The definition of Alzheimer’s disease (AD) now considers the presence of the markers of amyloid (A), tau deposition (T), and neurodegeneration (N) essential for diagnosis. AD patients have been reported to have increased blood–brain barrier (BBB) dysfunction, but that has not been tested within the ATN framework so far. As the field is moving towards the use of blood-based biomarkers, the relationship between BBB disruption and AD-specific biomarkers requires considerable attention. Moreover, other factors have been previously implicated in modulating BBB permeability, including age, gender, and ApoE status. A total of 172 cognitively impaired individuals underwent cerebrospinal fluid (CSF) analysis for AD biomarkers, and data on BBB dysfunction, demographics, and ApoE status were collected. Our data showed that there was no difference in BBB dysfunction across different ATN subtypes, and that BBB damage was not correlated with cognitive impairment. However, patients with BBB disruption, if measured with a high Qalb, had low Aβ40 levels. ApoE status did not affect BBB function but had a dose-dependent effect on the Aβ42/40 ratio. These results might highlight the importance of understanding dynamic changes across the BBB in future studies in patients with AD. The definition of Alzheimer’s disease (AD) has undergone a progressive shift from a clinical to a biological construct, with biomarkers of amyloid status and tau pathology now being essential for in vivo diagnosis . Indeed,While many different CSF biomarker ratios have been evaluated to increase diagnostic accuracy , since tAlthough Aβ is found in the blood at the same concentration of CSF (0.1–0.5 nM), Aβ deposits are largely found in the interstitial compartments within the brain . The relThe role of the alteration of the blood–brain barrier (BBB) in AD patients has been widely investigate . The accPrevious studies found that AD cases with evidence of concomitant cerebrovascular pathology have high Qalb as a sign of impaired BBB function, and Qalb correlated positively with CSF biomarkers of angiogenesis and endothelial dysfunction rather than AD-specific biomarkers ,31, whilMoreover, there is conflicting evidence of an association between Qalb and cognitive impairment for standard neuropsychological measures such as the MMSE  or the MoCA , and between Qalb and annual change on the MMSE and CDR  . InteresThe aim of our study was to investigate the relationship between biomarkers of BBB damage and AD-specific CSF biomarkers. We assessed the relationship between BBB biomarkers , serum and CSF IgG, and the IgG index), CSF markers of AD , age, sex, ApoE status, and cognitive status. Moreover, we compared biomarkers of BBB disruption between groups characterised using the ATN biomarker framework.According to the ATN criteria, 57 patients showed a normal biomarker profile (A-T-N-), 21 met the criteria for Alzheimer’s pathologic change (A+T-N-), 70 were classified as Alzheimer’s disease , and 24 as non-Alzheimer’s pathologic change  [Groups were not statistically different in age, sex, MMSE scores, or ApoE status, as shown in We found no difference in any of the BBB biomarkers  among the four groups , nor amop = 0.014), but not with Aβ42 or the Aβ42/40 ratio. Patients were then divided according to the integrity of their BBB using the standard age-related Qalb cut-off of eight as having normal Qalb (<8) or high Qalb (>8). We found that the negative correlation between Aβ40 and Qalb was only significant  in patients with high Qalb, while in patients with normal Qalb this was not observed . Age was negatively correlated with serum albumin , with lower levels of serum albumin seen in elderly individuals. Females had lower CSF proteins , CSF albumin , CSF IgG , and Qalb . A correlation matrix across all variables in shown in MMSE scores were not correlated with the BBB markers studied but were positively correlated with Aβ42 levels, with lower MMSE associated with lower levels of Aβ42 , as seen in The Kruskal–Wallis group comparison among different ApoE groups divided by graded risk factor showed that carrying a more high-risk genetic profile did not affect BBB biomarkers but was associated with lower levels of Aβ42/40 ratio in a dose-dependent manner (H (4) = 9.973, p < 0.001), see A PCA was then performed to reduce the dimensionality of our dataset and reveal the latent structure of the relationship between the different factors in our data. A five-factor model was able to reflect the data efficiently  could reflect sex-related differences in CSF protein  content and Qalb, with males showing higher levels of BBB disruption. The second component (RC2) reflects AD-specific CSF biomarkers, with higher t-tau and p-tau correlating negatively with Aβ42/40 ratio, as expected. The third component (RC3) might reflect the complex interplay between Aβ species, ApoE status, and cognition. The fourth component (RC4) seems to mirror the well-known age effect on serum albumin, with older age being associated with lower serum albumin. The fifth component (RC5) could represent a systemic inflammatory response. To note, RC1, RC2, and RC4 are highly correlated, as seen in In light of the recent advent of biomarkers to measure ATN status in blood, a deeper understanding of the impact of BBB dysfunction on AD biomarkers is warranted. Beyond its importance in determining the fine-grain balance between the concentration of AD-specific biomarkers in the blood and in the CSF for diagnostic purposes, this might also extend to a better evaluation of success rates of therapeutic interventions if a future shift to blood-based biomarkers is implemented as a surrogate for CSF and PET imaging to determine clinical trial endpoints. We found no difference in any of the BBB biomarkers  among the four groups divided by their ATN profile, thus confirming previous data based on the clinical stratification of patients . In lineOur data also showed that only when the BBB is damaged, if measured by a high Qalb, Aβ40 levels decrease. Albumin is the most abundant protein in blood, with a concentration of 640 μM, but has a markedly reduced concentration in the CSF of typically 3 μM . At CSF BBB biomarkers were not impacted by the ApoE genotype, but our data support a modulation of the Aβ42/40 ratio with ApoE status. This seems to follow a dose-dependent effect, with ApoE phenotypes at higher risk of developing AD being associated with a lower Aβ42/40 ratio. This result is in line with the previous literature on the association between carrying at least one ApoE4 allele and lower levels of amyloid but not higher levels of tau in the CSF , but extIn this study, ageing was only negatively correlated with serum albumin levels and not with Qalb, but the age range was small (60–80 years) when compared to other studies that have investigated the effect of ageing on Qalb across the lifespan, so in our data the variability might not be enough to detect those differences. On the other hand, we were able to confirm that there are sex differences in Qalb, which is in line with previous data showing males as having higher Qalb values, and therefore higher BBB dysfunction . The corn = 21 to n = 70), and a bigger sample might be needed to confirm these findings. Moreover, other biological measures acquired for research purposes such as brain MRI, FDG, amyloid or tau PET, and blood-based biomarkers of AD were not available for this dataset.Our work has several limitations. Firstly, all participants had perceived memory complaints and objective low memory scores at testing, and data on healthy controls without cognitive impairment were not acquired. This hugely hinders the interpretation of the results since a physiological baseline would be advisable. Nevertheless, we were able to compare patients with memory impairment within the ATN framework, with a cohort of ATN negative individuals, and proved that Qalb impairment is independent from their ATN profile. Secondly, the numbers in each ATN subtype were relatively small , magnetic resonance imaging (MRI), and CSF analysis. Exclusion criteria were the presence of a major psychiatric disorder, autoimmune, paraneoplastic and infectious disorders, chronic or acute polyneuropathy, radiological evidence of recent ischemic lesions, or any know condition that could alter CSF results due to the CSF sampling procedure such as lumbar spinal stenosis or malignancies.g at +4 °C) for 10 min. The supernatant was pipetted off, mixed to avoid potential gradient effects, aliquoted in 1 mL portions in polypropylene tubes, and stored at −80 °C pending biochemical analyses. The level of CSF biomarkers Aβ40, Aβ42, t-tau, and p-tau phosphorylated at Thr-181 were determined using a sandwich enzyme-linked immunosorbent assay (Fujirebio). Genotyping for ApoE was performed using real-time PCR on the LightCycler instrument (Roche) [CSF and Serum Biomarker Analysis: CSF collection was performed in the morning on fasting patients. The first 12 mL of CSF were collected in a polypropylene tube and directly transported to the local laboratory for centrifugation (2000×  (Roche) . Blood-c (Roche) .p-value < 0.05.Statistical analysis: Statistical analysis was conducted with MATLAB (ver R2019a) and JASP . According to the normality of the data, between-group differences within the ATN framework were analysed using either a frequentist one-way analysis of variance (ANOVA) or Kruskal–Wallis H test, followed by Bonferroni post-hoc correction. Based on ApoE genotype, a value was assigned according to the graded risk factor associated to each genotype, with consecutive values given to E2/E2, E2/E3, E3/E3, E2/E4, E3/E4, and E4/E4 according to Reiman et al. . Sex corOur data showed that BBB dysfunction does not change across different ATN subtypes and that is not correlated with cognitive impairment. However, BBB damage, if measured by Qalb, is associated with low Aβ40 levels, which might potentially have implications for the fine-grain balance between different amyloid species. BBB disruption was not modulated by ApoE status, which, however, showed a dose-dependent effect on the Aβ42/40 ratio. Further studies are needed to replicate these findings including a cohort of cognitively unimpaired individuals and to investigate how blood-based AD-specific biomarkers are impacted by a disruption of the BBB. We hope that this data will highlight the need for further research in this field, which might be crucial for diagnostic and therapeutic purposes.


