The results indicated that psychological contract breach acts a partial mediating role in the inverted U-shaped relationship between perceived overqualification and knowledge hiding, while leader-member exchange acts as a moderator. The finding manifests that overqualification encourages employees to hide knowledge, but their possibly vigorous aspects will be displayed when a certain limit is exceeded. This study benefits organizations by advising them to dialectically treat and properly place overqualified employees and contributes to the research on overqualified employees’ knowledge management by offering a new explanation and complete understanding of perceived overqualification and knowledge hiding, with specific focus given to the psychological states of employees.Although the negative effects of a sense of overqualification on organizations and individuals have been examined, it is debatable whether overqualified employees hide knowledge. Relying on the social comparison theory and psychological contract theory, this paper tried to investigate the non-linear relationship between perceived overqualification and knowledge hiding  Perceived overqualified (PO) has a variety of negative effects on organizations and individuals . KH has become prevalent in organizations, and has become an important obstacle to organizational innovation and growth , and ord growth , organizd growth , and orgd growth ], along d growth . It is nbreach of psychological contract (PCB) can encourage negative behaviors of employees, such as employee workplace deviance . LMX defines the degree of contact between the employee and their leadership  means that an individual’s education level, knowledge, experience, and skills are higher than that required by the post . It has According to this theory, the motivation for employees to hide their knowledge lies in their competitive advantage or ability when compared with others. Under the condition of low and middle PO, the ability of employees is only a little more than the job requirements, so employees have a strong sense of crisis and fear that their colleagues will surpass them. Therefore, they will deliberately hide their knowledge to speed up their self-improvement and gain competitive advantages . But oveH1: PO and KH have an inverted U-shaped relationship. In the low and middle (middle and high) level of PO, PO is positively (negatively) related with KH.Psychological contract breach (PCB) is an informal agreement within an organization and it refers to employees’ cognition caused by the enterprises’ failure to perform its promise . AnotherH2: PO is rightly associated with PCB.PCB can lead to a series of negative behaviors. The breach of psychological contract will facilitate counterproductive workplace behavior  and negaH3: There is an inverted U-shaped relation between PCB and KH.In conclusion, employees’ PO can promote their PCB, which has a reverse U-shaped relationship to KH. In the condition of low and medium levels of PO, according to psychological contract theory, employees deem that the agreement between them and the organization has been broken, the degree of PCB of employees becomes stronger, and their KH gradually increases. In terms of high and medium levels of PO, the degree of PCB of employees becomes stronger. But their intention to hide knowledge is weak, because employees, especially those with comparatively high abilities, will pay more attention to the collective interests, and the KH of employees gradually decreases. Therefore, we suggest as follows:H4: PCB mediates the inverted U-shaped relation between employee PO and KH.The differences of KH among different overqualified employees may be related to the interaction between employees and their leaders . Leader-On the contrary, staff with low-quality LMX acquire limited resources and are usually confined to normal working relationships . In the H5: LMX moderated the reverse U-shaped relationship between PO and KH. Compared with high-quality LMX, the positive and negative relationships between PO and KH were stronger in low-quality LMX.At present, the phenomenon of overqualification of employees is very serious in China , especiaThe scale applied to this study is relatively mature, and necessary adjustments and modifications are made to some items of the scale according to the actual situation. All scales were measured by Likert five-point scoring method.Perceived overqualification has adopted the scale of Knowledge hiding was measured with a three-dimensional scale adopted by Psychological contract breach employed the scale used by Leader-member exchange referred to the 7-item tool of Referring to previous studies, we took the gender, age, education, tenure, position, and company nature of employees as control variables .2/df = 11.586, RMSEA = 0.149, CFI = 0.478, TLI = 0.426, IFI =0.480, SRMR = 0.143; Self-reporting is an important method in organizational behavior investigation which needs to deal with method bias problem . ConsideThis study tested the convergent validity of the variables, and 2/df = 2.805, RMSEA = 0.062, CFI = 0.913, TLI = 0.902, IFI =0.914, and SRMR = 0.051) is significantly superior to others, which indicates that variables have good differential validity.To test the construct validity of each factor, combined with AMOS 24.0 , we testAdditionally, we tested the discriminant validity of variables, which means whether the square root of AVE of variable is greater than the correlation coefficient among the variables . From Taβ = 0.293, p < 0.001), PO and KH , P and CB has a remarkably active correlation with KH . Moreover, the variance inflation factors (VIF) of the respective variables are between 1 and 2 , and it is generally believed that there is no multicollinearity problem , and the square of PO was passively related to KH  when the term of square of PO was added, with a better model explaining , which indicated the reverse U-shape of PO and KH, and H1 was verified.In β = 0.266, p = 0.000) and KH , and H2 was verified. Further observation showed that PCB was dramatically positively related with KH , and the square term of PCB was significantly negatively related with KH , indicating that there was a reverse U-shaped relation between PCB and KH, and H3 was verified. Meanwhile, it was found that the square term of PO  still had a noteworthy effect on KH after commanding PCB, and the coefficient becomes smaller, indicating that PCB had a sectionally mediating effect on the reverse U-shape connection between PO and KH, and H4 was verified.We examined the mediating effect of PCB between PO and KH. As shown in β = 0.096, p = 0.031), and the interaction term of square term of PO and LMX had a remarkable effect on KH  with the significantly increased model fitting coefficient , which indicated that LMX took a moderating influence on the inverted U-shaped connection between PO and KH, and H5 was verified.To test the moderating effect of LMX on the PO and KH, this paper first standardized the PO and LMX, and constructed the interaction term to exclude the adverse effect of multi-line collinearity. Secondly, referring to This paper inspected the mediator effect of psychological contract breach (PCB) on the reverse U-shaped relation between perceived overqualification (PO) and knowledge hiding (KH), and tested the moderating effect of leader-member exchange (LMX). The results manifested that PO is in a reverse U-shaped relation with KH, and low-middle (middle-high) level of PO positively (negatively) affects KH. Moreover, PCB, which can be promoted by PO, had a reverse U-shaped contact with KH, and also partially mediated the inverted U-shaped connection between PO and KH. LMX moderated the reverse U shaped relation between PO and KH. Compared with high-quality LMX, the positive and negative effects of PO on KH are stronger in terms of low-quality LMX.Our study