This guideline, from a European Society of Organ Transplantation (ESOT) working group, concerns the management of kidney transplant patients with HLA antibodies. Sensitization should be defined using a virtual parameter such as calculated Reaction Frequency (cRF), which assesses HLA antibodies derived from the actual organ donor population. Highly sensitized patients should be prioritized in kidney allocation schemes and linking allocation schemes may increase opportunities. The use of the ENGAGE 5  Although kidney transplantation rates have increased in many countries in recent years, highly sensitized patients typically spend longer waiting for a transplant, or may never receive one. This guideline is aimed at healthcare professionals who are faced with a patient with HLA antibodies, to provide advice regarding the most appropriate way to achieve a successful transplant.The guideline does not include patients undergoing non-renal or multi-organ transplants, and does not consider pediatric recipients in detail.https://esot.org/wp-content/uploads/2022/07/WS06_Full-doc_07202022.pdf.This article provides a summary of the guideline; the full guideline can be accessed at: A working group (WS06) was convened by the European Society of Transplantation (ESOT) as part of the Transplant Learning Journey Project, including healthcare professionals from across Europe with expertise in the field, patient group representatives and a member of the Centre for Evidence in Transplantation, University of Oxford, United Kingdom.• Definition of sensitization• Comparison of practices across Europe for transplanting sensitized patients• The place of kidney exchange programs for sensitized patients• Desensitization strategies• Outcomes after HLA incompatible transplantation• Strategies for access to kidney transplantation for highly sensitized patientsSix areas of interest were defined and are listed below:www.transplantlibrary.com), Medline and Embase and consisted of a mixture of free text and controlled vocabulary terms.For each, a standard systematic search strategy was predefined, using the PICO model to formulate clinical questions. Bibliographic searches were developed for each of the clinical questions by experienced staff from the Centre for Evidence in Transplantation. Systematic searches were conducted in the Transplant Library , when a draft of the final document was presented and discussed, with further refinements following this.Different members of the working group drafted each chapter, which was then reviewed by the whole working group. The initial recommendations were presented at an ESOT webinar open to all, on the 29th June 2021, and again The detailed methodology, including the search strategies used and search dates, is presented in the full guideline .We have presented below a brief summary of each chapter listed above, along with our recommendations. Recommendations were graded according to the strength of the recommendation [strong  or weak High levels of donor-specific HLA antibodies (DSA) present at transplantation are associated with a high incidence of hyper-acute rejection ,4, and cHistorically, complement-dependent cytotoxicity (CDC) was the gold standard measure of HLA antibodies and the degree of sensitization was expressed as a percentage of panel reactive antibodies (%PRA). This %PRA was defined by the percentage of panel donors reactive with the patient serum in CDC. The %PRA was a relatively inaccurate assessment of sensitization, but often a PRA > 85% was considered the threshold for a highly sensitized patient  was introduced in several laboratories ,10. DonoClinically irrelevant antibodies (including autoantibodies) reactive with other structures on lymphocytes can interfere in the outcome of both a CDC and a FCM crossmatch ,13 leadiSolid phase assays were introduced more recently . Single The clinical relevance of antibodies detectable in SAB assays is still a matter of debate . IndividAlthough no absolute thresholds can be defined, it is generally accepted that the highest MFI values predict a positive CDC crossmatch, although exceptions exist as some high MFIs are associated with a negative CDC . As the Most centers use a cut-off MFI of 1,000–1,500  but therRecently, an attempt has been made to introduce more reliable parameters for the definition of the degree of sensitization based on the antibody specificities present in the patient and the HLA phenotypes of the actual organ donor population. Different names are now circulating for this novel parameter: vPRA  , cPRA c and cRF • A parameter, which is based on the HLA frequencies of the actual organ donor population, such as vPRA, cPRA or cRF, should be used to estimate the chance that a sensitized patient can be transplanted with a compatible donor without the need for any special treatment (1C).• When defining unacceptable mismatches in highly sensitized patients on the basis of (weak) antibody reactivities in single antigen bead assays only, one should consider the poorly defined risk of antibody-mediated rejection (ABMR) in the light of a prolonged waiting time and associated mortality and morbidity (2D).• Further standardization of single antigen bead assays and their interpretation is recommended (1C).• Better HLA matching on the basis of antibody epitopes rather than antigens and a restricted transfusion policy will probably diminish the number of highly sensitized patients, but more data are needed.http://www.accord-ja.eu/background). Eurotransplant (https://www.eurotransplant.org/) and Scandiatransplant (http://www.scandiatransplant.org/) each allocate donor organs for groups of countries. Larger donor pools would be expected to increase the likelihood of identifying a compatible donor for those who are hard to match. A survey of transplant practices around Europe was carried out during September and October 2021 for the purposes of this guideline, and the results form Both deceased and living donations are coordinated on either a national basis, or on behalf of a group of countries (https://www.odt.nhs.uk/transplantation/kidney/kidney-offering-and-matching/) or by the development of an Acceptable Mismatch (AM) program  was established in 2016. The program has contributed to aspirations for future developments, including modelling of European KEPs with the aim of future optimization (Kidney exchange programs (KEP) in Europe began in Switzerland in 1999 , and themization .No European country has a published national consensus on their optimal recommended management pathway for highly sensitized patients, although several European centers have published their protocols and outcomes following HLAi transplantation –38. The • We recommend an active policy of prioritizing highly sensitized patients for organ transplantation, using cPRA/cRF (1C).The simplest form of a KEP is a two-way exchange involving two incompatible pairs who swap their donors to achieve a compatible transplant for both recipients . The cloUnfortunately, for highly sensitized patients with a wide range of anti-HLA antibodies or for blood type O recipients, it is very hard to find a compatible match for each pair involved in a closed loop.The option of a non-directed altruistic (or unspecified) donor (NDAD) who is willing to donate his/her kidney with no intended recipient, avoids the need to “close the loop.” The NDAD’s kidney is matched with the recipient of an incompatible pair whose living donor donates to another incompatible recipient, initiating a domino-paired kidney exchange. The chain ends with the donor of the last pair donating to a recipient on the waiting list or waiting for another suitable match, starting another sequence of paired donations later , thus becoming a bridge donor. This model is potentially associated with an incremental risk of donor reneging. The occurrence of broken chains has been reported to be as low as 1.5%, with the most common causes for broken chains being bridge donor medical issues (0.46%), donors electing not to proceed (0.34%) and broken chains resulting from the kidney being declined by the recipient surgeon (0.23%) .The first deceased donor-initiated chain was reported by Furian, et al in 2019 . In the List exchange is another form of KEP, proposed by Delmonico et al. , to prevOther novel KEP schemes take place in the setting of “chronological incompatibility” and constitute the advanced donation programs where a living donor donates his/her kidney at his/her convenience to a recipient of an incompatible pair in need of transplant while his/her intended recipient will receive the reciprocal compatible kidney later on, when he/she actually needs a transplant .ABO or HLA compatible pairs may also be included in a KEP, in order to increase the pool and provide benefits (such as better age or HLA matching) for the compatible recipient. A recent report from the National Kidney Registry linked to data from the Scientific Registry of Transplant Recipients identified 154 compatible pairs involved in kidney exchange programs, seeking to improve their HLA matching through an exchange. These patients obtained a transplant from younger donors, with higher estimated glomerular filtration rate and body mass index and a better score on the living kidney donor profile index as compared with their original donor .Another strategy to improve results is combining exchange programs with desensitization. ABO incompatible transplantation in the absence of DSA provides excellent transplantation results, so ABO incompatible living donors against whom recipients have lower anti-blood group antibody titers can be included in a KEP. This strategy has been successfully applied in the Australian program and at the John Hopkins Institute ,45.Trans-organ paired exchange represents the most innovative concept of KEP. It might be used, for example, when a living kidney donor who is not eligible for renal donation but can donate his/her liver to a liver recipient of a pair whose donor is ruled out from liver donation but is suitable for kidney donation. Torres, et al published the first case of trans-organ exchange, attracting many criticisms related to the surgical risk of donation that is very different for different organs .In 2005, Segev, et al.  showed, However, despite the implementation of KEP strategies, in the United States, patients with a PRA of 99.9% remain the most disadvantaged transplant candidates with prolonged waiting times and high waiting list mortality . In fact• Access to the donor pool should be increased through greater use of:- Increased access to and harmonization of kidney exchange programs with greater and standardized sharing of outcomes (1C)- Inclusion of unspecified kidney donations (if these are performed) in kidney exchange programs (1C)- Inclusion of compatible pairs and deceased donor organs in kidney exchange programs (1C)• Entry into a kidney exchange program is the preferred initial option over desensitization given the better transplant outcomes and cost-effectiveness, unless there is a need for desensitization, there is clinical urgency or a low chance of a transplant (1C).If the strategies listed above have not yielded, or are unlikely to yield, a transplant, desensitization may be considered. There are several ways to desensitize HLA-immunized patients.In a randomized trial , it was Rituximab (anti-CD20) can be used to desensitize patients prior to transplantation. This drug aims to decrease the rebound effect linked to decreased levels of immunoglobulins in the plasma. Efficacy is monitored using the expression of CD19 on B cells. Currently, the two methods used to desensitize patients are either a combination of anti-CD20 antibodies and high-dose IVIgs (2 g/kg over 2–4 days) , or a coIt is possible to decrease the synthesis of proteins (DSAs) using proteasome inhibitors such as the first-generation drug, bortezomib. This drug was tested in a study with such a complex design (including the testing of many other drugs) that it is difficult to clearly see its role in desensitization . Second A logical approach to desensitization is to block the activity of complement in order to decrease the effect of antibodies such as DSAs. The anti-C5 monoclonal antibody, eculizumab, was the first to be tested in this indication. A randomized study was designed for living donor recipients and compared the use of eculizumab for 3 months post-transplantation with a control group who received desensitization . Unfortu®). Imlifidase is currently the only approved therapy for use in the EU for desensitization treatment of highly sensitized adult kidney transplant patients with a positive crossmatch against an available deceased donor. It cleaves all IgGs, both intra- and extra-vascularly, without regard to their specificity, with an immediate action that lasts around 5–7 days; this drug cannot be re-dosed due to immunogenicity (2 (49 ml/min/1.73 m2 for those with ABMR and 61 ml/min/m2 for those without ABMR)  .An additional desensitization strategy is the manipulation of the cytokines involved in B cell activation. In this indication, tocilizumab, an anti-IL6 receptor monoclonal antibody has been giving promising results in a randomized trial, used in addition to current desensitization protocols . Antibod• The most efficacious desensitization strategy is to start with rounds of plasma exchanges/immunoadsorption together with IVIG or B-cell depletion with anti-CD20 monoclonal antibodies (1C).