Autonomic dysfunctions are prevalent in several cerebellar disorders, but they have not been systematically investigated in spinocerebellar ataxias (SCAs). Studies investigating autonomic deficits in SCAs are fragmented, with each one focusing on different autonomic dysfunctions and different SCA subtypes.Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement, we conducted a systematic review of the literature to assess the presence of autonomic dysfunctions in various SCAs. PubMed served as the primary database, and the Rayyan web application was employed for study screening.We identified 46 articles investigating at least one autonomic function in patients with SCA. The results were analyzed and categorized based on the genetic subtype of SCA, thereby characterizing the specific autonomic deficits associated with each subtype.This review confirms the presence of autonomic dysfunctions in various genetic subtypes of SCA, underscoring the cerebellum's role in the autonomic nervous system (ANS). It also emphasizes the importance of investigating these functions in clinical practice.The online version contains supplementary material available at 10.1007/s00415-023-11993-8. Autonomic dysfunctions arise from abnormalities in the autonomic nervous system (ANS), a complex regulatory system responsible for maintaining physiological homeostasis. The ANS consists of the sympathetic and parasympathetic nervous systems, which innervate various visceral organs, each serving distinct functions –3. ThrouAutonomic dysfunctions can result from diverse factors, including hereditary predispositions, acquired conditions, or even adverse effects of pharmacological treatments , or can The role of the cerebellum in the ANS is extensively supported by animal and human studies that have identified many connections between brainstem nuclei and higher-order autonomic centers, enabling both direct and indirect influences on autonomic pathways –13. EarlThe central role of the cerebellum in regulating the ANS and the occurrence of autonomic deficits in cerebellar-related diseases directed our investigation toward the relationship between SCAs of different genetic origins and autonomic dysfunctions. SCAs are rare, genetically inherited neurodegenerative cerebellar disorders that lead to motor, cognitive, and social-affective deficits –25. AccoThe present review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement 2020 , an updaThe following autonomic dysfunctions have been investigated: postural/orthostatic hypotension, postural/orthostatic dizziness, postural/orthostatic syncope, gastrointestinal dysfunctions, urinary dysfunctions, sexual dysfunctions, sweating disorders, cardiac autonomic dysfunctions, thermoregulatory disorders, and heat/cold intolerance. More specifically, gastrointestinal dysfunctions included constipation, diarrhea, nausea, and nonspecified gastrointestinal dysfunctions; urinary dysfunctions included incontinence, dysuria, bladder dysfunctions, voiding difficulties, frequency, urgency, nocturia, retention, and nonspecified urinary disturbances; sweating dysfunctions included hypohidrosis, hyperhidrosis, and nonspecified sweating dysfunctions. When articles did not specify the type of autonomic dysfunction, deficits were reported as “aspecific autonomic dysfunction,” indicating the presence of an autonomic impairment of unspecified nature. In addition, to better characterize the presence of autonomic dysfunctions in genetically different populations, articles were divided based on the specific genetic diagnosis. SCA subtypes for which little evidence was found were combined into a single group to give an overview of the preliminary literature present for less prevalent SCAs. Articles that investigated autonomic functions in more than one type of SCA were cited more than once within each patient group examined. Each article was screened to collect data on genetic diagnosis, experimental or descriptive measures of autonomic dysfunctions, and demographic and clinical characteristics of the study population . When available in the text, percentages, means, and standard deviations of the measures of interest were collected. Alternatively, when applicable, these measures were independently calculated from data reported in the articles using an Excel spreadsheet. Additionally, the percentage prevalence of each autonomic dysfunction was calculated for each SCA subtype based on the sum of patients found to be impaired compared with the total sample in which the specific autonomic dysfunction was investigated in the various studies. The calculation was performed via an Excel spreadsheet.Importantly, in a few papers, autonomic dysfunctions were divided into different subtypes. For example, different subtypes of urinary dysfunctions were sometimes reported, specifying the exact number of patients affected by each subtype. However, since a patient could be affected by multiple subtypes of urinary dysfunction, it was not possible to estimate how many patients in total suffered from overall urinary dysfunction. In cases such as this, the results of the most impaired subcategory have been indicated, but the data have not been included in the prevalence calculation to maintain its truthfulness. Furthermore, in some studies, specific autonomic dysfunctions were analyzed in a smaller number of subjects out of the total study sample. In these cases, prevalence analyses were carried out on the exact number of subjects in which the dysfunction was investigated.The bibliographic search conducted in PubMed identified 393 articles. After Rayyan importation, 66 duplicates were removed, and two investigators (N.U. and L.S.) independently reviewed the studies based on titles and abstracts. This first inspection involved 327 articles, 269 of which were excluded according to the inclusion and exclusion criteria. After full-text reading, the two investigators removed an additional 26 studies, resulting in a total of 32 included articles. The inspection of these studies (text and bibliography) led to the addition and screening of 16 relevant articles, of which two were excluded in accordance with the eligibility criteria. The final analysis involved 46 articles  in accordance with the selection criteria. The PRISMA updated flowchart synthesizing the search and screening process is presented in Fig. In the following paragraphs, the results are reported according to the genetic subtype of SCA. The order of presentation is determined by the number of studies that have investigated each subtype. In each paragraph, autonomic deficits are listed in order of prevalence. Detailed urinary, gastrointestinal, and sweating dysfunctions are shown in Tabs S1, S2, and S3 in the supplementary materials, respectively. The other autonomic dysfunctions were not described in detail as they lack characterization through multiple symptoms or distinct sub-disorders.The autonomic dysfunctions found in patients with SCA3 are shown in Table The autonomic dysfunctions found in patients with SCA2 are shown in Table The autonomic dysfunctions found in patients with SCA1 are shown in Table The autonomic dysfunctions found in patients with SCA6 are shown in Table For most of the other genetic subtypes of SCA, few studies were identified, which did not allow for their individual characterization. Here, we report the results for SCA17, SCA31, SCA7, SCA4, SCA5, SCA8, and SCA10, presented in Table The primary objective of this systematic review was to investigate the presence of autonomic deficits in patients with diverse genetic subtypes of SCA, aiming to further elucidate the role of the cerebellum within the ANS and to identify potential deficits currently underexplored in clinical practice. The results enabled us to characterize the peculiar autonomic disturbances of each SCA subtype while also assessing preliminary findings for less commonly investigated SCAs. However, despite the abundance of findings available, not all studies investigated the whole body of autonomic dysfunctions, with many focusing on the investigation of specific deficits. As a result, the estimated prevalences provided in this review may be more reliable for certain autonomic dysfunctions, drawing from the results of numerous studies, while others are less reliable. Subsequently, the results obtained for different genetic subtypes will be discussed, followed by a comparison between different SCAs to explore the potential involvement of the cerebellum in determining the observed autonomic deficits.Autonomic dysfunctions have been extensively investigated in SCA3 patients, leading us to identify 26 relevant studies , the alterations in autonomic nuclei and autonomic fiber tracts found by Gierga and colleagues (2005) in SCA2 patients could explain the cardiac autonomic and gastrointestinal disorders reported in this patient population. The disturbances in cardiac autonomic functions could arise from a lack of cerebellar control, supported by recent evidence that correlates the HRV response with the activation of the right cerebellar cortex in patients with different SCA subtypes . FurtherIn conclusion, based on the identified autonomic dysfunctions and the supporting evidence for the role of the cerebellum in these dysfunctions, it can be asserted that SCAs frequently present with autonomic deficits that may arise from cerebellar degeneration. These deficits warrant further investigation to facilitate their identification and treatment in clinical practice.To our knowledge, this systematic review represents the first investigation into autonomic dysfunctions in patients with different genetic subtypes of SCA. However, it is essential to acknowledge some limitations. Despite employing strict inclusion and exclusion criteria, the inclusion of both case reports and studies with varying methodologies might introduce bias in the estimated prevalences. However, given the limited number of studies investigating these dysfunctions in patients with SCAs, imposing stricter criteria could result in missing important results. Additionally, the data provided by the authors in the studies did not allow further classification of patients according to sex, precluding the assessment of potential differences in autonomic deficits between men and women in the various investigated SCAs.Considerations pertaining to extracerebellar alterations in SCAs warrant thorough examination. Particularly, SCAs like SCA2, SCA3, and SCA1 exhibit extensive damage not only within the cerebellum but also across the brainstem, pons, basal ganglia, thalamus, and midbrain . NotablyNotwithstanding the aforementioned limitations, this review marks a significant step toward identifying the potential role of the cerebellum in the onset of autonomic deficits, a domain previously overlooked in this population and one that will need further investigation.With regard to the cerebellum's role in autonomic deficits, further exploration is warranted, especially in relation to cardiac autonomic dysfunction. The majority of the studies that assessed cardiac autonomic dysfunction in various SCAs focused on measures of HRV, an index of cardiac parasympathetic control . HoweverSupplementary file1 (DOCX 49 KB)Below is the link to the electronic supplementary material.