contributes to the existing literature in three ways. Firstly, we expanded the study on the relationship between PO and KH. In the study of antecedents of knowledge hiding, PO has not been fully discussed. In terms of study on the relationship between PO and KH, the positive impact of PO on KH  and theiSecondly, previous literature has analyzed the influence mechanism of the PO and KH from the perspective of emotion , while wThirdly, there is little research on the boundary conditions of KH of overqualified employees, which our study contributes to by confirming the moderating effect of LMX on the reverse U-shaped relationship between PO and KH. This study confirmed the significant positive correlation between employees’ PO and KH under high-quality LMX. And under low-quality LMX, PO had a more significant negative effect on KH, indicating that low-quality LMX had a stronger effect on the KH of PO, which provides a new perspective to interpret the boundary condition for the relationship between PO and KH.There are a large number of employees with excessive qualifications in the workplace . OrganizFirstly, this paper reveals an important phenomenon: employees with different levels of PO have different attitudes toward KH, that is, those high-quality talents in the organization do not always hide knowledge, and we are should treat the phenomenon of overqualification rationally. On the one hand, employees’ PO can lead to KH, which generally means that organizations need to focus on the matching of employees’ qualifications and positions. For competent people, excessive training provided by organizations may sometimes bring adverse results . ManagerSecondly, managers need to understand the psychological state of employees and reasonably grasp employees’ psychological contract with organizations. On the one hand, managers need to understand and take targeted strategies to repair the psychological tacit with overqualified employees , when thEventually, managers should properly control the distance between themselves and their employees. Managers should pay attention, trust, and support to employees with low or high levels of PO. However, although relevant research affirmed that maintaining a good relation between employees and leaders can bring positive effects for individuals and organizations , we shouThis paper adopts the method of employee self-evaluation, which inevitably has the problem of common method deviation. In the future, a variety of research methods  can be considered to increase the scientific nature of the research. Next, we only examined the roles of PCB and LMX. Whether other factors  affect PO and KH remains to be explored. It remains to be probed whether other theories  can better account for the non-linear relation between PO and KH. Future research can further explore the relationship from these aspects. Then, although we have found the non-linear relationship between PO and KH, there is still a lack of discussion on the hidden dimension of employee rationalization. Future studies can further analyze the reasonable KH of overqualified employees. Moreover, this paper found that the connection between PCB and its outcome variables is not simply linear, and future studies can test the possible non-linear relationship according to this idea.The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.HQZ performed the research design and outline of the manuscript. LZL contributed to drafting the manuscript and analyzing questionnaire data. AHC collected the data and improved the empirical analysis. XMS revised the draft. All authors contributed to the article and approved the submitted version.This article was financially supported by the the Soft Science Project for Sichuan Science and Technology Agency (grant number 2022JDR0024) and Sichuan Grassroots Public Cultural Service Research Center (grant number JY2019B04).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Dear Editors,The need of organs for transplantation is increasing worldwide, though the availability of them does not meet the existing demand . In someWe report a case of paracorporeal donor heart resuscitation followed by successful heart transplantation after 7 h of cold storage.Burkholderia cepacia, Staphylococcus aureus) resulted in a higher waitlist status priority for HTx.Fifty-five years old male patient was diagnosed with dilated cardiomyopathy at 2012 years. He underwent a HeartMate3 left ventricular assist device  implantation as a bridge to heart transplantation (HTx) in January 2018. His clinical status was complicated by a deep driveline infection. After initial antibiotic therapy, the patient underwent two driveline site debridement surgeries in May and September 2019. Recurrent infections with resistant pathogens  no abnormalities, 2 days in ICU and 1 day on ventilator. Donor and recipient had the blood group O (I) Rh-positive and AB (IV) Rh-positive status, respectively. Our institution is sole transplant center in the country, and donor hearts are often retrieved from distant regions to be transplanted  circuit of recipient and assess its function after reperfusion. The ascending aorta of donor heart was connected in end-to-end manner to splitted systemic flow line Following 6 min of reperfusion of the heart, stable sinus rhythm was restored, using the 15 J shock from external biphasic defibrillator. The standard CPB roller pump was used , with the mean aortic pressure between 68 and 84 mmHg and coronary blood flow between 800–860 ml/min at a temperature of 35C. The graft was conditioned with 45 μg/kg Levosimendan infusion in the coronary perfusion and cytokine adsorption  with a blood flow of 200–300 ml/min was applied from recipient’s CPB circuit. Periodic arterial and venous blood samples were taken from donor heart during perfusion period of 54 min. After organ evaluation, the decision was made to use the heart for transplantation based on surgeon observation, good visual contractility, stable sinus rhythm, and lactate values. The donor heart was arrested with 1 L of regular normothermic blood cardioplegia solution just before transplanting. It took two cardioplegia doses before resuming systemic blood flow in the heart. The bi-atrial technique of performing orthotopic cardiac transplantation has been utilized. In 52 min, the heart rhythm was restored. Transplantation and preoperative care proceeded according to the standard procedures of our center. The next day after HTx the remnants of driveline and ICD with leads were extracted. Prolonged suppressive antibiotic therapy with Colistin, Meropenem and Vancomycin in combination with Cansidas was used up to 4 weeks.2of cardiac index. Echocardiography parameters confirmed normal biventricular function: S1LV lateral 21 cm/s, S1LV medial 7.8 cm/s, S1RV 7.6 cm/s, LVEF 68%. For over 9 mon post-transplant, there is no evidence of rejection or cardiac dysfunction.Total cold ischemic time was 430 min and duration of coronary perfusion was 54 min. This resulted in a total cross-clamp to cross-clamp time of 534 min . Venous With prolonged ischemic time, the donor graft can be transported for longer distances and increase the chances of gaining a matching donor graft. We report a successful heart transplantation following donor heart resuscitation after 7 h of cold storage using recipient’s cardiopulmonary bypass circuit prior to organ implantation. This maneuver allowed to start resuscitation of the donor organ by means of controlled warm reperfusion, medical treatment and cytokines adsorption. It is remarkable that this scenario was covered by recipient’s physiologic conditions, i.e., patient’s multiorgan system reserve capacity.To the best of our knowledge, this is the first reported case of paracorporeal resuscitation of donor heart after 7 h of cold static storage using recipient’s cardiopulmonary bypass circuit to be successfully transplanted.