• As yet to be defined protocols including proteasome inhibitors and other anti-plasmocyte antibodies with costimulation blockade, B-cell immunomodulation targeting IL-6 as well as cleavage of IgG donor-specific antibodies with imlifidase are highly promising new strategies that deserve further investigation.Results from HLAi are often compared with those from compatible transplants, but many HLAi patients will never have the option of a compatible transplant, as the chance for the most highly sensitized to receive a deceased donor kidney, or matching in a KEP is essentially nil ,61. It i• A comparison of mortality rates between HLAi and those who remain without a transplant• A comparison of morbidity between HLAi and those who remain without a transplant• A comparison of quality of life between HLAi and those who remain without a transplantWe have therefore considered the following:There are only four studies comparing mortality in those who have undergone HLAi with those who remain on the waiting list, and these are detailed in However, it might have been possible that the survival benefit shown for HLAi was due to the approach in this (expert) center, so in 2016, a study by Orandi  considerThe results from these studies have been partly contradicted by a UK registry study, which found no difference in survival when comparing 213 HLAi patients with 852 well-matched controls who remained on the waiting list . It is un = 3,701), or who received a deceased donor transplant (n = 907). They found that patient survival was significantly better for those undergoing an HLAi transplant, compared with either control group  with a group of matched controls of those who were waiting for a transplant , but interestingly, had lower rates of hospitalization subsequently .There are no studies that compare morbidity in those undergoing HLAi with those who remain on the waiting list. This is an important gap in our knowledge, particularly given the statements above regarding survival. There is one study by Orandi , which cp < 0.001). Another study that compared 27 HLAi patients with 69 ABOi patients, found no significant difference in viral, bacterial or fungal infections between the two groups, although of note, 6% of the ABOi group had PJP, compared with none of the HLAi group  sliding scales or priority points programs; 2) an allocation system based on AM HLA antigens rather than the avoidance of unacceptable ones and 3) achieving HLA compatibility using living donor transplant options, such as ABO incompatible transplantation or KEP.ex vivo in FCM is a good predictor of subsequent ABMR lesions and graft loss in 50% and 30% of recipients, respectively  offers are also permitted due to their lower immunogenicity –79. Rema• National demographics: the incidence of blood groups and HLA types varies across different countries, and will therefore affect the chances within a KEP• The size of the pool: the larger the pool the greater the chances of a match, although there is probably a maximum size beyond which there is no incremental advantage• Recipient characteristics: for example, those who are very highly sensitized  will have a low or even negligible chance in a KEP, for the same reasons that they will have a low chance of receiving a deceased donor transplant• KEP algorithm: each KEP will have its own algorithm, which will affect the chances an individual has for a match in the scheme. This should be considered when entering a patient into the scheme.KEP are discussed earlier but some important points with respect to risk stratification are:https://www.odt.nhs.uk/living-donation/tools-and-resources and at https://www.odt.nhs.uk/transplantation/tools-policies-and-guidance/calculators/, which addresses the likelihood of a deceased donor transplant for sensitized patients.The easiest way to address these factors is to access an online calculator which incorporates the factors into a probability of a match, ideally with confidence intervals. An example from the UK scheme is given at Finally, an important point to consider is that entry into a KEP should not be considered as a definitive solution. Figures from the UK KEP show that the incremental chance of a match after 6 or 7 “runs” is low , and thuThe Eurotransplant AM program fully prioritizes the allocation of compatible donor kidneys to highly sensitized patients >85% cPRA), focusing on finding acceptable matches rather than to prohibit matches . The mai% cPRA, f• To define the humoral risk in kidney transplantation, the use of the ENGAGE 5 strata system is recommended (1C).• Prioritization policies should be linked across countries for equity of access (1C).• The Eurotransplant Acceptable Mismatch program should be expanded to other European countries (that do not have this type of matching) to improve donor/recipient matching (1C).• All kidney exchange programs should develop calculators to help assess the probability of an organ match (1C).• Therapeutic options (including HLA- or ABO- incompatible transplantation) should be reconsidered if there are no organ offers for a patient in a kidney exchange program (1C).• Work to develop schemes to help patients with very high cPRA or cRF who may not be transplanted in kidney paired donations or under deceased donor priority schemes should continue.• The role of induction immunosuppression in relation to sensitization and its role in long-term outcomes should be further explored.• Whether better risk stratification, thorough immunological evaluation and avoidance of HLA-DSA can improve outcomes should be determined.We have given a suggested algorithm for approaching patients with HLA antibodies in


AD-MCI patients showed lower MMSE and FWT scores  than non-AD-MCI. The best predictor of amyloid plaques’ presence at amyloid-PET imaging was the encoding sub-score of the FWT (AUC = 0.84). Both FWT and MMSE had low/moderate accuracy for the detection of pathological CSF Aβ42, t-Tau and p-Tau181 values, with higher accuracy for the t-Tau/Aβ1–42 ratio. In conclusion, the FWT, as a single-domain cognitive screening test, seems to be prompt and moderately accurate tool for the identification of an underlying AD neuropathological process in patients with MCI, supporting the importance of associating biomarkers evaluation in the work-up of patients with dementing neurodegenerative disorders.New diagnostic methods have been developed for the early diagnosis of Alzheimer’s disease (AD) with the primary purpose of intercepting the transition-phase  between normal aging and dementia. We aimed to explore whether the five-word test (FWT) and the mini-mental state examination (MMSE) are predictive for the early diagnosis of MCI due to AD (AD-MCI). We computed ROC analyses to evaluate the sensitivity and specificity of MMSE and FWT in predicting abnormal CSF (t-Tau, p-Tau Alzheimer’s disease (AD) represents one of the most common causes of dementia ,2,3, witFor more than 20 years, AD remained a probabilistic clinical pathological syndrome , but witNevertheless, there is still no agreement about which cognitive screening instruments could be more sensitive and specific enough to detect AD in the early phases of disease . SeveralThe aim of the present study was to explore whether the FWT, as a screening neuropsychological test appointed for the episodic memory assessment, may be prompt, valid, and predictive clinical marker of AD compared with the MMSE. Moreover, the ultimate purposes consist in investigating the feasibility of the screening tests administration to guide the early clinical differential diagnosis, to verify the probability of intercepting the earlier stages of the Alzheimer’s disease and, consequently, to increase the patients’ possibility of being eligible for the disease-modify therapies.This study included a consecutive sample of 96 participants from the outpatient neurological Centre of Cognitive Disturbances and Dementia (CDCD) unit of the ASST Spedali Civili Hospital , across a four-year interval (2017–2021). We enrolled all patients that reported memory complaints without functional impairment in activities daily living, that underwent FTW and MMSE testing at baseline, and that underwent amyloid-PET imaging or CSF analysis. After cognitive evaluation and biomarker assessment, patients were divided in two groups: AD-MCI and non-AD-MCI.The study was approved by the Ethic Committee of the ASST Spedali Civili di Brescia Hospital (protocol NP4818) and was conducted in accordance with the statements of the Declaration of Helsinki.Participants underwent the cognitive assessment with two screening instruments, the five-word test  and theIn order to carry out the FWT test, a list of 5 words in their Italian translation , printed on a sheet of A4 paper, was shown to the patients, who were asked to read and, later on, to point and name out loud each item when the matching semantic category cue was verbally given by the examiner. Then, after removing the sheet the subjects were requested to recall the words; when one or more words were not spontaneously recalled, the semantic category cue was given in order to stimulate the item’s retrieval. An immediate recall score (IRS) was obtained by adding the number of spontaneously retrieved items to those that were retrieved thanks to the semantic cue. If the subjects failed again to recollect any words, the sheet would be shown and removed again until the missing items were identified and retrieved (max. 3 repetitions) to ensure the possibility to proceed with the second phase; this step had no impact on the individual IRS ,33. DuriFurthermore, we assessed functional independence using the basic (BADL) and instrumental activities of daily living (IADL) questionnaires ,35. We aTo investigate amyloid burden, PET amyloid imaging was acquired using 370 MBq (10 mCi) of 18F-florbetapir or 18F-flutemetamol, following the procedures provided by the ligand manufacturer, as previously reported  were recruited in the present study of whom 53 (55.2%) were classified as AD-MCI , and 43 (44.8%) as non-AD-MCI , according to clinical, CSF and amyloid PET results. Groups were comparable for age and years of education , with a median (IQR) score of 23.4 (20.7–24.9) in the AD-MCI and 25.4 (23.0–26.3) in the non-AD-MCI group.We observed a significant difference in MMSE scores between groups (p < 0.001)  [3.0 (2.0–4.0) vs. 4.0 (3.0–5.0)], in the delayed recall score (DLR) [3.0 (1.0–4.0) vs. 4.0 (3.0 - 5.0)], and in the total recall score (TRS) [3.0 (2.0–4.0) vs. 4.0 (3.0–5.0)] between AD-MCI and non-AD-MCI, respectively  see .Regarding biomarker assessment, 75.0% of patients underwent CSF analysis, 17.7% amyloid-PET imaging and 7.3% performed both. As expected, we observed significant differences in CSF biomarkers between groups see .1–42 and MMSE and FWT  scores and a negative correlation between CSF t-Tau and p-Tau181, and MMSE and FWT  scores . We observed larger correlation coefficients between CSF parameters and FWT DRS than IRS (see A Spearman rank-order correlation was run to assess the relationship between CSF biomarkers and MMSE and FWT scores. There was a significant positive correlation between CSF Aβ IRS see .p = 0.011); on the contrary, patients with abnormal CSF Aβ1–42 values showed significant lower values in MMSE scores, FWT DRS and FWT TRS . Regarding CSF t-Tau and p-Tau181, we observed significant differences only for MMSE scores in patients with abnormal t-Tau levels (p = 0.017), and differences in both tests in patients with abnormal p-Tau181 levels .We also analyzed MMSE and FWT scores relatively to the presence/absence of an abnormal biomarker according to laboratory cut-offs, regardless of group . We founp = 0.004], while MMSE, FWT DRS and TRS performed similarly in predicting abnormal CSF Aβ1–42 levels, CSF p-Tau181 levels and, to a minor extent, also CSF t-Tau levels , p < 0.001] , vels see . We obse001] see .1–42, t-Tau and p-Tau181) and the presence of amyloid plaques at amyloid-PET imaging.MCI can be considered as the transition phase between normal aging and dementia . The evaAging is one of the main risk factors for the development of dementia ,49, whil1–42, and a negative one between MMSE and CSF t-Tau and p-Tau181. Furthermore, we found a positive correlation between immediate, delayed and total recall scores at the FWT and CSF Aβ1–42, and a negative one with CSF t-Tau and p-Tau181, suggesting that better episodic memory performance  and higher sensibility to the semantic cue are linked to an reduced disease burden [Regardless of group, we found that cognitive performance measured with both MMSE and FWT correlated with CSF biomarkers, expanding the literature about the relation between FWT and biomarkers. Specifically, in accordance with a previous study in an MCI population , we foune burden ,47. We aIn addition, abnormal amyloid-PET imaging was only associated with lower memory encoding ability  , while n1–42, t-Tau and p-Tau181 pathological values seem to be detected with low/moderate accuracy by the MMSE and FWT, with higher accuracy for DRS and TRS sub-scores. However, considering the CSF t-Tau/Aβ1–42 ratio > 1, the specificity increases especially for the DRS FWT (from 42% to 64%) and TRS FWT (from 46% to 68%), in accordance with previous studies [Regarding CSF biomarkers, Aβ studies .These findings provide evidence for a differential effect of abnormal amyloid deposition evaluated with CSF or amyloid PET imaging on memory scores.We acknowledge that the present study entails some limitations. First, the number of patients that underwent CSF analysis is unbalance to those who underwent amyloid-PET imaging and only a few underwent both. Second, this is a single center study with a limited number of patients, and results should be confirmed in larger multicenter cohorts. Further research should verify these results expanding the sample and comparing the FWT scores in patients that underwent both CSF and amyloid-PET imaging analyses, and compared to a group of healthy controls.42, t-Tau and p-Tau181 values.In summary, taken together our data suggest that the discriminatory proprieties of the encoding subscore of the FWT is accurate for detecting amyloid plaque pathology at PET imaging. Moreover, MMSE and FWT scores have only moderate accuracy for detecting abnormal levels of CSF AβIn conclusion, the MMSE and FWT screening tests seem to be rapid but only moderately accurate tools for the identification of an underlying AD neuropathological process, highlighting the importance of associating biomarkers evaluation in the work-up of patients with dementing neurodegenerative disorders.


In vitro osteoclast methods require constant treatment with macrophage colony stimulating factor (M-CSF) to support precursor survival and addition of the differentiation agent receptor activator of NF-κB ligand (RANKL). Constant exposure to granulocyte macrophage colony stimulating factor (GM-CSF) suppresses human osteoclast formation in vitro. Addition of the chemokine monocyte chemotactic protein-1 (MCP1) to such cultures dramatically increases osteoclast formation and overcomes GM-CSF mediated suppression. We investigated the effect of M-CSF, GM-CSF and the combination of M-CSF and GM-CSF treatment on the expression of chemokines in human CD14+ cells in culture. Of assayed chemokines, MCP1 was the most abundant in terms of mRNA transcript and protein in M-CSF treated cultures and was suppressed by GM-CSF. MCP1 protein accumulated up to 50 ng/mL in culture medium, greatly exceeding other assayed chemokines. C-C chemokine receptor-2 (CCR2) is the receptor for MCP1: the formation of osteoclast-like cells was inhibited by constant exposure to the CCR2 antagonist RS102895, in part by decreasing expression of RANK, the receptor for RANKL. Chemokines are a diverse family of small secreted proteins, first identified via leukocyte chemotaxis, that mediate signalling between cells via G-coupled trans-membrane receptors ,2,3. MonIn mice, MCP1 knockout reduced osteoclast size , while kThe effect of M-CSF, GM-CSF and RANKL on the abundance of chemokines in human osteoclast precursor cultures is an important piece of information. In this paper we present data on CD14+ mononuclear cells purified from human blood, used as effective osteoclast precursors in order to further understanding of the biology of chemokines as they relate to human osteoclast differentiation.7 cells in a final volume of 100 μL. The suspension was incubated at 4C for 15 min and then cells recovered by centrifugation  and washed. Cells were then resuspended and run through a column in a magnetic field. In this method, while the magnetic field is present, CD14+ cells are retained, and non-bound cells are washed through. After adequate depletion of the CD14- cells, the magnetic field was removed and CD14+ cells were collected. The yield of CD14+ cells was 18.9% (±4.5% standard deviation) of total PBMC measured from 13 different blood isolations.All cell culture reagents and vessels were from Life Technologies . All peptide hormones were from Peprotech . Cells were routinely cultured in Dulbecco’s modification of Eagle’s medium (DMEM) augmented with 10% heat inactivated fetal bovine serum (FBS). Healthy volunteer donors gave blood samples with informed consent in protocols approved by the Griffith University Human Ethics Committee . Blood was taken directly into BD Vacutainer CTP Cell preparation tubes  that contain heparin to isolate peripheral blood mononuclear cells (PBMC) by centrifugation. Blood was centrifuged in a swing-out rotor for 15 min at 1500 relative centrifugal field (RCF) in a Beckman Allegra centrifuge . The mononuclear cell layer was removed and washed in 15 mL of phosphate buffered saline (PBS) and recovered by centrifugation at 300 RCF. Cells were counted using a hemocytometer slide and following the recommended amounts used for magnetic bead separation. The washed mononuclear fraction was incubated with magnetic-tagged anti-CD14 antibody and subsequently purified by magnetic selection to produce a CD14+ enriched fraction of PBMC according to the instructions of the manufacturer . Briefly, 20 μL of CD14 Microbeads were used per 10CD14+ cells were cultured in plastic multi-well plates and in DMEM with 10% FBS augmented with various combinations of growth factors, essentially as described in Day et al. . Cells w5 cells/cm2 with 5 mL medium and cultured for several days. Medium (100 μL) was taken without replacement for assays at time points. Some experiments with pre-treatment and post-treatment were conducted in 12-well plates with 1 mL of medium and CD14+ cells plated at 2 × 105 cells/well. Culture medium samples were assayed using BioRad Bioplex Human Cytokine 27-plex Immunoassay for chemokines and cytokines according to the manufacturer’s instructions . Bioplex is a Luminex  immunobead based multiplex assay system that provides simultaneous assays of multiple target molecules with a high dynamic range and appropriate calibration tools provided by the manufacturer ethyl]-spiro -2(1H)-one, described by Mirzadegan et al.  and know5 copy of GAPDH or per million copies of 18S, depending on which particular housekeeper was used in that experiment. Primers used in this study are shown in The RNA was isolated from cultured cells in multi-well plates using TRI-ZOL reagent  as per the manufacturer’s instructions. cDNA was produced using the Improm-2 reverse transcriptase system  using both random primers (300 ng) and oligo dT (300 ng). RT-PCR was carried out using the Bio-Rad iQ iCycler real-time PCR system using SYBR Green 1 supermix (Bio-Rad) using primers and conditions as previously described ,9,24,25.Osteoclast-like multinucleated giant cells were considered as cells with three or more nuclei. Two indexes were used to measure the formation of such osteoclast-like cells. Osteoclast area is the area of a microscope field occupied by osteoclast-like multinucleated giant cells as a percentage of the total area of the field. Typically, five fields are measured per culture. Osteoclast fusion index is the proportion of nuclei found within multinuclear giant cells with three or more nuclei as a percentage of the total number of nuclei in microscopy fields. Typically, between 500 and 5000 nuclei are counted to establish the fusion index. Since nuclei do not divide in osteoclasts, the fusion index is a measure of the total accumulated fusion events in the culture. Hoffman modulation contrast microscopy was used to view live cells. Cells were also fixed and stained with 4′,6-diamidino-2-phenylindole (DAPI) to visualise nuclei, tartrate resistant acid phosphatase (TRAP) to identify TRAP+ cells and rhodamine-phalloidin to identify F-actin rings as described previously . We did t-tests. In all figures, error bars are standard error of the mean. Data are derived from either duplicate or triplicate cell cultures performed simultaneously. Occasionally, as described in figure legends, data are presented as relative mRNA levels, meaning that all differences are presented relative to a particular treatment used as a comparison. The Pearson’s chi-squared test was used to compare counts of nuclei within MNC and non-MNC populations. In measures of inhibition of osteoclast formation, the hypothesis is one-tailed, in that suppression is expected for osteoclast phenotypic measures. Gene expression and other phenotypic measures are correlated tests of the null hypothesis tested by linear regression against log of concentration for each parameter.For real time PCR, the difference between the cycle threshold (CT) of the gene being examined and the reference housekeeper gene was referred to as ΔCT. Data were considered on occasion as fold difference using the delta-delta CT (ΔΔCT) method. CT, ΔCT and ΔΔCT values