Purpose: The driver mutations of gliomas have been identified in cerebrospinal fluid (CSF). Here we compared the concordance between CSF and tumor tissue for integrated diagnosis in gliomas using next-generation sequencing (NGS) to evaluate the feasibility of CSF detection in gliomas.Patients and methods: 27 paired CSF/tumor tissues of glioma patients were sequenced by a customized gene panel based on NGS. All CSF samples were collected through lumbar puncture before surgery. Integrated diagnosis was made by analysis of histology and tumor DNA molecular pathology according to the 2021 WHO classification of the central nervous system tumors.Results: A total of 24 patients had detectable circulating tumor DNA (ctDNA) and 22 had at least one somatic mutation or chromosome alteration in CSF. The ctDNA levels varied significantly across different ages, Ki-67 index, magnetic resonance imaging signal and glioma subtypes (p < 0.05). The concordance between integrated ctDNA diagnosis and the final diagnosis came up to 91.6% . We reclassified the clinical diagnosis of 3 patients based on the results of CSF ctDNA sequencing, and 4 patients were reassessed depending on tumor DNA. Interestingly, a rare IDH1 R132C was identified in CSF ctDNA, but not in the corresponding tumor sample.Conclusion: This study demonstrates a high concordance between integrated ctDNA diagnosis and the final diagnosis of gliomas, highlighting the practicability of NGS based detection of mutations of CSF in assisting integrated diagnosis of gliomas, especially glioblastoma. Central nervous system (CNS) tumors present survival challenges for people because of high incidence and high mortality. Glioma is the most common primary CNS tumor, accounting for 80.8% of the malignant CNS tumors in adults . IntegraIDH1 and IDH2), 1p/19q codeletion, EGFR amplification, amplification of chromosome 7 and loss of chromosome 10 (+7/−10), TERT promoter alteration, ATRX deletion, TP53 positive, and CDKN2A/B deletion are usually used for molecular diagnosis and stratification of most patients with gliomas [The method of integrating histological and molecular characteristics heightens the objectivity of CNS tumor diagnosis and prompts the precision treatment for the CNS tumors. Mutations including IDH ( gliomas .Cell-free circulating tumor DNA (ctDNA) is shed by tumor cells, and can be found in circulating blood, along with normal cells . The cliIn this study, we performed a comprehensive genomic analysis based on targeted next-generation sequencing (NGS) of DNA from the tumor tissue and CSF ctDNA of 27 glioma patients, aiming at determining the feasibility of ctDNA from CSF in Chinese population. We compared the mutational and diagnostic concordance between the tumor tissue and CSF and revealed the utility of CSF ctDNA for advancing and complementing the diagnosis of gliomas.We prospectively collected the clinical data of 27 glioma patients admitted to Nanjing Jinling Hospital  from December 2020 to July 2021. All patients were confirmed with glioma by magnetic resonance imaging (MRI). CSF samples (4–8 mL) were collected from the lumbar puncture before surgery. Tumor tissue samples were collected from surgical specimens, and part of removed tumor tissue was used for histopathologic diagnosis. 4 CSF samples were collected from patients with encephalitis as normal controls. In accordance with the Declaration of Helsinki, written informed consent was obtained from all individuals or guardians, and the study was approved by the Institutional Review Board of Nanjing Jinling Hospital (2022DZKY-093-01) and local regulations.Cell-free DNA (cfDNA) was extracted using an Apostle MiniMax High Efficiency cfDNA Isolation Kit (APOSTLE) according to the manufacturer’s instructions. Fresh tissue DNA was extracted using a QIAamp DNA Tissue & Blood Kit (Qiagen) according to the manufacture’s recommendations. Extracted cfDNA and DNA were quantified by Qubit4.0 (Life Technologies) according to the manufacturer’s protocols. Quantitative levels of CSF cfDNA were measured in haploid genome equivalents per milliliter (hGE/mL), which was calculated as the result of total cfDNA concentration and the mean allele fraction of somatic mutations.NGS libraries of tumor tissue DNA were prepared with KAPA HyperPlus Kit (Roche). cfDNA libraries were established with the xGenTM Prism DNA Library Prep Kit (IDT). After hybridization of DNA and cfDNA libraries, the captured libraries were amplified by polymerase chain reaction (PCR) according to the manufacturer’s protocols. As described previously, we performed the targeted sequencing by NGS with the panel containing 131 genes and 4 chromosomes (131 + 4 panel) related to brain tumors (see All CSF and tumor samples were measured for single nucleotide variants (SNVs), copy number variants (CNVs), and chromosomal variations. Initial sequencing reads were trimmed and filtered with fastp (v.2.20.0) and then aligned against the human reference genome (hg19) using BWA-mem (v.0.7.17). The realignment of local regions was performed to improve the accuracy of variant detection around Indels using ABRA2.We used VarDict (v.1.5.7) for variant detection. The results of variant detection were annotated to multiple databases using SnpEff and ANNOVAR software, including dbNSFP (v.4.2a), COSMIC (v.9.6), gnomAD (v.3.11), ClinVar and more. According to annotation results, the filtration was further conducted, and the background baseline was used to filter systematic false positive results. In combination of Intervar results and pathogenic interpretation from databases including ClinVar, somatic and germline mutations were finally identified. Threshold for mutation calling is ≥0.5% variant allele frequency (VAF) in CSF and ≥1.0% VAF in tumor tissue.The original sequencing depth was counted, which was standardized to exclude the influence of the amount of sequencing data. The CNVkit algorithm was used to construct the background distribution model of the copy number with normal genomic sequencing. CNVs were determined based on the difference between the copy number in each region of the sample and the background model. The structural variations of chromosomes were determined according to the copy number distribution of the whole chromosome arm. CNVs were analyzed by CNVkit (dx1.1). We used CSF from the patients with non-tumor diseases as the baseline, and mutations that were identified as false positive were excluded.Hematoxylin and eosin staining and immunohistochemistry (IHC) were performed on 4 µm-thick sections using standard protocols. IHC stains contains Ki-67 (Roche), and IDH1 R132H (Agilent).All cases were comprehensively diagnosed and reviewed by the two pathologists (NYL and NW) using histology, as well as molecular features of tumor DNA according to the 2021 WHO CNS classification criteria and the Consortium to Inform Molecular and Practical Approaches to CNS Tumour Taxonomy group in 2018–2020 –16.p-values lower than 0.05 were considered as statistically significant. To assess the possibility of CSF ctDNA test for glioma diagnosis, we performed a receiver operating characteristic (ROC) curve analysis. Consistency of diagnosis was examined by Kappa measure of agreement. The Kappa values were categorized as “almost perfect” (0.8–1.0), “fair to good” (0.4–0.8), or “poor” (below 0.2) [Statistical analysis and plots were conducted by Graphpad Prism 5 and IBM SPSS statistics software version 19. Quantitative ctDNA differences across different characteristics of patients were assessed using Mann-Whitney U test. Spearman’s rank correlation coefficient test was used to evaluate the association between the tissue DNA and CSF ctDNA with SNVs and CNVs. low 0.2) .n = 13), astrocytoma (n = 6), pilocytic astrocytoma (n = 2), pleomorphic xanthoastrocytoma (n = 2), oligodendroglioma (n = 1), ganglioglioma (n = 1), glioneuronal tumor (n = 1), and gliosarcoma (n = 1). The median age of the participants was 54 years (range 15–78 years) and 66.7% (n = 18) were male. These patients were confirmed pathologically and classified into histological grade I (n = 3), grade II (n = 6), grade III (n = 4), grade IV (n = 14). After surgery, 63.0% (n = 19) patients received standard Stupp treatments, including adjuvant radiotherapy and temozolomide (TMZ) chemotherapy, 11.1% (n = 3) patients received adjuvant radiotherapy. The characteristics of these patients were presented in Twenty-seven gliomas were recruited for the CSF and tumor biopsy tissue collection. Histopathological subtypes included glioblastoma , ventricular involvement (p = 0.303), tumor number (p = 0.644) and location (p = 0.762), as well as involvement of cerebral cortex (p = 0.769) (We measured the ctDNA level among 27 CSF samples and found the ctDNA level showed significant differences across age (= 0.769) .TERT was the most prevalent gene. TERT promoter mutations were detected in 12 (50%) of patients via CSF, while were identified in 16 (66.7%) of patients though tumor tissue. The concordance with the TERT mutation status between CSF and tumor tissue yielded 83.3%. The mutations were detected both in tumor tissue and CSF samples, apart from FLT4 and CIC mutations. Abnormal gene copy number was found in 10 patients for CSF ctDNA. The coincidence between CSF and tumor tissue for CDKN2A/B deletion was 87.5%. However, 8 patients presented with PTEN deletion in tumor DNA but not detected in CSF ctDNA. Alterations of chromosome chr 1p, chr 19q, chr 7, or chr 10 were detected in 11 CSF samples.Twenty-seven patients were all detected using 131 + 4 panel, and 3 of them were excluded on the grounds of their undetectable ctDNA concentration. We identified at least 1 tumor-related gene mutation or chromosome variant from CSF ctDNA of 22 patients, consistent with the number of tumor tissue DNA samples . In contr = 0.64, p < 0.0001; r = 0.86; p < 0.0001; TERT, TP53, EGFR, PTEN, PIK3CA, and PDGFRA .17 Case 11, a 59 year-old woman, was diagnosed as anaplastic pleomorphic xanthoastrocytoma, WHO Ⅲ, in September 2019, and recurred in March 2021 with histologically confirmed as GBM, WHO IV  PCR, and next-generation sequencing . Sanger In our study, 3 patients  with grade II diagnosed by histology showed ctDNA concentration below the detection limit. In general, people with CNS tumors harbored a very low ctDNA level in CSF, although it was much higher in CSF than in blood , 25. FurTumor cell turnover is accelerated as tumor size and degree of malignancy, resulting in release of apoptotic and necrotic tumor cells into the circulation –28. We dTERT mutation, TP53 mutation, PTEN mutation, EGFR mutation, and CDKN2A/2B loss. The molecular features were consistent with those of an adult glioma cohort previously reported [TERT mutation is recognized as the most prevalent molecular marker for GBMs [H3F3A G35R and K28M variants had been found in two gliomas separately, but no IDH mutations were detected in these two patients. It has been reported that mutations in H3F3A and IDH1 were mutually exclusive [In our cohort, the hotspot aberrations detected involved reported . TERT mufor GBMs . H3F3A Gxclusive . The diaxclusive . GeneralIDH1 mutation in CSF ctDNA from case 11, whereas a negative result was found in tumor DNA. The samples we collected were prospectively obtained by removing a small section of the tumor. Intratumor heterogeneity may explain why sampling bias leads to a difference in biomarker discovery and not every driver or passenger mutation will be discovered in each cancer cell [IDH1 in gliomas and IDH1 R132H mutations are the most predominant mutation which indicates a better prognosis and may be effective for inhibitor treatment [IDH1 mutation with R132C was found. Previous studies showed that R132C had a more favorable prognosis than R132H for gliomas with a much higher enzymatic metabolism efficiency [EGFR amplification, +7/−10, and TERT promoter mutation are alterations frequently observed in adult IDH-wildtype GBM [TERT promoter mutations in CSF ctDNA. Weller et al. found TERT promoter mutations combined with +7/−10 had the worst progression-free survival and OS compared with those having TERT promoter mutation alone [CSF ctDNA detection exhibited some attractive features in help to observe the molecular biomarkers. We found a rare cer cell , 37. CSFcer cell . IDH1/2 cer cell , 40. Argreatment . In our ficiency . EGFR amtype GBM . Notablyon alone . ImportaPrecise tumor subtyping and grading are essential for prognosis and treatment of patients. Our present study demonstrated that CSF ctDNA may be a reliable alternate tumor source for integrated molecular diagnosis of gliomas, especially for GBMs, supported by previous studies , 46. In In conclusion, CSF ctDNA detection for integrated molecular diagnosis of gliomas especially GBMs is feasible. NGS-based detection of CSF ctDNA could be used as a supplement for integrated diagnosis of gliomas. In the future, CSF ctDNA sequencing will form genetic profiling for each patient, assisting in targeted therapy and enhancing the therapeutic effect.


Donor derived infections (DDIs) in pediatric kidney transplant recipients remain challenging to diagnose and can result in serious morbidity and mortality. This review summarizes the current guidelines and recommendations for prevention, diagnosis, and treatment of unexpected DDIs in pediatric kidney transplant recipients. We provide a contemporary overview of DDI terminology, surveillance, epidemiology, and recommended approaches for assessing these rare events with an emphasis on the pediatric recipient. To address prevention and risk mitigation, important aspects of donor and pediatric candidate evaluations are reviewed, including current Organ Procurement and Transplantation Network (OPTN) and American Society of Transplantation (AST) recommendations. Common unexpected DDI encountered by pediatric transplant teams including multi-drug resistant organisms, tuberculosis, syphilis, West Nile Virus, toxoplasmosis, Chagas disease, strongyloidiasis, candidiasis, histoplasmosis, coccidioidomycosis, and emerging infections such as COVID-19 are discussed in detail. Finally, we consider the general challenges with management of DDIs and share our experience with a novel application of next generation sequencing (NGS) of microbial cell-free DNA that will likely define a future direction in this field. Kidney transplantation remains the treatment of choice for children with advanced or end-stage kidney failure. However, transplantation is not without risk. Balancing immunosuppression with risk of infection remains an ongoing challenge, especially in the context of donor derived infections (DDIs). DDIs can cause serious morbidity and mortality for solid organ transplant (SOT) recipients. These complications can be even more devastating in pediatric SOT recipients and early recognition, timely reporting, close monitoring, and appropriate management are essential to help mitigate these risks.Unexpected donor derived disease (DDD) was recently reported to occur in 0.18% of SOT recipients in a review of patients from 2008 to 2017 , as outlined in policy 15.4 . With revia SOT and it remains essential for clinical transplantation teams to remain vigilant to this risk, react quickly to potential DDI, and start treatment immediately.In contrast, “unexpected” transmission of various types of pathogens from donor to recipient occur when donor infection is previously unknown, undetected, and/or incompletely treated . For theThe Ad Hoc Disease Transmission Advisory Committee (DTAC) reviews any potential donor derived transmission events in a blinded fashion. After committee review, the probability of a transmission event is determined and classified as proven, probable, possible, unlikely, excluded, intervened upon without documented transmission (IWDT), positive assay without apparent disease transmission, or not assessable as defined in The most recent published report from DTAC describes all reports received between 2008 and 2017 in both pediatric and adult SOT donors and recipients. Of the 2,185 reports during that time period, 335 (15%) were considered a proven or probable donor transmission event with an overall rate of unexpected donor derived disease at 0.18% of all transplant recipients .A prior report of DDD limited to pediatric donors and recipients showed overall similar results. Between 2008 and 2013, there were 103 reported potential DDIs reported to involve pediatric donors or recipients. With regard to pediatric recipients, 11 cases were classified as proven or probable events  using United States Centers for Disease Control and Prevention (CDC) recommended HIV laboratory algorithm-Hepatitis B Virus (HBV) surface antigen (HBsAg), hepatitis B core antibody , hepatitis B surface antibody (HBsAb)-Hepatitis C Virus (HCV) antibody (anti-HCV), hepatitis C ribonucleic acid by nucleic acid test (NAT).As of July 1, 2022, the OPTN updated its policy on pre-transplant evaluation for all candidates less than 12 years of age with regard to HIV, HBV, and HCV testing. Pediatric patients less than 12 years old must be tested any time prior to transplant and the testing must be documented, but it does not have to occur at time of hospital admission for transplant. This policy change was made given incidence of these infectious are very low in this age group and there is a large concern for overdrawing blood, especially immediately prior to a solid organ transplant -certified laboratory or equivalent. For a candidate to be eligible for an organ transplant, the following testing is required  by OPTN per policy 15.2, updated in 2020 :-Human ansplant .-Screening for periodontal disease and treatment prior to transplant-Tuberculosis (TB) with purified protein derivative (PPD) or interferon-gamma release assay and chest x-ray-CMV IgG antibody-EBV with EBV viral capsid antigen IgG, IgM and/or EBV nuclear antigen (EBNA) IgG-Herpes simplex virus IgG-Varicella IgG-Mumps IgG, Rubella IgG, Rubeola IgG-Human T-lymphotropic virus 1 & 2 with IgG serology in candidates from endemic areas-Syphilis with any of the following: treponema pallidum particle agglutination, treponema pallidum immunoassay, rapid plasma regain, venereal disease research laboratory-Toxoplasmosis with IgG antibodyIn endemic areas, other testing should be strongly considered such as strongyloides serologies, coccidiodes serologies, histoplasmosis serologies/urine antigen tests, chagas serologies, malaria screening with a malaria blood smear, West Nile Virus serologies or NAT, and/or hepatitis A total antibody, hepatitis A IgM  guidelines from 2019 and KDIGO Clinical Practice Management Guideline on Evaluation and Management of Candidates for Kidney Transplantation 2020, recommend the following candidate infectious disease screening:is A IgM , 12. ThiThe Coronavirus Disease 2019 (COVID-19) pandemic presented new challenges for infectious disease screening and recommendations are evolving. Based on a review from 2021, the current recommendation is to wait at least 4–6 weeks after recovering from COVID-19 prior to transplantation. It is also recommended that patients undergo pulmonary function testing (PFT) and have SARS-CoV-2 IgG and RT-PCR once fully recovered . If a caAll eligible vaccinations should be administered to patients on the waiting list potentially on an accelerated schedule, if necessary. A 4-week delay of transplantation is recommended after any live vaccinations . Many peWhile it is impossible to eliminate the potential for transmission of DDIs, a thoughtful and thorough approach to donor evaluation can mitigate risk. Various screening protocols have been proposed, although the sensitivity and specificity of these approaches are unknown . In geneSpecific details from the donor medical and social history can provide insight into the risk of DDI transmission. In the case of a deceased donor, the cause of death should be a starting point. For example, a donor who dies from trauma is a much lower infectious risk than one who died from complications of intravenous drug use . AlternaDetails in a social history can help provide risk stratification, although the reliability of this information can be variable in between living and deceased donors. In the setting of a deceased