Pathological cerebral conditions may manifest in altered composition of the cerebrospinal fluid (CSF). Although diagnostic CSF analysis seeks to establish pathological disturbances in the brain proper, CSF is generally sampled from the lumbar compartment for reasons of technical ease and ethical considerations. We here aimed to compare the molecular composition of CSF obtained from the ventricular versus the lumbar CSF compartments to establish a relevance for employing lumbar CSF as a proxy for the CSF bathing the brain tissue.40, Aβ42), and total tau (T-tau) and phosphorylated tau (P-tau) forms). The latter analysis was extended to include paired porcine samples obtained from the lumbar compartment and the cerebromedullary cistern closely related to the ventricles.CSF was collected from 46 patients with idiopathic normal pressure hydrocephalus (iNPH) patients during their diagnostic workup (lumbar samples) and in connection with their subsequent CSF diversion shunt surgery (ventricular samples). The mass-spectrometry-based proteomic profile was determined in these samples and in addition, selected biomarkers were quantified with ELISA , amyloid-β  levels in the ventricular compartment. In the porcine samples, all biomarkers were most abundant in the lumbar CSF.In total 1231 proteins were detected in the human CSF. Of these, 216 distributed equally in the two CSF compartments, whereas 22 were preferentially (or solely) present in the ventricular CSF and four in the lumbar CSF. The selected biomarkers of neurodegeneration and Alzheimer’s disease displayed differential distribution, some with higher  and some with lower (NfL, AβThe overall proteomic profile differs between the ventricular and the lumbar CSF compartments, and so does the distribution of clinically employed biomarkers. However, for a range of CSF proteins and biomarkers, one can reliably employ lumbar CSF as a proxy for ventricular CSF if or a lumbar/cranial index for the particular molecule has been established. It is therefore important to verify the compartmental preference of the proteins or biomarkers of interest prior to extrapolating from lumbar CSF to that of the ventricular fluid bordering the brain.The online version contains supplementary material available at 10.1186/s12987-022-00405-0. Cerebrospinal fluid (CSF) surrounds the brain and fills the central ventricles and is thus in direct contact with the brain. The molecular composition of CSF may therefore reflect biochemical changes in intracranial fluid compartments, e.g., CSF and/or interstitial fluid. Accordingly, CSF analysis is commonly used as a surrogate measure for pathological conditions in the brain, and proteins detected in the CSF are used as biomarkers of disease both as a screening tool and as a supplementary information to diagnostic investigations –6. CSF uThe lumbar CSF compartment is anatomically remote from the ventricular compartment. This distance results in a gradient of protein concentration along the neuroaxis; the CSF total protein, albumin and IgG content increase progressively from the ventricles to the lumbar sac with a 60% higher protein content in lumbar CSF than in ventricular CSF in humans , 18 and One of the difficulties in studying differences between brain and lumbar CSF is the limited access to ventricular CSF. Lumbar CSF can be collected during a wide range of diagnostic lumbar punctures and even from central nervous system (CNS)-healthy individuals, e.g., patients undergoing procedures with spinal anesthesia. However, it is generally only possible to collect human ventricular CSF from patients undergoing neurosurgery either as intracranial invasive diagnostic procedures or as treatment of neurological disorders. Thus, the CSF compartmental profile cannot be studied in CNS-healthy individuals. Idiopathic normal pressure hydrocephalus (iNPH) is a frequent neurological disease in the elderly population , 21, wheout). An abnormally high Rout and/or improved gait function following tap-test increase the diagnostic accuracy, but normal tests do not preclude iNPH . Patients were diagnosed with iNPH at the Danish Dementia Research Centre, Copenhagen University Hospital—Rigshospitalet, Denmark, according to the international guidelines from 2005 [ude iNPH . The lum − 80 °C . WrittenDanish mixed breeds of Yorkshire, Danish Landrace and Duroc pigs (n = 18) with a mean weight of 24.02 ± 4.37 kg and an estimated age of 10–13 weeks were included. CSF collection was performed with the pigs placed in lateral recumbency under general anaesthesia. Anaesthesia was induced with an intravenous injection of propofol  and subsequently maintained with isoflurane , 1–2.5 vol% inhalation in a circle system after an intramuscular premedication with 1 ml/10 kg of a custom-made Zoletil 50 Vet-mixture  dissolved in 6.25 ml xylazine , 2.5 ml ketamine  and 2.5 ml butorphanol ). Additional 0.07 ml acepromazine  and 0.5 ml methadone  were administrated intramuscularly at the time of premedication. Upon shaving of all puncture sites and cleaning with 70% ethanol, 1 ml CSF was collected from cisterna magna  and from the lumbar cistern (the lumbar site between L7 and S1) with 90 mm and 75 mm spinal needles, respectively. Both CSF samples were obtained with < 5 min interval approximately 3 h after induction of anesthesia. Animals were excluded if puncturing failed at one site. To avoid bias, the order of CSF collection was randomly controlled for each animal. All animal experiments were performed on pigs employed for veterinary student training of abdominal surgical procedures and according to the legislation for animal protection and care, animal permission no. 2016-15-0201-00957 approved by the Danish Animal Experiments Inspectorate.g for 1 min. 20 µl of solvent A was then added to equilibrate the tips followed by sample loading. Finally, 20 µl solvent A was used to wash the Evotip and 100 µl was added to avoid drying.Human CSF sample preparation was performed on an Agilent Bravo Liquid Handling Platform (Agilent) according to an optimized version of previously published protocols , 29. Bri. The peptides were separated on an 8 cm Pepsep column  at 1 μl/min flow rate with a 21 min gradient. The heated capillary temperature was set to 275 °C, the spray voltage to 2650 V and the funnel radiofrequency to 40 Hz. The mass spectrometer was operated in a data-independent mode (DIA) with a full MS