are normally distributed parameters, and are suitable for analysis of variance (ANOVA) and The CD14+ fraction of PBMC was isolated from fresh blood and plated directly in medium supplemented with either 25 ng/mL M-CSF or 25 ng/mL M-CSF and 40 ng/mL RANKL, in order to verify the formation of multinucleated osteoclast-like cells. Such cultures produced abundant osteoclast-like cells with prominent F-actin rings and numerous nuclei A,B at 7 p = 0.003). MCP1 mRNA levels were not intermediate in combined M-CSF and GM-CSF treatments, showing that GM-CSF suppression of MCP1 still occurs in the presence of M-CSF . For CCL1, CCL3 and CCL4, the combined treatment with GM-CSF plus M-CSF, resulted in intermediate mRNA levels, suggesting some suppression of GM-CSF induction, relative to GM-CSF alone treated cells.Human CD14+ mononuclear cells were isolated from fresh blood and plated in medium supplemented with either M-CSF, GM-CSF or a combination of M-CSF and GM-CSF to test the effect of culture pre-treatment on the expression of chemokines. Relative mRNA levels for various chemokines were determined. These data show that GM-CSF induces the expression of chemokines CCL1, CCL3 and CCL4 while M-CSF induces expression of MCP1 A–D. CCL1of M-CSF B, at leaThe chemokine CCL5  was expressed at very low levels in all experiments (data not shown). Although CCL1 mRNA was about 45 times more abundant in GM-CSF treated cells compared to M-CSF treated cells A, the abThe data suggest, in CD14+ monocytes, that: (1) M-CSF induces MCP1 and suppresses CCL1 and the MIP1 family members, MIP1α (CCL3) and MIP1β (CCL4); and (2) that GM-CSF suppresses MCP1 while inducing CCL1, CCL3 and CCL4. Therefore M-CSF and GM-CSF compete to produce a different chemokine outcome in human osteoclast precursor cultures.The expression of chemokine receptors from CCR1 through to CCR8 was assessed using RT-PCR in CD14+ human mononuclear cells pre-treated with either M-CSF, GM-CSF or M-CSF combined with GM-CSF. Chemokine receptors CCR3, 4, 6, 7 and 8 had very low transcript abundance and will not be considered further. For those receptors where assays were calibrated , assays suggest a rank order of mRNA abundance of CCR5 > CCR1 > CCR2b mRNA transcript in this experiment . AlthougIn this series of experiments, CD14+ cells were isolated from peripheral blood and then stimulated to form osteoclasts using RANKL in the presence of M-CSF after three different pre-treatments. Cells were pre-treated for five days with either M-CSF, GM-CSF (25 ng/mL) or M-CSF plus GM-CSF (both at 25 ng/mL) prior to media change then subsequent treatment under conditions to generate macrophage-like cells  or osteoclast-like cells (RANKL plus M-CSF). A zero-time sample was taken, representing the state of the cells immediately after the 5 day pre-treatment. The effect of pre-treatment of cells on gene expression was followed day 1, 3 and 5 after media change . CCL1 mRIn M-CSF pre-treated cells, MIP1α (CCL3) mRNA was of low abundance at zero time , but shoChemokine protein levels were assessed in similar cell experiments. Cells were cultured as above with pre-treatment with either M-CSF, GM-CSF or GM-CSF plus M-CSF combined treatment. After five days, MCP1 accumulated to around 7 ng/mL in the medium of M-CSF treated cells, but to a lesser extent in cells pre-treated with GM-CSF A. The meWe determined the relative rank order of abundance of MCP1 and other chemokines secreted in media by CD14+ cells in culture. Furthermore, we tested whether there was any difference in chemokine accumulation between M-CSF alone and M-CSF plus RANKL treatment. These cultures were set up so that sufficient media were present to permit repeated sampling of 100 μL aliquots and that media did not require changing during the assay period. CD14+ cells were purified as before and plated in media supplemented with either M-CSF alone or the standard combination of M-CSF plus RANKL. Aliquots were taken at third-daily intervals and assayed for chemokines. MCP1 was clearly very abundant in culture media, with levels up to 50 ng/mL A, exceedIn contrast to MCP1, other assayed chemokines accumulated more slowly and to a much lower concentration B–D. MostRANKL had a specific effect to increase MIP1β between 7 and 9 days, coinciding with a drop in MCP1 concentration. In contrast, CCL5 accumulated to low levels, reaching around 270 pg/mL at day 9 D. The peThe data shows that MCP1 protein is abundant in media from cultured CD14+ cells, greatly exceeding that of other chemokines assayed. RANKL did not alter MCP1 accumulation but had a late effect on accumulation of CCL3, CCL4 and CCL5.The compound RS102895 is a small molecule antagonist of chemokine receptor CCR2 with IC50 values reported as 360 nM and 17.8 μM for CCR2b and CCR1, respectively . The amoThe key observation in this study is the surprisingly large amount of MCP1 made by human osteoclast precursor cultures during treatment with M-CSF. These data show that conditioned media from human osteoclasts may contain up to 50 ng/mL MCP1. Prior work suggested that RANKL was responsible for MCP1 increase; however, this work using CD14+ human cells suggests that M-CSF is the primary driver of MCP1 expression. Several authors using human, mouse and rat cell systems and different culture conditions, report that adding exogenous MCP1  to osteoclast cultures leads to larger, more abundant osteoclasts with higher nuclear counts ,13,14. OOf human osteoclast models, crude PBMC preparations isolated from blood by density gradient centrifugation are the easiest to prepare but are a mixed culture of many cell types including some that suppress osteoclast formation, such as T-cells. Colony-forming unit granulocyte-macrophage (CFU-GM) and colony-forming unit macrophage (CFU-M) have high osteoclastic potential but are rather difficult to prepare, requiring cloning in methocult of hematopoetic stem-like cells from umbilical cord blood . Such ceWe did not observe any meaningful difference in any chemokine concentration or gene expression comparing M-CSF treated cultures with M-CSF and RANKL treated cultures. Any differences that were observed occurred at 9 days of culture, after osteoclast formation, and coincident with the cultures reaching the end of their capacity to grow. Osteoclasts have limited lifespans and apoptotic material accumulates in cultures as osteoclasts expire. The apparent RANKL difference in MIP1α (CCL3), MIP1β (CCL4) and RANTES (CCL5) protein concentrations at day nine see  may be aHow the MCP1 enhancement of osteoclast formation occurs is not yet understood, but may relate to either precursor expansion, cell fusion or osteoclast life span. MCP1 protein in culture medium reaches ng/mL levels quickly in the presence of M-CSF. The lack of difference in MCP1 concentrations between M-CSF and M-CSF plus RANKL treated cultures suggests that the large amount of MCP1 produced is a component of macrophage lineage differentiation under the influence of M-CSF. In other words, more mononuclear osteoclast precursor cells might differentiate or survive better in the presence of MCP1 induced by M-CSF. Osteoclast numbers are increased on treatment with MCP1 and enhanced bone resorption has been observed with MCP1 supplementation ,9,10. SuThe CCR2 antagonist RS102895 blocked CCR2 mediated early effects in vivo in a mouse fracture healing model resulting in delayed healing and much lower numbers of TRAP+ osteoclasts in the fracture site . Likewis


Despite recent advances in diagnostic procedures for neurological disorders, it is still difficult to definitively diagnose some neurodegenerative diseases without neuropathological examination of autopsied brain tissue. As pathological processes in the brain are frequently reflected in the components of cerebrospinal fluid (CSF), CSF samples are sometimes useful for diagnosis. After CSF is secreted from the choroid plexus epithelial cells in the ventricles, some flows in the brain, some is mixed with intracerebral interstitial fluid, and some is excreted through two major drainage pathways, i.e., the intravascular periarterial drainage pathway and the glymphatic system. Accordingly, substances produced by metabolic and pathological processes in the brain may be detectable in CSF. Many papers have reported changes in the concentration of substances in the CSF of patients with metabolic and neurological disorders, some of which can be useful biomarkers of the disorders. In this paper, we show the significance of glucose- and neurotransmitter-related CSF metabolites, considering their transporters in the choroid plexus; summarize the reported candidates of CSF biomarkers for neurodegenerative diseases, including amyloid-β, tau, α-synuclein, microRNAs, and mitochondrial DNA; and evaluate their potential as efficient diagnostic tools. Cerebrospinal fluid (CSF) is produced by the choroid plexus (CP), which is situated in the lateral, third, and fourth ventricles. CP with a vascularized stroma is covered by a monolayer of epithelial cells interconnected by tight junctions a 1,2]. ,2. 1,2].+, 2.9 mM K+, and 130 mM Cl−, showing 305 mOsm/L and pH 7.27 . A. A39]. And Aldoc , which and Aldoc . These fRegarding the association between CSF fructose levels and the pathogenesis of neurodegenerative diseases, some findings have been reported. A non-targeted and mass spectrometry-driven approach showed that CSF levels of fructose, as well as mannose and threonic acid, were significantly higher in 34 patients with early-stage Parkinson’s disease (PD) than in 35 healthy controls . In addiThe normal serum reference levels of uric acid are 2.5–7.0 mg/dL 149–417 μM) in men and 1.5–6.0 mg/dL (89–357 μM) in women , whereas–417 μM iInterestingly, an inverse association between serum urate levels and the risk and progression of neurodegenerative diseases, including AD, vascular dementia, and PD, has been reported ,55, suggThe CSF lactate level has been reported to be 1.3–2.4 mM . The lacn = 101) increased compared with controls (n = 60) and were correlated with both clinical disease progression and neurodegeneration biomarkers, such as tau proteins and dopamine [n = 92) and frontotemporal dementia (FTD) (n = 27) than in individuals without dementia (n = 28) [A CSF-based study showed that lactate levels in patients with PD (rols n =  and weredopamine . However(n = 28) .It is known that the neurotransmitter metabolites of dopaminergic neurons in the CSF of PD are diagnostically useful . CSF 5-hChanges in glucose- and neurotransmitter-related CSF compounds in some disorders are summarized in n = 33) were significantly correlated with T-tau, P-tau, and the mini-mental state examination score [n = 43) compared with controls (n = 42) [Several kinds of age-associated CP alterations are prominent in AD, and they are considered to be linked to an increasing Aβ burden in CP ,64,65. Ton score . A systeon score . In addion score . It was (n = 42) , indicat(n = 42) .n = 28) and FTD (n = 12) [n = 8). Lower miR-328-3p levels could differentiate patients with AD from patients with FTD and controls and showed a significant correlation with lower Aβ1-42 levels. These findings suggest that miR-328-3p is involved in the AMPK signaling pathway, which is linked to amyloid-β and tau metabolism in patients with AD. However, it remains to be clarified which gene is regulated by the miRNAs. Various types of information on miRNAs may allow for the effective treatment of multifactorial diseases. Accordingly, the precise mechanism of miRNA–target interactions and the regulation of the targets by miRNAs should be clarified to develop successful miRNA-based drugs for the treatment of AD. In addition, recently, Hoshi et al. [Recently, many data on changes in the expression of