donor, the social history may be limited and/or incomplete. Elements of a social history that suggest increased risk of blood borne viral pathogens include high risk sexual contacts ; men who have sex with men; persons who have had sex in exchange for money or drugs; and persons who have had sex with a person who uses injection drugs nor non-medical reasons), birth to a mother infection with HIV, HBV, or HCV (for donors under 2 years old), persons use injection drugs for non-medical reasons, inmates of a correctional facility for more than 3 days in the past 12 months, persons treated for gonorrhea, chlamydia, or genital ulcers, or persons who have been on hemodialysis in the preceding 12 months . Donors While use of organs at increased risk of infection transmission at adult centers has the potential to decrease wait times, use of PHS increased risk organs in pediatric recipients is uncommon . The pedAny available information about the clinical and physical assessment of the donor needs to be assessed. This can be done by review of available records (such as imaging studies) or direct observation of the donor and organ by a procurement team. Physical evidence of infection includes unexplained rash, abscesses, ulcers and lymphadenopathy. Recent injection drug use can be inferred by the presence of track marks or skin-popping ulcerations. Cross-sectional imaging can also reveal evidence of active infection, such as metastatic foci, lymphadenopathy, or granulomatous disease .The review of infectious disease-related laboratory data is of utmost importance. OPTN policy requires that all donors undergo testing of commonly transmissible infectious diseases . In addiIn deceased donors, all microbiological culture data should be reviewed and ideally a minimum of 48 hours of no culture growth should be available prior to the acceptance of an organ. Ideally, any known active bacterial or fungal infection in the donor should be treated and resolved before transplantation  is not routinely done for decreased donors due to the length of time required for traditional screening methods. Use of the purified protein derivative (PPD) test is not practical due to the time needed to assess a response . Even ifAs noted in the candidate evaluation, COVID-19 presents new challenges for infectious disease screening and recommendations are evolving as new variants arise, changes in therapeutic options, and vaccination availability. SARS-CoV-2 NAT positive organs had similar outcomes compared to COVID-19 NAT negative donors on 30-day patient and graft survival . More inAdditional considerations for pre-transplant deceased donor testing includes testing for endemic infections such as strongyloidiasis, coccidioidomycosis, histoplasmosis, chagas disease, malaria, West Nile, and/or hepatitis A , 29.It is important to consider the limitations of infectious disease screening in donors. There are limitations to diagnostic tests and donor history, especially in the time-sensitive setting of a deceased donor offer. There is always risk of undetected infection for transplant candidates and recipients. Understanding some of these limitations can help inform risk and management of DDIs.Currently, either serologic or nucleic acid testing (NAT) can be performed to detect viral infections. Historically, much of this evaluation was done using serologic testing given it is less expensive. However, NAT is now being used more commonly due as the technology is becoming more affordable and available.Serologic testing is limited by poor sensitivity early after initial infection, as seroconversion may not occur during the acute phase of an infection. This “window period” can result in a false-negative result and be a high risk period for donor transmission . NAT mitThe potential for hemodilution in deceased donors is another important limitation of NAT or serologic tests . Many poThere can be a wide spectrum of presentation of unexpected DDIs and clinicians need to have a high index of suspicion. It is possible that DDIs are an under recognized cause of morbidity and mortality . Early rRecognize Potential Infections-Recipients with atypical post-transplant course-Recipient with early post-transplant bacterial or fungal infection-Multiple recipients of organs from the same donor with the same infection-Atypical infection for the early post-transplant periodConsider DDIs in the following settings:Report and Communicate-Team should NOT wait until donor origin is confirmed.-Report should be made NO later than 24 hours after initial concern.-In the USA, reports should be made to UNOS via the Patient Safety Portal and to the patient safety contact for the transplant centers and OPOs. The patient safety contact is listed in DonorNet.-Inform all key members of the team, including but not limited to the transplant team, risk management, and media relations.-Make direct contact with any involved patients to ensure they are asymptomatic and current clinical status must be obtained.-Provide timely updates and information to your patients.As soon as any team member considers a potential DDI, a report should be made to all involved organ procurement organizations (OPOs), transplant centers, and transplant authorities:Collect and Retain Specimens and Data-Contact pathology – retain any fresh residual specimens, biopsies, and/or discarded tissue from the donor and recipient.-Contact microbiology – retain any cultures and/or residual specimens that were sent for testing.-Contact molecular diagnostics lab – retain any sequences and/or specimens sent for PCR or similar testing.-Contact HLA – retain any donor and/or recipient blood, lymph nodes, cells or tissues, particularly pre-transplant specimens.As soon as you are informed of the potential disease transmission event, collect the following key specimens and data:Test and Manage-For most viral pathogens you will need direct testing of the virus with PCR/NAT or antigen testing.-Serologies may be unreliable in post-transplant period, particularly if there was significant blood transfused.-Provide appropriate therapy based on presumed pathogen.Continue routine monitoring for disease transmission.Perform appropriate testing of the recipient for potential DDI:The American Society of Transplantation Infectious Disease Community of Practice has published general guidelines for the approach to donor-derived infection evaluation in 2019. These guidelines outlined in below involve steps for recognizing potential infections, reporting and communicating, how to collect and retain specimens and data, and tests and management .RecogniWhile every effort is made to treat any known positive cultures prior to transplantation, given the urgency of donation at times, cultures can become positive after transplantation. If a documented bacteremia is found, the recipients should also receive a course of antibiotics targeting the pathogenic organism for at least 7–14 days  prophylaxis which could theoretically also prevented transmission. More studies are needed to understand the risk of positive urine cultures for kidney transplant recipients. Regardless, at this time it is still recommended to treat positive urine culture, especially of virulent or more resistant organisms .Special attention must be paid to any multi-drug resistant organism (MDRO) identified in the potential donor as the risk mitigation is extremely difficult and/or unmanageable . MDROs aIn a multicenter, retrospective, cohort study of 658 adult SOT recipients with deceased donors, it was found that MDRO significantly increased early post transplantation infection risk, although did not appear to affect long term graft or recipient survival the 1-year mark . In thisMycobacterium tuberculosis. The risk of DDI with TB varies depending on geographical location. In non-endemic areas, risk factors for donor derived TB include donors who were born outside the US or prolonged residence outside of the US , history of incarceration, homelessness, alcohol and/or substance abuse, healthcare worker, and known history of TB/latent TB infection (LTBI) (Tuberculosis (TB) is caused by the bacterial pathogen, n (LTBI) . Active n (LTBI) .As noted in the donor evaluation portion of this review, it remains challenging to screen for TB given the time limitations of deceased organ donation typically does not allow for PPD placement and there are challenges with interferon gamma release assay. Interferon gamma release assay most often does not result until post-transplantation and had indeterminate results at least 30% of the time .The median time to presentation of donor derived TB in kidney transplant recipients was 2 months post-transplantation and carries a high risk of morbidity and mortality. In a review from 2018 on adult SOT, graft loss was seen in 18.2% of patients, all-cause mortality was 25%, and specifically TB attributable mortality was 44%. The clinical presentation is usually persistent fever and often has an insidious course, which requires a high index of suspicion for diagnosis. Allograft involvement is common in non-lung SOT, presentation commonly included extrapulmonary TB or disseminated TB presentations  is an arborvirus transmitted to humans by way of bites from infected mosquitos, typically of the d States . In heald States . The mosd States . Risk ofd States .