range from 350 to 1650 m/z at a resolution of 60,000 at 200 m/z. The AGC target was set to 300% with an injection time of 50 ms. The AGC value of the targeted MS2 experiment was set to 1000%. Thirty-two windows of variable sizes were defined for target MS2 (tMS2) acquisition and subjected to high-energy collisional dissociation (HCD) fragmentation with a normalized collision energy at 30%. Each tMS2 scan was acquired at a resolution of 30,000 with a maximum ion injection time (IT) of 100 ms for a scan range of m/z 349.5–1650.5.The samples were injected into an Exploris 480 Thermo Fischer Scientific system using Evosep One (Evosep Biosystem). A preset chromatographic method was used corresponding to 60 samples per dayThe MS raw files were processed with Spectronaut version 15 . A previously generated CSF spectral library was imported from MaxQuant software analyses. The library contained 2733 protein groups and 17,301 peptides. DIA files were searched against the library using default parameters except for the normalization, which was set to local. Dynamic mass and retention time tolerances (for both MS1 and MS2) were applied. Q-value cutoff was set to 1% both at precursor and protein level using a mutated decoy method . The cal40, Aβ42, total tau (T-tau) and phosphorylated tau (P-tau) were measured with Lumipulse technology , as previously described [The CSF samples were analyzed at the Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden. CSF concentrations of Aβescribed . CSF neuescribed . S100B wStatistical analyses were carried out using R v. 4.1.0 . Continuous data were presented as mean and standard deviation (SD) or median and interquartile range (IQR)/range depending on normality, while categorical data were presented as n, proportion, and percentage. Proteomics data were assessed for availability in both lumbar and ventricular CSF, to assess which proteins are available (above level of detection) in each compartment. A principal component analysis (PCA) plot with complete case analysis , and a PCA plot where missing data was imputed for proteins available for at least 10 pairs (available in both lumbar and ventricular CSF in at least 10 patients) were conducted . PCA addTo determine the proteomic profile of lumbar versus ventricular CSF, we employed lumbar samples obtained from 46 iNPH patients during their diagnostic work-up paired to ventricular samples obtained at subsequent neurosurgical procedure . Mass spectrometry-based proteomic analysis of these samples identified 1231 unique proteins in either sample  and no other clinical biomarkers associated with age or BMI  was 40, P < 0.05, F42, P < 0.001, Fig. Curiously, all the six tested clinical biomarkers analyzed from near-simultaneously lumbar and cisternal sampling from the porcine model displayed higher levels in the lumbar compartment  were detected at higher levels in the lumbar compartment, whereas tau (T-tau and P-tau) was lower in lumbar than in ventricular samples from iNPH patients/human samples. Although the lumbar elevation of Aβ42 and NfL align with a previous study, the elevated ventricular concentration of tau, here observed, conflicts those earlier findings [To quantify the compartmentalized distribution of commonly employed clinical biomarkers, we employed standard ELISA-based quantification. The obtained levels of these CSF proteins aligned with earlier published reports on these biomarkers .nd P-tau , 56, 57.nd P-tau . Curiousnd P-tau . This cofindings . The samThe craniospinal fluid dynamics are complex and incompletely understood and complex. In- and outflow of blood with the cardiac cycle is a driving force from the cranial compartment towards the spinal compartment in systole which is reversed in diastole. Flow dynamics thus contain flow patterns in both caudal and cranial direction . DiffusiRats with congenital hydrocephalus (H-Tx) display changes of proteomic and metabolic CSF profile compared to normal rats . Whetheren route from the ventricles to the sample vials. Although sampling was performed during different clinical/surgical procedures, 43/46 (93%) were obtained before the effect of treatment  and thus represent the pre-treatment period.Another limitation is the heterogeneity of the sampling of ventricular CSF. The CSF samples were collected upon shunt insertion (n = 39), ETV (n = 1), tap test through shunt chamber (n = 3) or during insertion of EVD (n = 3). The major limitation of this issue is that some samples have been collected through plastic tubing or syringes and others not. This could affect the results as some proteins, amyloid beta specifically, stick to plastic surfaces and thus would not be collected in the sample vials. However, of the 46 samples collected only one sample (ETV) was not in contact with plastic ware In conclusion, our findings demonstrate that although the CSF presents with an overall protein gradient along the neuroaxis, each protein/biomarker distributes in a specific manner in these two CSF compartments. One therefore cannot directly extrapolate biomarker levels measured in lumbar samples to ventricular CSF without a known lumbar/cranial index for the molecule. However, future research and clinical work may benefit from the comprehensive list of protein distribution in these two CSF compartments.Additional file 1: Figure S1. Normality test for biomarkers measured in human and porcine CSF (both compartments) by immunoassays. Figure S2. Pearson’s correlation coefficients to investigate the influence of BMI (A) and the age of the patients (B) on biomarker levels. BMI: body mass index; LCSF: lumbar cerebrospinal fluid; VCSF: ventricular cerebrospinal fluid. Figure S3. No signs of blood contamination in CSF from one CSF compartment over the other. Visual assessment of data obtained by mass spectrometry. Data demonstrate that lumbar and ventricular samples contains similar levels of erythrocytes, platelets and coagulation factors. a.u.: arbitrary units; LCSF: lumbar cerebrospinal fluid; VCSF: ventricular cerebrospinalvæske. Table S1. Clinical biomarkers in porcine CSF. The table summarizes levels of S100B, NfL, Aß40, Aß42, T-tau and P-tau in porcine CSF measured by ELISAs.Additional file 2. Complete list of proteins detected in lumbar and ventricular CSF samples from iNPH patients quantified with mass spectrometry and divided into ‘higher lumbar’, ‘higher ventricular’, ‘similar’ and ‘unclear’.