microRNAs (miRNAs), which are small and non-coding RNAs enriched in exosomes, in the CSF of patients with neurodegenerative diseases were summarized in a review paper . In the (n = 12) . Expressi et al.  reportedn = 13) exhibited higher pS129-α-synuclein/α-synuclein ratios than patients with FTD (n = 26). In addition, a systematic review of CSF biomarkers for PD involving at least 20 patients with PD showed that oligomeric α-synuclein might be helpful in the separation of patients with PD from controls [n = 9) were found to have lower α-synuclein levels than patients with corticobasal degeneration (CBD) (n = 9). Patients with a synucleinopathy (PD and MSA) exhibited lower total α-synuclein levels and higher pS129-α-synuclein/total α-synuclein ratios than patients with tauopathies  and CBD).CSF total α-synuclein has been studied as a surrogate biomarker of synucleinopathies ,78. Mostcontrols . Patientn = 151) and patients with MSA (n = 17) from controls (n = 20). CSF S100B most favorably discriminated patients with PD and dementia with Lewy bodies (DLB) (n = 45) from controls (n = 20). Rojas et al. [n = 50), indicating the usefulness of the CSF NfL/phosphorylated tau ratio for the diagnosis of PSP. In addition, the CSF levels of chromogranin A in patients in PD (n = 119) and MSA (n = 18) groups tended to be lower than those in a control group (n = 31), but there was only a significant difference between MSA and control groups [n = 157), PD with dementia (n = 29), DLB (n = 11), MSA (n = 26), and PSP (n = 21) compared with controls (n = 47) and patients with AD (n = 124), emphasizing the importance of synaptic dysfunction in these parkinsonian disorders [n = 57) compared with controls (n = 65) [n = 8). miR-204-3p, miR-873-3p, and miR-6840-5p were most significantly up- or down-regulated in a PSP early-onset group [Delaby et al.  evaluates et al.  reportedl groups . CSF neuisorders . The exp(n = 65) . Recentlet group . The resRecently, there has been increasing evidence showing that viable mitochondria in extracellular fluids act as an inflammatory signal . In addiIn this paper, we first showed the significance of CP as the production site of CSF and the transport of intravascular nutrients and metabolized substances into CSF mainly based on papers recently published. As CSF is mixed with ISF, including substances physiologically and abnormally metabolized in neurons and glia through two major drainage pathways (the IPAD pathway and the glymphatic system) and via the ependymal cell layer, the analyses of CSF may be useful to explore biomarkers in order to diagnose neurological diseases. As summarized in


Disrupted tau proteostasis and transneuronal spread is a pathological hallmark of Alzheimer’s disease. Neurodegenerative diseases remain an unmet medical need and novel disease modifying therapeutics are paramount. Our objective was to develop a mechanistic mathematical model to enhance our understanding of tau antibody pharmacokinetics and pharmacodynamics in animals and humans. A physiologically-based pharmacokinetic-pharmacodynamic (PBPK-PD) modeling approach was employed to support the preclinical development and clinical translation of therapeutic antibodies targeting tau for the treatment of Alzheimer’s disease. The pharmacokinetics of a tau antibody was evaluated in rat and non-human primate microdialysis studies. Model validation for humans was performed using publicly available clinical data for gosuranemab. In-silico analyses were performed to predict tau engagement in human brain for a range of tau antibody affinities and various dosing regimens. PBPK-PD modeling enabled a quantitative understanding for the relationship between dose, affinity, and target engagement, which supported lead candidate optimization and predictions of clinically efficacious dosing regimens. Alzheimer’s disease is a central neurodegenerative disease and the leading cause of dementia. With an aging global population and the lack of effective disease modifying therapies, age-related neurodegenerative diseases are an increasing public health concern. In a collaborative effort, a systematic analysis was performed to assess the global burden of neurological disorders . AuthorsThe complex nature of neurodegenerative disease makes it challenging to develop effective therapies. Over the last decade, there has been a considerable amount of investigation into passive immunotherapy strategies for the treatment of neurodegenerative disease. Therapeutic antibodies have been developed against proteins that aggregate under pathological conditions, such Aβ and tau, for the treatment of AD. However, the clinical success with these therapies has been limited . Aducanua priori predictions that deviate from observed data can shed light on additional phenomena in the system that has not been mechanistically described. Hence, mechanistic models can be a useful tool for integrating and transforming data into actionable knowledge to provide guidance for drug development programs.There has been a rising interest in the application of mechanism-based pharmacokinetics-pharmacodynamics (PK-PD) modeling approaches, such as physiologically-based pharmacokinetic (PBPK)  and quanThe objective of our research was to develop a mechanistic mathematical model to enhance our quantitative understanding for pharmacokinetics and pharmacodynamics of tau antibodies in animals and humans. To demonstrate the application of PBPK modeling to support preclinical and early clinical development, we have expanded upon a previously published brain PBPK model for antibodies to include tau protein dynamics. Tau is a protein found predominately in neurons and is responsible for the stabilization of microtubules. Under pathological conditions, tau becomes hyperphosphorylated, dissociates from microtubules, aggregates, and spreads transneuronally throughout the brain, which thought to be a primary driver of dementia . There aA multi-species  physiologically-based pharmacokinetic (PBPK) model for antibody therapeutics was originally developed by Shah and Betts in 2012  and subsin vitro  experiment . The intin vitro . However52 pg/ml . We used52 pg/ml . Note th52 pg/ml . The con52 pg/ml . Using a52 pg/ml . Therefox represents the concentration of ptau (Cptau) or antibody ptau complex (CmAb_ptau) in one brain ISF and four CSF compartments: lateral ventricle (LV), third-fourth ventricle (TFV), cisterna magna (CM), subarachnoid space (SAS). We have assumed no distribution of target and antibody-target complex between compartments.The following target binding equations were introduced into the model to describe the interaction between antibody and phosphorylated-tau (ptau).n = 5–6 per group) were group housed and provided access to food and water ad libitum. Animals were kept on a 12/12 h light/dark cycle with constant room temperature (22 + 2 °C) and humidity (∼50%) and acclimated for at least 7 days prior to surgery. On the day of surgery, rats were anesthetized using isoflurane . Lidocaine was also used for local analgesia and carprofen for peri/post-operative analgesia. Animals were implanted with cannula into the cisterna magna and jugular vein for CSF and blood collection respectively. Animals were then implanted with a microdialysis probe  via stereotaxic surgery targeting the hippocampus at the following coordinates: antero-posterior = −5.3 mm to bregma, lateral = −4.8 mm to midline and ventral = −8.0 mm to dura, the tooth bar set at 0 mm. After surgery, animals were single-housed with ad libitum access to food and water. Approximately 24 h after surgery, brain ISF sampling was initiated for up to 28 h collection. On each ISF sampling day, microdialysis probes were connected with tubing  to a microperfusion pump . Microdialysis probes were perfused with aCSF containing 147 mM NaCl, 3.0 mM KCl, 1.2 mM CaCl2 and 1.2 mM MgCl2, and 0.15% BSA at a flow rate of 0.75 μL/min. After stabilization (2 h), microdialysis samples were collected for 60-minute periods by an automated fraction collector  into polypropylene (300 μL) mini-vials. On day 1, ISF was collected at baseline for 2 h . Then, rats were administered with either 10, 50, or 100 mg/kg of antibody A at a dose volume of 2 ml/kg IV and ISF collections continued for 6 h. At the end of the first ISF collection day, rats were disconnected and remained undisturbed in their home cage until the next day. On day 2, ISF was collected at timepoints 24–28 h post dosing. Following collection, all ISF samples were stored at −80C. In addition to brain ISF, serum and CSF samples were collected at baseline (∼2 h prior to treatment) . For each serum sample, blood was collected via the jugular vein cannula into serum separator vials and kept at room temperature for 30 min before processing for serum . Serum samples were then snap frozen on dry ice. For CSF, samples were collected via the cisterna magna cannula and snap frozen on dry ice. At the end of microdialysis experiment, rats were euthanized with CO2. Terminal CSF and blood were collected and snap frozen. Brains were collected and verified for probe placement.Rat microdialysis studies were conducted by Charles River Laboratories, South San Francisco (SSF) in accordance with the Institutional Animal Care and Use Committee (IACUC) of Charles River laboratories SSF. Sixteen male Sprague Dawley rats . Rhesus microdialysis studies were conducted in-house on four monkeys. Monkeys were implanted with a silicone, 5 French Cisterna Magna Port (CMP) catheter  attached to a titanium port body . The catheter tip (5 mm) was surgically implanted into the cisterna magna . ImplantThe microdialysis flow rate was set to 0.5 μL/min utilizing a Harvard CMA 402 micro syringe pump and a 2.5 ml Hamilton syringe. Microdialysis probes (CRL-PP-PE-180-040—1000kDA-manufactured by CRL-Netherlands) were perfused with Hamilton syringes containing artificial CSF (CMA-Ref P000151) and 0.15% BSA  solution. The solution was filtered using a 0.22 μm filter (Millex GP-Ref SLGP033RB). The probes were connected to the Hamilton syringe with PEEK tubing and the collection tube (Eicom/Richell low protein binding tubes -polypropylene) on wet ice was placed ∼30 cm below the head cap. ISF collection was driven by gravity. For the study only one site was used for ISF collection. For three monkeys the same site was used for all the ISF collections days 0–21. For one monkey the same site was used for all ISF collections days 0–10 and then another site was used for days 15 and day 21 due to no patency in the original site. Collection tubes were then placed on dry ice following the 30 min collection. On study days monkeys were chaired and at 8:00 a.m. probes were inserted. A 2 h probe equilibration period was done. Dosing of antibody A at 40 mg/kg IV  was performed at 10:00 a.m. on day 0. Monkeys were in the lab on days 0 and 1 for a period of 8 h. For all study days after day 1 when serum, CSF, and brain ISF were collected, monkeys stayed in the lab for a period of 4 h. For all microdialysis sessions the first 2 h were used for probe equilibration and then the brain ISF samples were collected 2 h post-probe insert. For CSF collections the area over the CMP was prepped prior to the microdialysis probe insert utilizing 6 ml Duraprep applicator . Area was allowed to dry 3 min and a single sterile Huber needle (Access Technologies) was placed and capped with a sterile injection cap. When a study time point was collected and the procedure was done under sterile conditions utilizing sterile gloves. The injection cap was removed and 0.9 ml of CSF was collected and discarded to allow for catheter volume (0.4 ml) plus an additional 0.5 ml waste. Following this a 500 μL study sample was collected and placed immediately