α-2b or Ribavirin (WNV incubation is between 3 and 15 days and methods for diagnosis include serology (IgM and IgG), NAT, or viral culture of serum and/or CSF . Infecteibavirin .Donor screening for WNV is not currently required by OPTN policies and practices vary based on center and OPO . CurrentToxoplasma gondii, which is considered a neglected parasitic infection by the CDC . General treatment recommendations include induction with pyrimethamine, sulfadiazine, and leucovorin for a minimum of six weeks followed by lifelong suppression in conjunction with infectious disease consultation .Trypanosoma cruzi. The majority of infections occur in endemic areas of South and Central America. In immunocompetent individuals, most infections are mild with fever or minimal symptoms. Without treatment, the infection is lifelong and can manifest later as severe gastrointestinal and/or cardiac disease in 20%–30% of patients  . IRIS caMoreover, there is often lack of definitive evidence to determine treatment courses, especially on topics such as secondary prophylaxis in the SOT population . The eviWith the advent of new technologies and diagnostic tools, there is the potential for earlier and more accurate diagnoses of infection to guide decisions about accepting an organ, understand potential risks of transplantation, determine need for post-transplant prophylaxis, and to guide management of DDIs. One example of new technology shaping SOT infectious disease care is next generation sequencing (NGS) of microbial cell-free DNA. There is evidence that NGS of (cf)DNA may result in de-escalation of antimicrobial therapy, shorten duration of therapy, and more rapidly identify microbes than conventional culture-based methods. This also would result in improved antimicrobial stewardship in the SOT population. However, there are still unanswered questions about the challenges of interpretation in immunosuppressed individuals and further investigation is needed .Encephalitozoon cuniculi) approximately 12 weeks status post deceased donor kidney transplantation. Our local OPO was alerted that two other patients who had received organs from same donor had posttransplant infections with Encephalitozoon cuniculi. Despite an array of tests to assess for Encephalitozoon cuniculi, our patient was only found to be positive on microbial cell-free DNA testing. Strong communication with our OPO and transplant teams led to the recognition of Encephalitozoon cuniculi in our pediatric patient prior to the development of fulminant disease. To date, our patient has had no evidence of recurrent microspordiosis and microbial cell free DNA has been used to help track the infection and guide management DNA in the case of pediatric kidney transplant recipient with unexpected donor derived microsporidiosis (nagement . This scAs always, it is essential that the transplant community continues to engage in high quality research and innovation to develop new technologies, directed therapies, and a more personalized approach for our SOT who remain at high risk of DDIs.While DDIs overall rare, the consequences can be devastating, especially in the pediatric kidney transplant population. Prevention is key and clinicians must maintain a high index of suspicion for a possible DDIs, especially in any patient who does not follow the expected post-transplant course. As transplant care providers, we must remain vigilant in staying up to date on emerging infections and highly resistant bacteria. Strong communication between OPOs and transplant teams will always be essential to helping recognize, manage, and/or treat DDIs. Furthermore, new innovative ways to rapidly identify potential pathogens prior to transplantation will be essential to help mitigate risk for candidates.


Intrathecal pseudodelivery of drugs is a novel route to administer medications to treat neurodegenerative diseases based on the CSF-sink therapeutic strategy by means of implantable devices. While the development of this therapy is still in the preclinical stage, it offers promising advantages over traditional routes of drug delivery. In this paper, we describe the rationale of this system and provide a technical report on the mechanism of action, that relies on the use of nanoporous membranes enabling selective molecular permeability. On one side, the membranes do not permit the crossing of certain drugs; whereas, on the other side, they permit the crossing of target molecules present in the CSF. Target molecules, by binding drugs inside the system, are retained or cleaved and subsequently eliminated from the central nervous system. Finally, we provide a list of potential indications, the respective molecular targets, and the proposed therapeutic agents. Neurodegenerative diseases (NDD) are a group of disorders of the central nervous system (CNS) that cause progressive death of nerve cells and loss of function in the brain and spinal cord. The CNS compartments are represented by the parenchyma of the brain and spinal cord, including the intracellular space with the intracellular fluids (ICF) and the extracellular space with the interstitial fluid (ISF), and the cerebrospinal fluid (CSF) space. The tissues of the CNS are separated from the systemic circulation by the blood-brain barrier (BBB) and blood-CSF barrier (BCSFB) ,2. ThesePathologically, besides cellular loss, most NDD exhibit molecular hallmarks such as beta-amyloid (Aβ), tau, α-synuclein, mSOD1, and TDP-43. Disease-distinctive proteins exist in different states that aggregate between them: soluble monomers aggregate together to form dimers and oligomers, that can form soluble protofibrils. In turn, protofibrils aggregate to form insoluble fibrils, that eventually deposit in the form of plaques or tangles . SolubleFrom this perspective, the central event in the pathophysiology of NDD is a proteostasis imbalance leading to protein aggregation overwhelming the proteostasis capacity of brain cells , and interfering with the ability of neurons to cope with pathogenic proteins ,11, whicIn parallel to proteostasis disbalance, a common feature in NDD is chronic immune activation, in particular of astrocytes and microglia, the resident macrophages of the central nervous system . The CSFThe administration of drugs targeting the CNS poses challenges. The main difficulty is related to poor drug penetration through the BBB, as multiple in vivo, in vitro, and in situ experiments have demonstrated . Small mIn recent years, a multitude of brain drug delivery technologies have emerged, including trans-cranial delivery, CSF delivery, BBB disruption, lipid carriers, prodrugs, stem cells, exosomes, nanoparticles, gene therapy, endogenous BBB carrier-mediated transport and receptor-mediated transport systems . As presInvasive techniques rely on implantable devices accessing the CSF-intrathecal (IT), intraventricular (IVT) or ISF intraparenchymal delivery. In contrast to the extensive use of the CSF for diagnostic purposes, the CSF has not been frequently regarded as a target biological fluid for therapies for CNS conditions because it requires invasive procedures and systems. Today, few drugs are delivered in the CSF, mainly because it is an invasive procedure not without risks. However, in recent decades, therapies addressed at the CSF have gained some momentum as a result of advanced treatments such as gene therapies and replacement enzymatic therapies which need IT or IVT delivery. While the IT drug delivery pathway is still regarded as an experimental approach in neurodegenerative diseases, this route has long been successfully used in the treatment of other pathological conditions and in the symptomatic therapy of pain and spasticity ,43,44,45Controlled drug delivery systems (DDS) seek to improve patient compliance by increasing therapeutic efficacy, extending drug release time and stability, increasing drug bioavailability, reducing side effects, and reducing dosage frequency. Moreover, DDS contribute to the safety of pharmaceuticals during their whole delivery period by serving as various kinds of protective barriers that enclose them, minimizing the loss of active ingredients and any harmful impacts on patients. They are typically constructed at nanometric and micrometric levels in order to combine several qualities such as site-specificity, endurance, or external stimuli sensitivity ,49,50.Intrathecal pumps are a good example of implantable DDS targeting the CNS. Intrathecal pumps consist of an electromechanical pump cased with a metal reservoir that stores the medication, and a catheter that is implanted in the spinal intrathecal space to deliver the medication from the pump to the CSF, thus accessing the CNS where the medication takes effect. Two types of pumps are available: a constant rate pump delivers the medication at a constant rate, and a programmable pump delivers the medication according to a rate determined by a computer program ,52. WhilDifferent approaches have been investigated with the aim of removing pathogenic proteins from the CNS, including inhibition of protein synthesis, and promoting protein degradation. Most therapeutic strategies addressed to enhance the clearance of brain proteins rely on clearing them from the periphery . DiseaseIndeed, several attempts have been made to treat neurodegenerative diseases using different CSF filtration systems. In ALS, extracorporeal CSF filtration was shown to successfully mitigate the neurotoxic capacity of CSF from subjects with sporadic Amyotrophic Lateral Sclerosis (ALS) in vitro  and in aIt is worth revising methods aimed at CSF dilution or enhancing CSF flow, as they are closely related to CSF-sink therapeutic strategy. In AD, mechanical dilution of CSF has long been a proposed therapeutic approach . CSF shuTherapeutics such as enzymes, antibodies, and even transport proteins , which are mostly intended to link with molecular targets to be removed from the organism, do not really need to be released in the fluid or tissue to action. In fact, binding to the molecular target can be achieved regardless of the compartment. With this in mind, IT pseudodelivery of drugs is a novel concept to administer drugs to treat CNS conditions relying on the CSF-sink therapeutic strategy , by meanFunctional nanoporous materials are an important class of nanostructured materials because of their tunable porosity and pore geometry  and their unique chemical and physical properties. Progress in developing a broad spectrum of nanoporous materials has accelerated their use for extensive applications in biomedical fields . NanoporIT pseudodelivery is the first DDS to be endowed with nanoporous membranes acting on the CNS ,74,75. DA target molecule should be present in the CSF (soluble). This should be identified as potentially “toxic” or “pathogenic” and involved directly (aggregating proteins) or indirectly (mediators) in the physiopathology of the disease.A drug acting specifically on the target molecule is needed. This can be an antibody, an aptamer, an enzyme, or any other compound that has specificity over the target molecule and either binds or cleaves the target molecule.A significant size difference should exist between the target and drug molecules. While other physicochemical features may also play a role (such as electrostatic charge), the size difference is the main feature driving the selective molecular permeability through nanoporous membranes.Not every target molecule or drug is suitable to be targeted/used via pseudodelivery. For a disease to be suitable to be treated using IT pseudodelivery, three conditions must be met:While the development of this therapy is still in the preclinical stage, it offers promising advantages over traditional routes of delivery. Being target-selective provides advantages over other CSF clearance systems since the level of other proteins —not involved in disease pathogenesis—would be preserved. It also provides important advantages over “standard” peripherally administered drugs, including the following: 1. Acting continuously, on the CSF directly, is expected to be much more effective than acting peripherally. 