Pythium insidiosum. Antimicrobials’ in vitro and in vivo results against P. insidiosum are inconsistent. Although antimicrobials are clinically useful, they are not likely to achieve therapeutic success alone without surgery and immunotherapy. New therapeutic options are therefore needed. This non-exhaustive review discusses the rationale antimicrobial therapy, minimum inhibitory concentrations, and efficacy of antibacterial and antifungal agents against P. insidiosum. This review further provides insight into the immunomodulating effects of antimicrobials that can enhance the immune response to infections. Current data support using antimicrobial combination therapy for the pharmacotherapeutic management of human pythiosis. Also, the success or failure of antimicrobial treatment in human pythiosis might depend on the immunomodulatory effects of drugs. The repurposing of existing drugs is a safe strategy for anti-P. insidiosum drug discovery. To improve patient outcomes in pythiosis, we suggest further research and a deeper understanding of P. insidiosum virulence factors, host immune response, and host immune system modification by antimicrobials.Human pythiosis is associated with poor prognosis with significant mortality caused by  Pythium insidiosum, a fungus-like aquatic oomycete microorganism, is a causative agent of pythiosis. The motile flagellate zoospore plays a significant role in initiating an infection. The zoospores of P. insidiosum adhere to the skin cut or wound sites and encyst on the surface of the injured tissue(s). The encysted spore develops a germination tube (hypha) that uses chemotaxis to find the host and infiltrate human blood vessels . Sur. SurP. i patient ,11. The 2 (IL-2) ,12,13. Eythiosis ,12,14. I pathway . Howeverical use ,16.P. insidiosum, the pathogenesis of the P. insidiosum infection in humans, or the management of human pythiosis with immunotherapy and surgical intervention. Finally, we conclude with general remarks on future strategic options for managing human pythiosis.This review focuses on evidence supporting and disputing the effectiveness of antimicrobials to expand the pharmacotherapeutic role of antimicrobials in the management of human pythiosis. We do not explicitly discuss the biology of P. insidiosum, identifying potential targets for antimicrobials is necessary for managing pythiosis. The microbial cell wall is a critical target for antimicrobials, and the cell wall of P. insidiosum is primarily composed of β-glucan and cellulose [P. insidiosum lowers the penetration of drug molecules and prevents drug access to targets inside the cell wall [COX2) in Thai P. insidiosum strains was 2.5-fold higher at 37 °C compared to the expression at 27 °C [ELI025, was highly up-regulated in P. insidiosum hyphae at 37 °C compared to hyphae grown at 28 °C and facilitated the evasion of the host antibody response [COX2 and ELIO25 can be candidate targets for controlling P. insidiosum infection.Antimicrobial therapy should achieve a clinical response by eliminating the invading microorganism(s) while minimizing cost, adverse effects, and antimicrobial resistance ,18. Whenellulose . Howeverell wall . The genat 27 °C . In addiresponse . COX2 anP. insidiosum in an in vitro study  [P. insidiosum keratitis patient was successfully managed with topical 0.2% linezolid and topical 1% azithromycin, administered hourly [in vitro by establishing minimum inhibitory concentration (MIC), which is the lowest concentration of an antimicrobial that inhibits visible growth of a microorganism [P. insidiosum discussed in our review.Several antifungal and antibacterial drugs have been examined for their susceptibility profile against ro study . They hae a day) . Recentld hourly . Antimicorganism ,27. Tablin vitro and in vivo efficacy, host immune response, and wrong choice of antimicrobial drug  included in the sterol biosynthetic pathways [P. insidiosum. These drugs cannot be exploited for rationalized and successful management of pythiosis [P. insidiosum to the antimicrobial drug [P. insidiosum and avoid being pumped out of the membrane [pathways . Howeverythiosis . Differeythiosis , increasial drug , redesigmembrane , and modmembrane .The innate immune system protects the host from various toxins and infectious agents, including bacteria, fungi, viruses, and parasites via phagocytosis and intracellular killing, recruitment of other inflammatory cells, and presentation of antigens . The innP. insidiosum enters and adheres to the host tissues, the soluble exoantigens from P. insidiosum trigger the Th2 response and lock the host immune system into a Th2 subset. Further, P. insidiosum protects itself from the host immune system by concealing inside the eosinophilic material formed by the eosinophil degranulation, which helps protect the P. insidiosum from being fully presented to the host’s immune system [P. insidiosum induced a TLR2-mediated innate immune response with a subsequent increase in the levels of the pro-inflammatory cytokines IL-6 and IL-8 [Once e system . Toll-lie system . Wongproand IL-8 . To combat the pathogen and prevent its spread, it is rational to administer antimicrobial drugs that interact with the host’s innate immune system to provide profound indirect effects and enhance pathogen clearance. Antimicrobial drugs have been shown to modify the immune responses to infection, guiding improved treatment strategies in human pythiosis .in vitro screening of antibacterial drugs has identified tetracycline, minocycline, tigecycline, azithromycin, clarithromycin, erythromycin, gentamicin, streptomycin, paromomycin, neomycin, linezolid, nitrofurantoin, quinupristin-dalfopristin, chloramphenicol, clindamycin, and mupirocin, which demonstrated inhibitory activity against P. insidiosum [Previous sidiosum ,63,64,65Tetracycline antibiotics such as tetracycline, tigecycline, and minocycline inhibit bacterial protein synthesis by binding with the bacterial 30S ribosomal subunit . Tetracyin vivo studies in rabbits, minocycline in combination with immunotherapy may be an effective therapeutic medical treatment of pythiosis to heal injuries [in vitro susceptibility of human, environmental, and animal P. insidiosum isolates to eight antibiotic classes and demonstrated that tetracyclines and macrolides inhibited the in vitro growth of P. insidiosum isolates at concentrations 10 to 100 times lower than those observed for previously studied antifungal drugs [P. insidiosum isolates. Loreto et al. also reported a similar in vitro susceptibility of P. insidiosum isolates to tetracyclines and their superior potency compared to amphotericin B, echinocandins, and triazole antifungals [Based on the injuries . Worasilal drugs . Also, tifungals .Macrolides are the group of antibiotics that inhibit bacterial protein synthesis by binding with the bacterial 50S ribosomal subunit. Common macrolides include erythromycin, clarithromycin, and azithromycin . Among tP. insidiosum in a rabbit model [in vivo activity of azithromycin (20 mg/kg/day twice daily) alone and combination with minocycline (10 mg/kg/day twice daily) against subcutaneous lesions. In an in vitro susceptibility study, the MICs of azithromycin and clarithromycin were less than 4 μg/mL for P. insidiosum isolates [Jesus et al. investigated the antimicrobial activity of azithromycin alone and in combination with minocycline against it model . The resisolates .Oxazolidinones such as linezolid inhibit bacterial protein synthesis by binding with the 50S subunit of the ribosome . The