on dry ice. The catheter was then locked using 0.4 ml sterile saline flush and the Huber needle was removed. For serum collection monkeys were bled using a butterfly and blood was taken from the saphenous veins. Blood was allowed to sit for 20 min then centrifuged at 10,000 rpm, 20°C for 5 min. A 200 and 300 μL serum sample was placed into a 1.4 ml alphanumeric tubes and immediately placed on dry ice. Feeding regimen for the study entailed on day 0, monkeys were fasted the night prior to dosing. Monkeys were given a Pedialyte 10% solution in study chairs at 2, 26, and 50 h timepoints. Monkeys were fed treats (grapes and bananas) after the 2 h, 26 h post time point in study chairs. Monkeys were fed again upon return to cages at ∼4:00 p.m. full ration on days 0 and 1. Upon return to their cages monkeys were fed a full ration. Enrichment (TV) was provided in the study room after the 4 and 28 h time points for ∼1 h.The concentration of Antibody A was measured by a bioanalytical method using an electrochemiluminescence based assay with a lower limit of quantitation (LLOQ) in rhesus monkey serum and CSF of 8.23 ng/ml. Briefly, 96-well flat-bottom Meso-Scale Discovery (MSD) Streptavidin Gold multi-array plates were blocked with 5% bovine serum albumin (BSA) in PBS followed by coating with biotinylated mouse anti-human Ig kappa light chain antibody in Modified ELISA Diluent buffer (MED) . For signal detection a sulfo-tagged mouse anti-human IgG CH2 domain antibody was used. Standards, controls, and sample dilutions followed by detection reagent were added in between sequential wash steps and incubations. Electrochemiluminescence signal proportional to captured Antibody A was captured on an MSD plate reader, Meso Sector S600. Concentrations of Antibody A were derived through comparison to a standard curve (0.41–300 ng/ml range) applying a 4 parameter non-linear regression fit. The method was qualified via assessment of accuracy, precision and dilutional linearity using spiked Antibody A samples serving as high, medium and low controls spanning the calibration curve. Assay run acceptance was determined by recovery of a minimum of 4 out 6 control samples  within 20% of nominal value, as well as visual inspection of calibrator curve readings.in vivo preclinical experimental studies investigating the PK of Antibody A were conducted. A rat microdialysis study was performed to investigate the PK of Antibody A at single IV doses of 10, 50, and 100 mg/kg. Concentrations of Antibody A were measured in serum, CSF, and brain ISF. The PK of Antibody A was then investigated in cynomolgus monkeys at single IV doses of 3, 10, 40, and 80 mg/kg. Only serum concentrations were measured. Lastly, a microdialysis study was conducted in rhesus monkeys at a single IV dose of 40 mg/kg. Concentrations of Antibody A were measured in serum, CSF, and brain ISF. For model validation, a priori predictions were performed for each of these experiments and compared to observed data.Three in vitro tau binding assay  over a duration of 3 months. A binding affinity of 0.131 nM was used to predict the change in free N-terminal tau in CSF, which was previously determined using an ng assay .The impact of affinity and half-life on the dynamics of tau concentrations in brain ISF was investigated using simulations from the model. Simulations were performed for four hypothetical antibodies (A1–A4) with varying levels of affinity . Simulations were also performed for four hypothetical antibodies (A1a–A1d) that have the same affinity (Kd = 0.1 nM) and different terminal half-lives . For both simulation scenarios, each antibody was simulated across a range of doses from 0 to 100 mg/kg and tau occupancy at 8 weeks was calculated. Tau occupancy was calculated as follows:CSF_BC is the concentration of Antibody A in CSF when accounting for concentration difference due to blood contamination and FBC is the fraction of blood contamination. HCT is the hematocrit, represented as a fraction. CCSF and CSerum are the concentration of antibody in the CSF and serum, respectively. CSF tau occupancy (TOCSF) and CSF tau occupancy when accounting for blood contamination (TOCSF_BC) were calculated as:D is the antibody affinity to tau. The percent error in antibody CSF concentration (PEC_CSF) was calculated as:To assess the potential impact of blood contamination on Antibody A concentration and tau dynamics in human CSF, the percent error in antibody and tau concentrations were calculated as a function of different levels of blood contamination. Blood contamination was calculated as:TO_CSF) was determined using the following equation:The precent error in CSF tau occupancy (PEC_CSF and PETO_CSF verses a range of FBC (0–10%) to visualize the impact of blood contamination on the error of antibody concentration and target occupancy. The PBPK model was utilized to predict the concentration of antibody in CSF and serum at 1-month post-administration, which were used in the percent error calculations.Figures were generated for PESimulations were performed to predict the PK-PD of Antibody A in humans. Five dose levels  and three antibody affinities to tau  were evaluated. The PK-PD profiles for Antibody A concentrations in serum, CSF, and brain ISF and the change in free CSF tau relative to baseline were simulated over 16 weeks.The Chang brain PBPK model was expanded to include tau dynamics. The target-mediated effects of tau on the disposition of tau antibodies were incorporated in four CSF compartments and brain ISF . Model pA priori model simulations were able to adequately describe the pharmacokinetics of Antibody A in preclinical rat and NHP models in serum, CSF, and brain ISF across several dose levels  patients was digitized from the literature . Model pThe validated PBPK-PD model can be applied to understand the impact of anti-tau antibody affinity optimization and half-life extension on dose regimen. The tau occupancy in brain ISF at 8 weeks after a single IV dose for four theoretical antibodies  with different binding affinities to tau  was simulated across a range of doses (0–100 mg/kg) . This enTau occupancy in brain ISF at 8 weeks after a single IV dose for four antibodies  with the same binding affinity to tau (0.1 nM) and different elimination half-lives  was simulated across a range of doses (0–100 mg/kg) . This enrd dose (week 8) and at steady-state. However, less frequent administrations, Q8W and Q12W, were unable to achieve high levels of target engagement (>90%) in the brain. Simulations of this nature have been valuable for anticipating the level of tau engagement at the site of action for various clinical dosing regimens of interest.To understand how different clinical dosing regimens would impact tau engagement in the CSF, we performed simulations for an anti-tau antibody (Antibody A1b) with an affinity of 0.1 nM and half-life of 40 days . We predFor neuroscience therapeutics, CSF concentrations are often used as a surrogate for concentrations at the site of action (brain ISF). CSF is typically collected in the clinic via lumbar punctures. However, there is concern of potential blood contamination with this collection methodology. We utilized a quantitative approach to assess the impact of blood contamination on antibody concentrations and tau dynamics in CSF samples . CSF conSimulations were performed to predict Antibody A concentrations in serum, CSF, and brain ISF as well as tau dynamics in CSF in humans administered a single IV dose of 1, 3, 10, 30, and 100 mg/kg . AntibodA priori predictions of antibody PK and tau dynamics in human serum and brain ISF/CSF were useful for understanding relationships between antibody affinity/half-life and target engagement, which may inform first-in-human dose selection and design of phase I clinical trials. We have only reported mean predictions as the level of variability around parameter values is unknown. Clinical population PK-PD data would help to inform inter-individual variability. The mechanistic PBPK approach offers an alternative to allometry for predicting serum exposures, but it’s unclear whether a PBPK approach would be more or less predictive than traditional allometric scaling for preclinical-to-clinical translation of drug exposures. A head-to-head comparison between the two different approaches across multiple antibody therapeutics would be valuable.We have demonstrated the application of PBPK-PD modeling to support various aspects of preclinical and early clinical development of antibody therapeutics for the treatment of Alzheimer’s disease and tauopathies. The model was validated using preclinical and clinical data for antibody pharmacokinetics in serum, CSF, and brain ISF as well as clinical data for tau engagement in CSF. Blood contamination may occur when collecting CSF via lumbar puncture. Blood contamination could change the concentration of drug and target in the CSF samples, which ultimately may alter the interpretation of target engagement. Therefore, it is crucial to quantitatively understand the level of blood contamination in each sample and perform an adjustment for the concentration of drug in CSF accordingly . SimulatSimplifying assumptions were made throughout the model development process. We describe tau using a single compartment. However, pathological tau is present intra- and extra-cellularly, undergoes complex aggregation processes and post-translational modifications, and spreads throughout the brain in a transneuronal fashion. We have assumed one-to-one binding, where an antibody is able to only bind to a single target. In reality, a single antibody could bind to two targets as well as multiple antibodies could bind to one oligomeric protein aggregate. We used the molecular weight of monomeric protein for conversions to molar units. Although this approach accounts for multiple antibodies binding to oligomeric tau, it makes the assumption that antibodies are able to bind to all tau monomers within an oligomer. This may not be appropriate as tau proteins within an oligomer could create steric hinderance by shielding the antibody binding site of other tau proteins. However, from a modeling perspective, its not entirely clear on how the molecular weight for aggregated proteins with multiple binding sites should be best considered, especially when working with protein aggregates of variable sizes. We assumed static target-mediated drug disposition (TMDD), where TMDD processes occur independently in each brain compartment. In other words, there is no distribution of target or antibody-target complex between brain compartments. A dynamic TMDD model would include additional kinetic processes, such as endosomal uptake/escape, FcRn binding, and paracellular transport of the target and antibody-target complex.The publicly available clinical data assessing the engagement of tau-antibodies in human CSF is currently only in PSP patients . ConsideAs more data begins to emerge from clinical trials investigating tau-targeted therapies in AD patients , a QSP mOur work exemplifies the utility of PBPK-PD modeling to address challenges faced in preclinical development and clinical translation of anti-tau antibody therapeutics for the treatment of Alzheimer’s disease. This modeling approach provides a foundation that can be further expanded to incorporate additional complexities of tau biology. This tau PBPK-PD model can also be refined as clinical data emerges to inform late stages of clinical development. However, the size of this platform model may limit its applicability. Minimal PBPK models of the brain provide a framework that can be more easily adapted to incorporate targets of interest and integrated with quantitative systems pharmacology models of neurological diseases.


