of 1% SDS. The samples were then vacuum transferred onto a nitrocellulose membrane. The membrane was rinsed once with PBS (0.1% Tween 20) and incubated for 30 min in PBST containing 2% BSA. The monoclonal antibody (mAb) Sha31 (1:8000 dilution in PBST + 2% BSA), which recognises residues 145–152 (YEDRYYRE) of PrP, was used for PrPSc/PrPres immunodetection [On completion of the PMCA procedure, Dot blot was used to analyse the presence of PrPetection . AntibodSc in the last round of each PMCA procedure. A volume of 20 µL of PMCA reaction product was mixed with 180 µL of 10% negative brain sheep homogenate before PrPSc extraction, as previously described [Sc detection was performed using Sha31 mAb conjugated to horseradish peroxidase (0.06 µg/mL), and ECL substrate (Pierce) was used to reveal peroxidase activity.TeSeE Western blot kit  was used following the manufacturer’s recommendations to analyse the presence of PrPescribed . PrPSc dsEVs isolated from CSF using both commercial kits were stored at 4 °C and used as inocula for the bioassay in transgenic Tg338 mice within the next 48 h. Intracerebral inoculations were performed under gaseous anesthesia using 50 µL syringes and 25-gauge hypodermic needles inserted into the right parietal lobe, delivering 20 µL of the inoculum to each animal. Nine mice were inoculated with CSF-derived sEVs obtained with Exiqon kit, 9 mice with CSF-derived sEVs obtained with Invitrogen kit and 9 mice with total CSF. In each group, 3 mice were inoculated with CSF or CSF-derived sEVs from a negative control sheep and 6 mice with CSF or CSF-derived sEVs from two positive scrapie sheep. To reduce post-inoculation pain, a subcutaneous injection of buprenorphine (0.3 mg/kg) was administered to each mouse before recovery to consciousness. After inoculation, mice were housed in filtered cages and their clinical status was monitored three times a week.Sc deposition was performed using the paraffin-embedded tissue-blot (PET-blot) method as previously described [C. The remaining PrPSc was denatured by incubating the membranes in a solution of guanidine thiocyanate 3 M. After blocking the membranes with 0.2% BSA, PrPSc was detected using the Sha31 mouse mAb  and a secondary alkaline phosphatase-conjugated antibody . Enzymatic activity was visualised using NBT/BCIP .Mouse brains were fixed in formalin, embedded in paraffin, cut into 4 µm sections, and mounted on glass slides for histological evaluation using haematoxylin and eosin staining. The level of vacuolisation was evaluated in each sample using a semiquantitative score with a scale of 0 (lack of vacuolisation) to 5 (very high presence of vacuoles). On the other hand, an analysis of PrPescribed . BrieflyTM Exosome Isolation Kit , total sEV RNA extraction was carried out using miRCURYTM RNA Exosome Isolation Kit-Biofluids (Exiqon) following the manufacturer’s recommendations. RNA from CSF-derived sEVs  was extracted using miRCURYTM RNA Isolation Kit—Cell and Plant  according to the manufacturer’s recommendations. In both cases, precipitated sEVs were lysed with the appropriate volume of Lysis Solution and subsequently spiked in with a 5.7 µL of a carrier/spike solution containing 0.5 µg MS2 RNA and 25 fmol cel-miR-39 and at the end of the process, RNA was eluted to 100 µL Elution Buffer and stored at −80 °C for miRNA expression analysis.After sEV precipitation from plasma using miRCURYTotal RNA was purified from 200 µL of CSF and 200 µL of CSF after sEV purification (sEV-depleted CSF) using Total RNA Purification Kit  according to the manufacturer’s instructions. Briefly, the samples were lysed with 600 µL of Lysis Buffer and carrier/spike solution was added as above. After adding 600 µL ethanol the lysate was passed through RNA binding columns, the bound RNA was washed and eluted to 50 µL Elution Solution and stored at −80 °C for miRNA expression analysis.−∆∆Ct method [t-test.Retrotranscription was performed using specific stem-loop TaqMan MicroRNA Assays  for each miRNA and TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). For each sample, retrotranscription for seven miRNAs and cel-miR-39, used as a “spike-in” normaliser, was performed in a pooled reaction containing 9 μL of template RNA. The sequence for each miRNA is shown in t method  using heSc and extracellular miRNAs. To what extent these potential biomarkers may associate with extracellular vesicle compartment in these biofluids remains largely unknown. Two of the most frequently studied biofluids in neurodegenerative conditions are CSF and blood plasma. We demonstrated that PrpSc/PrPres is present in CSF sEVs from scrapie sheep but was not detected in sEVs from plasma, despite the latter containing more sEVs. The CSF-derived, PrpSc-containing sEVs did not demonstrate infectivity in vivo. Both plasma and CSF displayed some miRNA alterations in natural sheep scrapie, the CSF showing increased number of altered miRNA species. Purification of sEV fraction from the said biofluids did not lead to better detection levels of studied miRNAs, even if the amount of biofluids used for sEV extraction was considerably larger. Both the presence of PrPSc and the altered levels of several studied candidate miRNAs in CSF could facilitate the development of biofluid-based biomarkers for the prion diseases in the future.Analysis of circulating biofluids could facilitate and/or complement current diagnostic markers for prion disease as these fluids are known to harbour PrP