supP. insidiosum keratitis, topical linezolid demonstrated superior efficacy and safety compared to azithromycin and tigecycline after prolonged treatment for more than 3–4 weeks [In a rabbit model of –4 weeks . Lincosamides, streptogramins, and phenicols inhibit bacterial protein synthesis by interacting with the 50S subunit of bacterial ribosomes . Among lP. insidiosum isolates. The microdilution-based MIC ranges (with geometric means) of lincosamides, streptogramins, and phenicols against P. insidiosum were reported to be 2 to >4 μg/mL, 1 to >2 μg/mL, and 8 to >16 μg/mL, respectively [Lincosamides, streptogramins, and phenicols have shown the ability to inhibit the growth of ectively .in vitro growth of one of the Thai P. insidiosum isolates [in vitro growth of P. insidiosum; however, they may not be clinically relevant due to the high MIC values [Aminoglycosides such as gentamicin, streptomycin, paromomycin, and neomycin bind to the bacterial ribosome and inhibit protein synthesis . Streptoisolates . AminoglC values . TherefoP. insidiosum mycelial growth was inhibited with nitrofurantoin (MIC range of 64 to >64 μg/mL) in an in vitro susceptibility test [Nitrofurantoin is used to treat urinary tract infections and works by attacking bacterial ribosomal proteins non-specifically, causing complete inhibition of protein synthesis . P. insiity test . in vitro susceptibility of Brazilian P. insidiosum strains showed that mupirocin could inhibit the growth of P. insidiosum isolates at MIC lower than 4 μg/mL [Mupirocin inhibits bacterial protein and RNA synthesis by reversibly inhibiting isoleucyl-transfer RNA . A study 4 μg/mL . P. insidiosum effects, it has been highly challenging to achieve consistently effective antifungal treatment in human pythiosis. Studies have focused on several antifungal medications, such as polyenes, azoles, allylamines, and echinocandins, for the adjunctive therapy in managing human pythiosis . DespiteP. insidiosum is lacking [Amphotericin B is a polyene antifungal that binds to ergosterol in the fungal cell membrane, which alters cell membrane permeability leading to the loss of intracellular components . Two Aus lacking ,85,86,87Studies have shown amphotericin B’s immunomodulatory properties, which activate the host’s innate immunity . Nitric Candida albicans is enhanced when combined with azoles [The primary mode of action of allylamines, such as terbinafine, is the inhibition of the enzyme squalene monooxygenase. Therefore, these drugs inhibit the fungal synthesis of ergosterol . Azoles,h azoles ,62. Howeh azoles .P. insidiosum isolates in vitro [in vitro susceptibility testing of a P. insidiosum isolate from the 2-year-old patient with a deeply invasive facial infection [P. insidiosum isolates was inhibited by terbinafine, and the efficacy of terbinafine increased against P. insidiosum isolates when combined with cetrimide, an antiseptic [In vitro susceptibility testing of P. insidiosum showed a MICs from 0.5 to 128 μg/mL for terbinafine, 2 to 32 μg/mL for miconazole, 4 to 64 μg/mL for ketoconazole, 1 to >128 μg/mL for itraconazole, 2 to >16 μg/mL for voriconazole, greater than 1 to >32 μg/mL for fluconazole, and >8 μg/mL for posaconazole based on the strains of P. insidiosum [Susaengrat et al. reported favorable responses to voriconazole and itraconazole in Thai vascular pythiosis patients . Synergiin vitro . A synernfection . The grotiseptic . Pediatrtiseptic . In vitrsidiosum ,14.Echinocandins, such as caspofungin, anidulafungin, and micafungin, act by inhibiting beta--D-glucan synthase, an enzyme that is necessary for the synthesis of beta--D-glucan, which is an essential component of the fungal cell wall . StudiesP. insidiosum effects were observed with caspofungin and terbinafine in vitro [P. insidiosum isolates ranged from 2 to 8 μg/mL [in vitro and in vivo activity against P. insidiosum [P. insidiosum [Synergistic anti-in vitro . The MIC 8 μg/mL . Howeversidiosum ,102. Cassidiosum . in vitro inhibitory activity against P. insidiosum [P. insidiosum isolates were 16 to 64 mg/L. Further, amorolfine hydrochloride produced alterations in P. insidiosum hyphae, with changes in the surface of hyphae, intracellular organelles, the cell wall, and plasma membrane of P. insidiosum.Amorolfine, a morpholine derivative, inhibits fungal ergosterol biosynthesis and leads to changes in the membrane permeability, which in turn causes fungal growth inhibition and cell death . Only residiosum . The MICP. insidiosum in humans, identifying novel therapeutic strategies is required to treat P. insidiosum infection in humans. Drug repurposing is a process for identifying new therapeutic indications different from the scope of the initial pharmacological indication [P. insidiosum activity using agar- and broth-based methods and revealed that P. insidiosum strains were susceptible to disulfiram with MICs ranging from 8 to 32 mg/Liter [P. insidiosum.Due to the limited success of pharmacological interventions against dication . For exadication . Using tdication . Disulfidication . Krajaejmg/Liter . Furtherin vitro and in vivo assays [Researchers utilize computational and experimental approaches to identify the promising candidates in the drug repurposing process . The como assays .P. insidiosum [P. insidiosum was observed for in vitro minocycline with amphotericin B, itraconazole, and micafungin and clarithromycin with micafungin [P. insidiosum [Using combination regimens of antibacterial plus antifungal or antibacterial plus antibacterial to achieve synergistic activity is one of the drug repurposing strategies against sidiosum . Synergicafungin . Susaengcafungin . Howeversidiosum . Studiessidiosum ,118. We In vitro studies have demonstrated the tetracyclines, macrolides, oxazolidinones, lincosamides, streptogramins, phenicols, aminoglycosides, polyenes, allylamines, azoles, and echinocandins reviewed in our papers inhibit the growth of P. insidiosum and have the potential implications for further research on their use in the management of human pythiosis. However, prolonged use of antimicrobials and prolonged treatment with antimicrobials is not warranted due to the side effects and threat of antimicrobial resistance. A practical pharmacological intervention guideline for human pythiosis remains to be discovered and is necessary to assist practitioner and patient decisions, lower treatment costs, and optimize patient outcomes. Despite the disease affecting the most vulnerable populations with higher mortality rates, pythiosis is not included in the Sanford Guide, which provides evidence-based recommendations for treating infectious diseases [Evidence supports using the antimicrobials reviewed in our article as a new therapeutic option in treating human pythiosis. diseases .P. insidiosum susceptibility testing for the management of pythiosis. Studies have shown the growth inhibitory effects of antimicrobials against P. insidiosum; nevertheless, studies regarding the mechanism of action of the antimicrobials against P. insidiosum are vital for clinical approval. Researchers must consider the pharmacodynamics principle involved in selecting the antimicrobials to assess the anti-P. insidiosum activity. In the future, human pythiosis could be managed with antimicrobials owing to their anti-inflammatory and immunomodulatory activities. Clinicians can optimize drug combinations based on the anti-P. insidiosum include glucan 1,3-beta-glucosidase, heat shock protein (Hsp) 70, and enolase [P. insidiosum and found up-regulated exo1 expression at 37 °C compared to 28 °C, thus suggesting a drug target against P. insidiosum [P. insidiosum and limit host damage. Metabolites have been isolated from Pseudomonas stutzeri and Klebsiella pneumoniaei, and these organisms have shown anti-P. insidiosum activity [Microbial virulence factors are molecules produced by microorganisms and may cause disease in the host  . The pot enolase . Keeratisidiosum . A new tactivity ,121. TheWith the evidence of the effectiveness of some antimicrobials in the management of human pythiosis, we suggest using new drug delivery systems to release the drug to the target site in the body and minimize the off-target accumulation of the drug. Antibiotics can be reformulated using nanotechnology-derived delivery systems to improve the targeting and specificity at the infected areas . Due to