Cryptococcus, mostly C. neoformans or C. gattii. Cryptococcal meningitis is the most frequent clinical manifestation in humans. Neutralizing autoantibodies (auto-Abs) against granulocyte–macrophage colony-stimulating factor (GM-CSF) have recently been discovered in otherwise healthy adult patients with cryptococcal meningitis, mostly caused by C. gattii. We hypothesized that three Colombian patients with cryptococcal meningitis caused by C. neoformans in two of them would carry high plasma levels of neutralizing auto-Abs against GM-CSF.Cryptococcosis is a potentially life-threatening fungal disease caused by encapsulated yeasts of the genus We reviewed medical and laboratory records, performed immunological evaluations, and tested for anti-cytokine auto-Abs three previously healthy HIV-negative adults with disseminated cryptococcosis.Peripheral blood leukocyte subset levels and serum immunoglobulin concentrations were within the normal ranges. We detected high levels of neutralizing auto-Abs against GM-CSF in the plasma of all three patients.Cryptococcus spp.We report three Colombian patients with disseminated cryptococcosis associated with neutralizing auto-Abs against GM-CSF. Further studies should evaluate the genetic contribution to anti-GM-CSF autoantibody production and the role of the GM-CSF signaling pathway in the immune response to The online version contains supplementary material available at 10.1007/s10875-023-01451-5. Aspergillus spp., Candida spp., Pneumocystis jirovecii, and Cryptococcus spp., with cryptococci accounting for almost half of all fungus-related deaths [C. neoformans or C. gattii. C. neoformans has a worldwide distribution and is the etiological agent responsible for about 95% of all cases of cryptococcosis, in both immunocompromised and apparently immunocompetent hosts [C. gattii has a restricted geographic distribution and generally affects apparently healthy individuals [Fungi are abundant in the environment, but only a few cause human disease , 2. Despd deaths . IFDs ocd deaths , 4, 5. Cividuals , 7. The ividuals .Anti-cytokine neutralizing auto-Abs, usually found in adult patients, are considered to constitute autoimmune phenocopies of IEI . Indeed,Mycobacterium spp. complex, Nocardia spp., Histoplasma capsulatum, and Cryptococcus spp. [Nocardia spp. or C. gattii with or without PAP manifestations [Anti-GM-CSF auto-Abs cause pulmonary alveolar proteinosis (PAP), a severe lung disease characterized by the accumulation of surfactant in the alveoli, with progressive respiratory failure and an increase in the risk of infection , probablcus spp. . In addistations –27. In tThis study was conducted according to the “Scientific Standards for Technical and Administrative Health Research” established in the Colombian Ministry of Health Resolution 008430 of 1993 and approved by the local review board of the Universidad de Antioquia (F8790-07–0010) and Necker Hospital for Sick Children, France. All patients or their family members provided written informed consent.Briefly, 96-well plates  were coated by incubation overnight at 4°C with 1 µg/mL rhGM-CSF or rhIFN-γ (R&D Systems). The plates were washed in PBS 0.005% Tween, blocked with 1 × PBS supplemented with 5% nonfat milk powder, washed, and incubated for 2 h at room temperature with 1:50, 1:250, and 1:1,000 dilutions of plasma from patients and healthy controls, or plasma from a patient with cryptococcal meningitis and a PAP patient previously shown to have high titers of anti-GM-CSF auto-Abs, as a positive control. The plates were washed and horseradish peroxidase (HRP)–conjugated Fc-specific IgG  was added to a final concentration of 1 µg/mL. Plates were incubated for 1 h at room temperature and washed. Substrate was added, and optical density was measured. The antibody specificity controls were plasma samples from a patient with high titers of auto-Ab against IFN-γ, and a patient with APS-1 and high titers of auto-Ab against IL-17A, IL-17F, IL-22, IFN-α, and IFN-ω.6 cells/well in 96-well V-bottom plates (Thermo-Fisher-Scientific), in 100 µL RPMI , supplemented with 10% fetal bovine serum (FBS)  or 100 µL RPMI supplemented with 10% plasma from patients or controls. PBMCs were left unstimulated or were stimulated with 5 to 80 ng/mL rhGM-CSF or 100 ng/mL rhIL-3 (Miltenyi-Biotec) for 30 min at 37°C, and the cells were then fixed permeabilized with a fixation/permeabilization kit (eBioscience). Extracellular labeling was performed with CD14-Pacific Blue and CD4-FITC . Cell viability was determined with the Aqua Dead Cell Stain Kit (Thermo-Fisher-Scientific), and STAT5 phosphorylation (p-STAT5 levels) was assessed by intracellular staining with Phospho-Flow PE Mouse Anti-p-STAT5 (pY694) antibody (BD-Biosciences). Data were collected with a Gallios flow cytometer (Beckman-Coulter) and analyzed with FlowJo software v.10.6.2 (Becton–Dickinson).Human peripheral blood mononuclear cells (PBMCs) from healthy controls were isolated from whole blood by Ficoll-Hypaque density centrifugation . The cells were counted and plated at 2 × 10Renilla luciferase for normalization (pRL-SV40). Then, cells were left unstimulated or were stimulated with IFNs (IFN-α subtypes (Miltenyi Biotec), IFN-ω (Peprotech), or IFN-β (Peprotech)) at 100 pg/mL (for IFN-α or IFN-ω) or at 1ng/mL (for IFN-β) for 16 h at 37°C in the presence of 10% of healthy control or patient plasma diluted in DMEM with 2% FBS. Luciferase activity was then assessed in the Dual-Luciferase® Reporter 1000 assay system, according to the manufacturer’s protocol (Promega). Raw luciferase induction was calculated as firefly luciferase activity normalized against Renilla luciferase activity, and this raw luciferase induction was normalized against the non-stimulated luciferase induction.The plasma blocking activity against type I IFNs  was determined with a luciferase reporter assay. Briefly, HEK293T cells, cultured in DMEM (Thermo Fisher Scientific) with 10% FBS were transfected in the presence of X-tremeGene9 transfection reagent (Sigma-Aldrich) for 24 h with a human ISRE-luciferase plasmid in the pGL4.45 backbone and a plasmid constitutively expressing Cryptococcus spp.  grew C. neoformans (Tables Patient 1 (P1) is an otherwise healthy 41-year-old man from Colombia (South America) who presented at the age of 34 years with progressive precordial pain with dysphagia, dry cough, moderate dyspnea, and a weight loss of 26 kg. After a convulsive episode, he was admitted to the emergency room, and physical examination revealed neck stiffness, a grade 2 systolic pulmonary murmur, and purple skin papules on his nose  for 2 weeks but developed respiratory failure due to loculated pleural effusion of the right hemithorax with secondary lung collapse, requiring a new pleurectomy with decortication. Throughout P2’s disease, radiological and BAL studies have consistently ruled out pulmonary alveolar proteinosis (not shown). The patient remains free of signs and symptoms of infection and is not currently receiving antifungal agents; she is, however, permanently paraplegic.Patient 2 (P2) is a 46-year-old otherwise healthy woman from Colombia (South America) who presented at the age of 40-year-old shoulder pain of four months’ duration radiating to the lower back, together with paresthesia of the lower abdomen and difficulty walking. Physical examination revealed a painful abdomen with bilateral positive Lasègue sign, sensory and motor deficit, a decrease in the muscle strength of both lower limbs, right Achillean reflex clonus, and bilateral positive Babinski sign. The patient suffered from permanent bilateral vision loss of unknown cause that had started 4 years previously, with lens opacification in the left eye and a corneal leukoma in the right eye. CE-MRI revealed extensive fluid collections in the right shoulder and lumbar spine, extending to the retroperitoneal, dural, and posterior mediastinum, along with spinal cord compression and instability and bone destruction between T9 and T11  of the lungs revealed an increase in interstitial involvement, with a “crazy-paving” pattern  regimen with rifampicin (R), isoniazid (H), pyrazinamide (Z), and ethambutol (E) for 2 months, followed by 4 months of R-H treatment with clinical improvement, but with dyspnea on exertion. P3 has not undergone whole-lung lavage or required oxygen supplementation, and she remains stable.Patient 3 (P3) is a 44-year-old previously healthy woman from Colombia who developed progressive whole-head headaches with photophobia, nausea, and vomiting in 2014. Physical examination was unremarkable except for neck stiffness with no neurologic focalization. Brain CE-MRI findings were consistent with meningitis . We also included plasma from a patient with high titers of anti-IFN-γ neutralizing auto-Abs, an APS-1 patient with high titers of anti-IL-17A, IL-17F, and IL-22 auto-Abs and two healthy individuals. All three patients had high levels of anti-GM-CSF auto-Abs (with P3 plasma showing the highest levels), in the range of the positive control, whereas none of the healthy individuals, the APS-1 patient, or the patient with auto-Abs against IFN-γ had auto-Abs against GM-CSF Fig. . AltogetHuman GM-CSF is produced by alveolar epithelial cells, monocytes/macrophages, activated T cells, B cells, NK cells, endothelial, epithelial, and fibroblast cells and promotes the development of bone marrow-derived macrophages and granulocytes , 29. AlvC. gattii than in those infected with C. neoformans [C. neoformans. One of the three patients (P3) was diagnosed with PAP nine months after developing disseminated cryptococcosis due to C. gattii. PAP is characterized by an accumulation of alveolar surfactant, resulting in respiratory impairment, an increase in the risk of pulmonary fibrosis, and opportunistic infections [Cryptococcus spp., Nocardia spp., and Aspergillus spp., may therefore be an early sign of a risk of developing PAP in the future [The