2. Immunoisolation of drugs impedes immune responses, fully avoiding immunologically mediated side effects reported with biological drugs systemically administered ,75.In contrast, potential adverse effects related to the intrathecal system implantation and functioning should be taken into consideration, with expected local complications similar to those seen with intrathecal pumps, such as CSF leak, hemorrhage, and infection, along with device-derived problems such as CSF flow obstruction or even device disconnection ,75.The field of disease-modifying therapies for NDD is one of the hottest topics in medicine nowadays. Despite a myriad of studies, no effective disease-modifying treatment is available at the present for most of these conditions  while thMonoclonal antibodies (mAbs) directed against misfolded proteins such as Aβ, Tau protein, or α-synuclein are a first choice when considering IT pseudodelivery, as they have been demonstrated to be effective when administered intravenously in many studies . MoreoveOther molecules binding pathogenic proteins can be of interest. For instance, human serum albumin (HSA) is a natural buffer of Aβ. A promising approach to AD prevention is to reduce the concentration of free Aβ by targeted stimulation of the interaction between HSA and Aβ. This approach can be implemented by pseudodelivering albumin alone  or in coAnother therapeutic possibility is to act on the enzymatic dysfunction, a relevant example being the switch from the non-amyloidogenic pathway to the amyloidogenic one in AD . In the Protein conformation stabilization and aggregation inhibition that targets the upstream of the insoluble aggregate formation would be a promising approach toward the development of disease-modifying therapies for most NDD, particularly for polyQ diseases. PolyQ aggregation inhibitors of different chemical categories, such as intrabodies, peptides, and small chemical compounds, have been identified through intensive screening methods ,117. AmoFinally, another clear target in NDD are molecules involved in inflammation such as anti-TNF-α. According to several reports, anti-TNF-α agents may affect amyloidosis in inflammatory/autoimmune diseases, such as rheumatoid arthritis and familial Mediterranean fever . Indeed,Drugs targeting the complement component C5, CD19 on B cells, and the inter-leukin-6 (IL-6) receptor, have been used for the treatment of patients with refractory inflammatory CNS diseases. Particularly, Tocilizumab, a humanized, monoclonal antibody against the IL-6 receptor, has been tested for neurologic indications, such as neuromyelitis optica  or primaLastly, a TREM2-activating antibody with a BBB transport vehicle enhances microglial metabolism in AD models  and tau IT pseudodelivery of drugs is a novel concept to administer drugs to treat NDD, based on the CSF-sink therapeutic strategy by means of implanted devices. The mechanism of action relies on the properties of nanoporous membranes enabling selective molecular permeability.Being an invasive procedure, the expected safety issues of IT pseudodelivery are related to device implantation and functioning. However, the promising advantages of IT pseudodelivery of drugs in terms of efficacy and drug-related safety would overcome the disadvantages.Potentially, there are a number of NDD where IT pseudodelivery might be of interest. While there is a theoretical rationale supporting these indications, in vitro and in vivo testing is still lacking for most of them; therefore, the proposal should be considered just a therapeutic hypothesis today.


Here, we present a successful right donor hepatectomy from a donor with a BMI of 36.5 kg/m2. Case Summary. A 39-year-old wife donated her right lobe of liver to her 43-year-old husband with nonalcoholic steatohepatitis-related chronic liver disease (CLD). His indications were refractory ascites, hepatic encephalopathy, acute kidney injury, recurrent elbow and urine infections leading to cachexia. She was initially rejected due to a high BMI but failed to lose weight over the next 2 months, and the need for a transplant in her husband was imminent. With no other potential living donors, we decided to proceed with donor evaluation as she had no other comorbidity. We were surprised to find normal liver function tests and a good liver attenuation index (LAI) of +16 on a computed tomography (CT) scan. Magnetic resonance (MR) imaging revealed a fat fraction of 3%. Volumetry confirmed a remnant of 37.9% and a potential graft-to-recipient weight ratio of 1.23. V/S ratio on CT scan  was <0.4 confirming subcutaneous fat obesity. Both surgeries were uneventful and both donor and recipient recovered well except recipient re-exploration on postoperative day (POD)-1 due to surgical bleeding. The donor was discharged on POD-6 and recipient was discharged on POD-15. At 3 weeks of follow-up, the donor's wound is clean and well-healed, and she is already back to doing her daily life activities without any pain with normal laboratory parameters.  Living donor liver transplantation (LDLT) has revolutionized the field of transplantation without compromising donor safety. Donor safety is of paramount concern to the transplant team. BMI >35 kg/m2. A small percentage of healthy individuals will not have visceral fat obesity and may not have steatotic livers. The CT scan and MR fat fraction estimation can confirm the findings. Biopsy may be avoided if MR fat estimation is <10% in obese donors. Intraoperative visualization in these donors remains the gold standard to decide the need for biopsy. Living donor hepatectomy may be safely performed in a select group of high BMI patients (>35 kg/m2) with pure subcutaneous fat obesity in the absence of other suitable living donors. Subcutaneous fat obesity should not be considered as a contraindication to liver donation even with a BMI >35 kg/m Living donor liver transplantation (LDLT) has revolutionized the field of transplantation since the 1990s, leading to a vast increase in the number of transplants worldwide without compromising donor safety. The donor is usually a healthy individual with minimal to no comorbidity, and donor safety is of paramount concern to the transplant team. The usual limits of donor acceptance in terms of age, body mass index (BMI), liver remnant, fat content of the liver , and other parameters are well established .2 while a BMI of 30–35 kg/m2 would still be evaluated for liver fat estimation [2 is mostly considered a contraindication to liver donation [2) who underwent an uneventful right donor hepatectomy without the need for a liver biopsy.Most centers would accept donors with a BMI <30 kg/mtimation . BMI >35donation , 4. Highdonation , 5. A bidonation , 5. HereStaphylococcus aureus that was not responding to treatment. He had undergone multiple orthopedic interventions with no benefit.The patient is a 43-year-old male patient with nonalcoholic steatohepatitis (NASH)-related chronic liver disease (CLD) since 3.5 years. His weight prior to transplant was around 68 kgs, and his dry weight (after paracentesis) was 63 kgs. His predominant symptoms were refractory ascites with frequent large volume paracentesis which was gradually increasing in frequency, and one episode of hepatic encephalopathy. His Na-MELD (sodium-model for end-stage liver disease) score was 15, and he was on the waiting list for deceased donor liver transplantation since the last 2 years (blood group B positive). He was found to have developed a grade-2 near-complete portal vein thrombosis in the last 4 months, extending almost up to the splenomesenteric confluence. His low hemoglobin required him to have repeated transfusions every 2 months since the last 1 year. He also had left elbow swelling which had persistent infection with methicillin-resistant 2 (weight 81 kgs and height 149 cms), he did not have any potential liver donors. As per our protocol, we advised her for a regimen of diet and exercise and to return in 2 months for reassessment. During these 2 months, the patient got admitted 3 times with repeated elbow infection and swelling. Major orthopedic surgery could not be performed and the elbow was deformed in a position of flexion and medial rotation. He was also getting recurrent Klebsiella infection in his urine, and his creatinine increased to 1.88 mg/dl. He was getting more and more cachexic, and the need for a transplant was becoming more imminent.At this time, he was counseled for living donor liver transplantation (LDLT) in September 2022, but he had no suitable donor. Apart from his 39-year-old wife, who had a matching blood group (O positive) but a high BMI of 36.5 kg/mHis wife returned to us after 2 months with no change in weight (81 kgs). Even with such a high BMI, we decided to evaluate her for donation. She had no significant medical or surgical history. To our surprise, her liver function tests, glycosylated hemoglobin (HbA1c), thyroid function, and