Parkinson’s disease (PD) is a growing burden worldwide, and there is no reliable biomarker used in clinical routines to date. Cerebrospinal fluid (CSF) is routinely collected in patients with neurological symptoms and should closely reflect alterations in PD patients’ brains. Here, we describe a scalable and sensitive mass spectrometry (MS)-based proteomics workflow for CSF proteome profiling. From two independent cohorts with over 200 individuals, our workflow reproducibly quantifies over 1,700 proteins from minimal CSF amounts. Machine learning determines OMD, CD44, VGF, PRL, and MAN2B1 to be altered in PD patients or to significantly correlate with clinical scores. We also uncover signatures of enhanced neuroinflammation in LRRK2 G2019S carriers, as indicated by increased levels of CTSS, PLD4, and HLA proteins. A comparison with our previously acquired urinary proteomes reveals a large overlap in PD-associated changes, including lysosomal proteins, opening up new avenues to improve our understanding of PD pathogenesis.  •Reproducible and scalable MS-based proteomics workflow for proteome profiling of CSF•Quantification of over 1,700 proteins collected from more than 200 individuals•Lysosomal and immune-related biomarker signatures specific to LRRK2 G2019S carriers•Overlapping PD- and LRRK2-associated alterations in CSF and urine proteomes Karayel et al. present a scalable, sensitive, and reproducible mass spectrometry-based proteomics workflow for profiling of cerebrospinal fluid and demonstrate its promise for biomarker discovery for idiopathic and familial forms of Parkinson’s disease. However, proteomic analysis of CSF has been challenging due to low protein concentration combined with the high dynamic range of protein abundances, resulting in low quantification precision, throughput, and limited proteome depth.In this study, we extended our pipeline of biomarker discovery to analyze more than 200 CSF samples from two independent cohorts in which we detected over 1,700 proteins from minimal CSF sample amounts. By employing co-variate (ANCOVA) analysis and machine learning, we identified unique protein signatures whose abundance was specifically changed in PD patients versus healthy controls and LRRK2 G2019S carriers versus non-carriers. Our study demonstrates that modern MS-based proteomics is a powerful technology for biomarker discovery in biofluids. It also provides potential biomarkers of PD as well as insights into biological pathways associated with PD and/or LRRK2 mutations.,,,,LRRK2 Cohort Consortium (hereinafter referred to as the LCC cohort). More information about the cohorts is summarized in To investigate how PD affects the CSF of patients and to identify potential biomarkers, we employed the “rectangular” biomarker discovery strategy, which aims to discover discriminating proteome signatures using rather large sample sizes in both discovery and validation cohorts.,,LRRK2. To maximize proteome depth and coverage, we generated cohort-specific hybrid spectral libraries by merging three sub-libraries: (1) a library constructed by data-dependent acquisition (DDA) consisting of 24 fractions of pooled CSF samples; (2) another DDA library consisting of eight fractions of extracellular vesicles enriched from pooled CSF samples; and (3) a direct-DIA library generated from the DIA analysis of all samples  strategy to perform proteome profiling of minimal amounts of CSFples see . Matchinples see B. On aveples see . Our DIAes see ,,E and 1F.es see ,,E and 1F.We further classified the identified proteins based on their human protein atlas (HPA) annotationTo assess quantification precision in our study, we investigated intra- and inter-assay variabilities of our automated workflow by repeated measurements of a pooled CSF sample C. This a,,Inconsistent sample collection and handling may result in systematic bias and hamper the discovery of true biomarkers. To ensure that the quantified changes in CSF proteins are due to disease-related pathological alterations, we assessed the quality of all samples according to previously established quality marker panels for coagulation-related proteins, platelets, and erythrocytes to identify samples with potential issues in pre-analytical processingWe found no correlation between erythrocyte counts and the number of erythrocyte-specific proteins in the CSF E. Our saWe additionally further excluded a single sample from the HBS cohort that clustered far away from all other samples in a principal-component analysis (PCA) and a single sample from the LCC cohort that was an outlier in proteome depth. Our subsequent bioinformatics analysis was based on 79 samples from the HBS cohort and 103 subjects from the LCC cohort to determine the impact of disease manifestation and LRRK2 mutation status on the CSF proteome E and S2FLRRK2 mutation status as confounding factors. We also included the different study centers as a confounding factor for the LCC cohort. For the HBS cohort, we further included the GBA mutation status. At 5% false discovery rate (FDR), we identified three significantly regulated proteins  in the CSF of PD patients compared with controls in the HBS and one (PRCP) in the LCC cohort  annotation enrichment analysis using the mean fold changes of PD versus HC.. Taken together, our CSF proteome data, when combined with ML algorithms, classified disease status and, more importantly, identified several promising PD-associated proteins, opening up interesting leads for future studies.Motivated by the presence—but small number—of commonly altered proteins in both cohorts, we tested how well machine learning (ML) could discriminate PD patients from controls by using the recently introduced open-source tool OmicLearn.LRRK2 G2019S mutation in the LCC cohort, we explored whether the CSF proteome is altered by LRRK2 mutation status. We again applied an ANCOVA with sex, age at sample collection, study center, and PD status as confounding factors. At a FDR of 5%, the mutation significantly altered the abundance of the proteins HLA-DRA, HLA-DRB1, HLA-DPA1, CTSS, PLD4, TKT, ITGB2, PRDX3, ITIH5, CNDP1, and FAH  to healthy individuals also revealed several HLA molecules, CTSS as well as ADAM10 and FAM234A (ITFG3) to be upregulated in pathogenic LRRK2 carriers, with HLA-DRB1 being the only protein reaching the significance level at a FDR of 5% G. Pathog,Cathepsins are proteases mediating protein degradation and turnover in endolysosomal compartments, and several of them have been implicated in inflammatory diseases, lysosomal storage, and neurodegenerative disorders such as PD.We previously analyzed 235 urine samples from two independent cross-sectional cohorts (Columbia and LCC), including two