three patients investigated here were otherwise healthy adults who developed disseminated cryptococcosis in association with high titers of anti-GM-CSF neutralizing auto-Abs. Anti-GM-CSF neutralizing auto-Abs block GM-CSF receptor signaling, thereby affecting the terminal differentiation of AM in the lungs and their ability to catabolize surfactant and to perform their host defense functions . Anti-GMoformans –25. Intefections . Early-ofections , or fromfections . By contfections , whereasfections . In adulfections . Lung ane future , 25, 36.M. tuberculosis killing by primary human monocyte-derived macrophages, Brison et al. recently demonstrated that GM-CSF enhances the control of M. tuberculosis, as GM-CSF blockade rendered macrophages more permissive to M. tuberculosis growth, whereas the addition of GM-CSF increased bacterial control [M. tuberculosis [M. tuberculosis infection. In addition, GM-CSF promotes human macrophage differentiation in vitro and fine tunes macrophage inflammatory state, enhancing mycobacterial control by activating antimicrobial pathways and amplifying an inflammatory environment mediated by IL-1β [M. tuberculosis. The endemic distribution of both pathogens (C. neoformans and M. tuberculosis) in Colombia may render patients with anti-GM-CSF neutralizing auto-Abs more susceptible to these diseases. Recent evidence suggests that additional host genetic factors, such as HLA haplotype, may result in the development of anti-cytokine auto-Abs and susceptibility to infections in the context of adult-onset autoimmune-induced immunodeficiency [Only one adult patient with disseminated cryptococcosis and high serum levels of anti-GM-CSF auto-Abs has, to our knowledge, developed pulmonary tuberculosis within 1 year of the diagnosis of cryptococcosis . P3 was  control . Furtherrculosis , 39. Antby IL-1β , 40. Theficiency . Sakaue ficiency . Howeverficiency . PulmonaSupplementary file1 (PPTX 922 KB) Supplemental figure 1: Anti-GM-CSF auto-Ab titers in plasmas, diluted 1/50, from the three patients (P1-P3), healthy European individuals , patients previously shown to have anti-GM-CSF  or anti-IFN-γ  auto-Abs, an APS-1 patient with auto-Abs against IL-17A, IL-17F, IL-22, IFN-α, and IFN-ω . Supplemental figure 2: Absence of neutralizing auto-Abs against type I IFNs in the plasma of the patients. Relative luciferase activity (RLA) ratio after stimulation with each of the IFN-α subtypes or IFN-ω at a concentration of 100 pg/mL, with a 1:10 dilution of plasma from seven healthy controls (negative control), an APS-1 patient (positive control), and the three patients with neutralizing anti-GM-CSF auto-Abs. Results are normalized against the RLA value obtained in the presence of plasma but absence of stimulation. Supplemental figure 3: Plasma from the three patients diluted 1:10 neutralizes up to 80 ng/mL GM-CSF. pSTAT5 levels assessed by flow cytometry on total PBMCs from a healthy donor stimulated with 5 ng/mL, 20 ng/mL, 40 ng/mL, or 80 ng/mL of GM-CSF or 100 ng/mL of IL-3, in the absence of plasma, or in the presence of a 1:10 dilution of plasma from two healthy controls (negative control), a patient with alveolar proteinosis PAP (positive control), or one of the three patients. NS: non-stimulated. Supplemental figure 4: STAT5 phosphorylation (p-STAT5), assessed by flow cytometry, upon stimulation with 5ng/mL rhGM-CSF (red) or 100ng/mL rhIL-3 (black) of control PBMCs in the presence of a 1:10, 1:100 or 1:1,000 dilution of plasma from two healthy individuals, three individuals previously described with neutralizing autoAbs against GM-CSF, or the three patients’ (P1-P3) plasma. NS: non-stimulated.Below is the link to the electronic supplementary material.


Gorham-Stout disease (GSD) is a very rare disorder characterized by massive osteolysis of poorly understood aetiology. The association between GSD involving the skull base and cerebrospinal fluid (CSF) leakage has been reported in the literature. However, few cases of CSF leakage and Chiari-like tonsillar herniation in GSD involving the spine have been reported.We present the case of a 20-year-old man with GSD involving the thoracic and lumbar spine, which caused CSF leakage and Chiari-like tonsillar herniation. The patient underwent four spinal surgeries for osteolytic lesions of the spine over a 10-year period. Here, we discuss the possible aetiology of the development of CSF leakage. Epidural blood patch (EBP) was performed at the T11-T12 level to repair the CSF leakage. After EBP treatment, rebound intracranial hypertension (RIH) developed, and tonsillar herniation disappeared 2 months later.GSD involving the spine with CSF leakage and Chiari-like tonsillar herniation is relatively rare. For patients who have undergone multiple spinal surgeries, minimally invasive treatment is an alternative treatment for CSF leakage. EBP can repair CSF leakage secondary to GSD and improve chronic brain sagging, with reversibility of Chiari-like malformations. Gorham-Stout disease, also known as massive osteolysis, Gorham’s disease, disappearing or vanishing bone disease, idiopathic osteolysis, and phantom bone disease, is a rare disorder of idiopathic and progressive bone resorption, with replacement by lymphatic or vascular and fibrous tissue proliferation. Imaging studies have demonstrated osteolytic lesions without new bone or periosteum reaction, which is significantly different from bone dissolution caused by inflammation or malignancy. The disease was first described by Jackson in 1838; a detailed clinical description was made by Gorham and Stout in 1955 . The cliCSF leakage can cause low intracranial pressure, presenting as postural headache. In some severe cases, brain sagging can develop, resulting in Chiari-like descent of the cerebellar tonsils . SpontanA 20-year-old man suffering from recurrent headache in the standing position for 10 months was admitted to our hospital. He had a history of GSD. In the past twelve years, the patient had undergone a closed thoracic drainage procedure due to chylothorax and 4 spinal surgeries for osteolytic lesions of the spine  images demonstrated smooth diffuse pachymeningeal enhancement and cerebellar tonsillar herniation, which was consistent with intracranial hypotension Figs.  and 4. TDuring the next two months, the patient’s headache gradually improved, and he returned to normal activities. However, two months after the EBP, the patient presented to another local hospital with severe headache, orbital pain, nausea and vomiting. He felt that the headache was aggravated in the lying position and relieved in the upright or sitting position. Lumbar puncture revealed a high opening pressure of 20 cmH2O. Based on the symptoms and lumbar puncture pressure, RIH was considered. Meanwhile, with increasing CSF pressure, the Chiari-like tonsillar herniation disappeared. The patient was treated with acetazolamide and mannitol to reduce cranial pressure. At the 1-year follow-up, his headache was in remission.GSD is a rare disorder for which the aetiology is not fully understood . The majPatients with GSD may have CSF leakage. We reviewed the cases of GSD with CSF leakage Table , which iCSF leakage can be treated with conservative methods, EBP or surgical repair. For patients with mild CSF leakage, conservative management, such as strict bed rest, intravenous hydration and analgesics, can be chosen . When coThe aetiology of the development of CSF leakage in our patient remains unclear. Dural injury is the main cause of CSF leakage. We propose 2 explanations for the CSF leakage observed in this GSD patient. One reason may be progressive osteolysis involving the dura mater, resulting in CSF leakage, which was similar to that proposed in the previously reported literature , 24.Alternatively, this patient had undergone multiple orthopaedic internal fixation operations in the past 10 years due to scoliosis caused by osteolysis. The last spinal surgery was revision and kyphosis osteotomy surgery involving the T10-L5 levels. The patient developed CSF leakage and intracranial hypotension syndrome six months after the last spinal surgery. This may be related to the weakening of the dura mater due to multiple revision surgeries. In spinal revision surgery, the anatomical structure of the surgical area is different from the normal structure, and the dura mater is widely adhered to the surrounding scar tissue, which increases the risk of dura mater breakage.Intraoperative posterior pedicle screw placement and pedicle screw malposition may carry a potential risk of occult dural invasion with subsequent CSF leakage and intracranial hypotension. Any behaviour or condition that increases intra-abdominal pressure after surgery, such as cough, constipation, and sneezing, can lead to a transient increase in intradural pressure. CSF can rupture and tear the weak dura mater, resulting in the leakage of CSF. In this case, symptoms appeared half a year after the last surgery, suggesting that they might be secondary to a delayed dural leak. However, regardless of the above causes of CSF leakage, EBP therapy is effective.It should be noted that after successful treatment of CSF leakage, RIH is sometimes encountered regardless of surgical repair methods or blood patch therapy , 29, 30.Cerebellar tonsillar herniation can be secondary to chronic CSF leakage , 25. In In summary, GSD is a rare disease. GSD involving the spine with CSF leakage is even more rare. For patients who have undergone multiple spinal surgeries, minimally invasive treatment of CSF leakage is an alternative treatment. CSF leakage is easily misdiagnosed. If early CSF leakage is not treated in a timely manner, cerebellar tonsillar herniation might occur. EBP can repair CSF leakage secondary to GSD and improve chronic brain sagging, with reversibility of Chiari-like malformations. It should be noted that after successful treatment of CSF leakage, RIH is sometimes encountered.