fasting lipid profile were normal. The computed tomography (CT) estimation calculated liver attenuation of 66.4 Hounsfield units (HU) (right lobe 66.1 and left lobe 66.7) and a mean splenic attenuation of 49.6 HU, giving us an LAI of +16.5 and +17.1 for the right and left hepatic lobes, respectively . We routth February, 2023. In the operating room, the donor liver looked healthy and nonsteatotic  Doppler showed excellent flows and the graft was pink and soft on inspection .Postoperative course was uneventful for the donor. She was extubated on the table and had no complications. Lactate normalized on postoperative day (POD) 2 and international normalized ratio (INR) was normal on POD-1. Liver function tests (LFT) gradually improved and the diet was started on POD-2. Gradually, she improved and was mobilized. She was discharged on POD-6 in stable condition. Her postoperative labs are summarized in 2 [2 in case the donor is very muscular or there are no other suitable donors [2 [Significant hepatic macrosteatosis is a known contra-indication to living donor hepatectomy , 4. Mostonors [2 , 4. The onors [2 . USG is onors [2 . MRI is onors [2 , 8. Highonors [2 . Donors onors [2 , 9. Theronors [2 , 9.2. With an LAI of +16 and a liver remnant of 37%, we proceeded to confirm our fat estimation by MR spectroscopy. When fat fraction evaluation was reported as 3% (mean) in both lobes and the volumetric analysis was acceptable, we agreed to accept the donor as she did not have any other comorbidity. We further revisited the CT scan to assess the V/S ratio. The CT showed a high amount of subcutaneous fat area (546 cm2) compared to the visceral fat area (202 cm2) at the level of L4. This gave us a V/S ratio of 0.37, which was lower than 0.4  indicating that our potential donor had subcutaneous fat obesity and not visceral fat obesity [In our patient, the recipient was getting sicker with no other suitable donor, so we proceeded to evaluate her even with a BMI of 36.5 kg/m obesity , 11. Hig obesity .The correlation between hepatic steatosis and obesity is well known. However, many studies have focused on the fat distribution and their relationship to liver fat estimates. Recent literature suggests that visceral obesity and V/S ratio have a direct positive correlation to hepatic fat content, while subcutaneous fat alone does not significantly impact it –17. Ther2.Our donor had a normal postoperative course without any complications. She was mobilized on POD-2, drain removed on POD-4, and discharged on POD-6. At 3 weeks of follow-up, her wound is clean and well-healed, and she is already back to doing her daily life activities without any pain. Her follow-up investigations are normal See . The rec2. When no other suitable donors are available, it may be worthwhile to evaluate potentials donors even with a higher BMI to assess for hepatic steatosis. A small percentage of healthy individuals will not have visceral fat obesity and may not have steatotic livers. The CT scan and MR fat fraction estimation can confirm the findings. A biopsy may be avoided if MR fat estimation is <10% in obese donors. Intraoperative visualization in these donors remains the gold standard to decide the need for biopsy. Living donor hepatectomy may be safely performed in a select group of high-BMI patients (>35 kg/m2) with pure subcutaneous fat obesity in the absence of other suitable living donors.In conclusion, subcutaneous fat obesity should not be considered as a contraindication to liver donation even with a BMI >35 kg/m


Post-operative CSF leak remains a significant problem following endoscopic skull base surgery, particularly when there is a high-flow intra-operative CSF leak. Most skull base repair techniques are accompanied by the insertion of a lumbar drain and/or the use of nasal packing which have significant shortcomings. Our aim was to review the results of a large series of endoscopic skull base cases where a high-flow intra-operative CSF leak rate was encountered and repaired to assess if modifications in technique could reduce the post-operative CSF leak rate.A retrospective review of a prospectively maintained database of skull base cases performed by a single surgeon over a 10-year period was performed. Data regarding patient demographics, underlying pathology, skull base repair techniques and post-operative complications were analysed.p = 0.006). This improvement in the rate of post-operative CSF leak was achieved without nasal packing or lumbar drains.One hundred forty-two cases with high-flow intra-operative CSF leak were included in the study. The most common pathologies were craniopharyngiomas , pituitary adenomas  and meningiomas . The CSF leak rate was 7/36 (19%) when a non-standardised skull base repair technique was used. However, with the adoption of a standardised, multi-layer repair technique, the post-operative CSF leak rate decreased significantly (4/106, 4% vs. 7/36, 19%, With iterative modifications to a multi-layered closure technique for high-flow intra-operative CSF leaks, it is possible to obtain a very low rate of post-operative CSF leak, without lumbar drains or nasal packing. The most significant advance in the management of skull base pathology over the last 20 years has been the introduction of endoscopic endonasal approaches (EEA) to lesions of the ventral skull base . These aHowever, a major initial shortcoming of EEA for skull base pathology was the associated high rate of post-operative cerebrospinal fluid (CSF) leak; in early published series, the incidence of this complication often exceeded 20% . CSF leaMultiple skull base repair protocols have been described, and a review of these techniques and their associated results has recently been published by our group . In addiAll endoscopic skull base cases performed by a single surgeon, where an intra-operative high-flow CSF leak was encountered between January 2012 and August 2022, were recorded prospectively in a clinical database and included in this study. Intra-operative CSF leaks were graded according to the Kelly grading system and cases with grade 3 leaks ‘large diaphragmatic and/or dural defect’ were included . For allOur skull base repair technique has undergone several modifications over the 10-year period studied. Our most commonly used technique has involved the use of 2 layers of fascia lata, NSF and a dural sealant. This ‘standardised technique’ involves multi-layer closure with the placement of an inlay  layer of endogenous fascia lata, with a NSF placed over the bony and dural defects. Following this, a further layer of fascia lata is placed over the NSF, and the entire construct is secured with BioGlue® tissue sealant, applied extradurally, as described previously  The ‘non-standardised technique’, rather than being a single, unified technique, reflects a time period when non-standardised techniques were used in the earlier part of the series and involved a variety of combinations of intradural placement of fat graft, synthetic dural substitute, Dura-Guard  or Duramend  or endogenous rectus sheath which was covered by a NSF and secured with BioGlue® or DuraSeal® .No nasal tampons or Foley catheters were used to buttress the skull base repair, and LDs were not utilised as an adjunct to any of the repair techniques.χ2 (with Yate’s correction where necessary) and Fisher’s exact tests were used for intergroup comparisons of categorical variables. Bonferroni corrections were used to correct for multiple comparisons. This project was approved by our institutional audit board and the requirement for individual patient consent was waived, as per institutional policy. The STROBE guidelines were followed in the drafting of this manuscript [All statistical analysis was performed in SPSS version 25 . The nuscript .During the study period (January 2012–August 2022), 541 EEAs were performed by a single surgeon. Of these, 142 EEA resulting in a high-flow CSF were performed. Table p = 0.006). Surgical approach used  had no impact on the rate of post-operative CSF leak. The standardised technique was used significantly more commonly as the series progressed , and the non-standardised technique was last used in 2020. Figure The rate of post-operative CSF leak with our standardised technique was 4/104 (4%). This was significantly lower than the post-operative CSF leak rate associated with the non-standardised technique which was 7/36 (19%). Table p = 0.372)  tissue sealant, which has a demonstrably higher tensile strength than other tissue sealants, may potentially be the factor that obviated the need for a buttress to reinforce the skull base repair . HoweverProphylactic LDs are used by many skull base surgeons in the post-operative period with the aim of reducing the risk of post-operative CSF leaks . Even suFrom our perspective, we feel that the placement of an inlay fascia lata graft as a dural substitute, beyond the margins of the bony/dural defect, is of critical importance. A fascia lata graft larger than the dural defect is placed intradurally, and opposed to the internal surface of the skull base. In our view, the placement of this inlay graft allows the natural brain/CSF pulsations to push the graft against the dura mater with each pulsation, as opposed to the risk of an extradural onlay graft migrating away from the defect. We have not observed any migration of the fascia lata away from the dural defect, which was the rationale for the creation of the ‘Bilayer Button’ by Luginbuhl et al. . While tThe strengths of this study include its prospective nature, and that consecutive cases were included, limiting the influence of any selection bias. The study is further strengthened by the use of robust, validated technique for the grading of intra-operative CSF leaks . The widWith iterative modifications to a multi-layered closure technique based on the use of a vascularised NSF, it is possible to obtain very low rates of post-operative CSF leak, even when a high-flow intra-operative CSF leak is encountered. This was achieved without the use of LDs and/or nasal packing, suggesting that these invasive adjuncts may not be necessary.