types of controls, healthy individuals, and LRRK2 G2019S carriers not manifesting the disease, and PD patients with and without the LRRK2 G2019S mutation, quantifying 2,365 urinary proteins in total.Correlating the ANCOVA q values of all common proteins quantified in CSF (HBS and LCC) and urine (Columbia cohort) of PD patients compared with the controls revealed several proteins regulated in both fluids D and 5E.We next investigated whether any protein level changes in the HBS cohort correlated with the severity of PD pathology as assessed by the Unified Parkinson’s Disease Rating Scale (UPDRS) , and S5.Furthermore, we compared the correlation scores of CSF proteins with those obtained in our previous study in urine. The comparison of Pearson coefficients for the UPDRS III scores (the only score available for urine) revealed 10 proteins  to have coefficients either higher than 0.4 or lower than −0.4 in both matrices B. Among Within the LCC cohort, Montreal Cognitive Assessment (MoCA) scores for all subjects were available I. IntereHere, we applied a scalable and highly reproducible MS-based proteomics workflow to CSF samples from two independent PD cohorts. To the best of our knowledge, our approach resulted in the deepest single-run CSF proteome acquired by MS to date, with about 1,400 proteins quantified per sample. In addition, our workflow is sensitive, using only 40 μL of CSF for sample preparation. It does not require depletion of highly abundant proteins such as albumin or any biochemical enrichment, and the amount of purified peptides is sufficient for several MS runs, thus allowing the re-measurement of individual samples if required. Despite the unmatched depth of our dataset, we have not yet identified some of the known and well-studied PD-relevant proteins such as α-synuclein and neurofilament light chain, although both proteins were present in the LCC cohort library, and neurofilament light chain was also present in the HBS cohort library. The high dynamic range of protein abundances in CSF limits the sensitivity of MS-based proteomics compared with antibody-based assays that are frequently used to study these low abundant PD markers. However, the analytical variation of our assay, with a median CV of 19%, was much better than the biological variation, with a CV of median 46%. Although single-analyte antibody-based assays like ELISAs often have even lower technical CVs, we found our workflow well suited to study disease-related biological differences on a proteome-scale.To reduce systemic biases in the analyzed samples and minimize the effect of pre-analytical variation, we performed a thorough quality assessment of every sample using our previously reported quality marker panels.,ANCOVA analysis considers confounding factors such as age, sex, or LRRK2 status and thus is well suited to stringently assess which protein changes are truly associated with PD status. This is important when analyzing cohorts with slightly imbalanced demographics between the compared groups like increased age of patients compared with controls, as frequently found in PD cohorts. When we employed ANCOVA to identify proteins that were disease-status-dependently regulated in both cohorts, OMD and CD44 stood out in both cohorts. OMD belongs to the small leucine-rich proteoglycans (SLRPs) and is involved in the organization and homeostasis of the extracellular matrix. CD44 is a cell surface glycoprotein involved in cell-adhesion and cell-cell interactions.,post mortem brain tissue of Alzheimer’s disease patients.,post mortem brain tissue of PD patients are elevated.,,,A GO term enrichment analysis revealed that proteins upregulated in PD patients were associated with lysosome-related terms, which agrees with previous data that lysosomal dysregulation is evident in PD patients and involved in disease pathogenesis. In fact, lysosomal enzymes have long been investigated for their potential as biomarkers in the diagnosis of neurodegenerative disorders including PD.Strikingly, several HLA proteins  were significantly increased in LRRK2 G2019S carriers compared with controls. The HLA locus is one of the key loci associated with susceptibility for PD.Comparing the analyzed CSF proteomes with our urinary proteome profiles in PD patients revealed 1,080 common proteins. This large overlap and the clear correlation of the corresponding protein intensities (Pearson r 0.49) are remarkable and presumably due to the shared blood plasma origin of both CSF and urine. Interestingly, OMD was upregulated in PD patients in both CSF and urine, suggesting that the pathophysiology of PD affecting the OMD pathway is not restricted to the brain. Furthermore, CTSS was upregulated in LRRK2 G2019S carriers in both body fluids. LRRK2 is known to be ubiquitously expressed in many organs, and the observed dysregulation of lysosomal enzymes including CTSS may be due to the hyperactivity of the mutated kinase. Moreover, we found VGF to be important in classifying PD patients from healthy controls by ML. Strikingly, we also reported this in our previous urinary-proteome-profiling study where VGF was the most important feature for classifying LRRK2 PD patients from NMCs, and its levels were strongly decreased in PD patients.,,We also identified several proteins including CHI3L1, FCGR3A, NCR3LG1, and ZP2  that correlate well with the subjects’ age at sample collection , as their levels correlate with disease activity and progression, likely reflecting microglia/macrophage activation.In conclusion, we have applied a highly reproducible and scalable MS-based proteomics workflow to perform proteome profiling of CSF in PD patients. We observed interesting proteome changes in PD patients and identified biomarker signatures that are specific to LRRK2 G2019S carriers. Further studies analyzing larger cohorts of patients will be required to confirm our findings and extend the panels of potential biomarkers. In a next step, clinical and targeted assays need to be developed to validate the biomarkers,The number of samples per disease group in each cohort in combination with patient heterogeneity and biological variation of the CSF proteome limits the power of the statistical analyses performed. Additional studies are needed to validate the identified biomarker candidates from this study using an independent cohort and potentially an orthogonal technology. Furthermore, clinical assays to measure these markers reliably in a routine fashion are still to be developed.mmann@biochem.mpg.de).Further information and request for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dr. Matthias Mann (This study did not generate new unique reagents.LRRK2 Cohort Consortium (LCC). HBS and the proteomics analysis of HBS samples conducted in the current study were approved by the Institutional Review Board of Brigham and Women’s Hospital. The LCC study was established in 2009, when the MJFF LCC brought together investigators from North America, Europe, North Africa, and Asia to study individuals with mutations in the LRRK2 gene. To be eligible to join the consortium, sites had to agree to share a core set of clinical data. Case report forms and standard operating procedures can be found at https://www.michaeljfox.org/news/lrrk2-cohort-consortium. Ethical review and approval was not required for the de-identified sample analysis in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study. Disease severity was assessed by the Unified Parkinson’s Disease Rating Scale (UPDRS), which ranges from 0 for no impairment to a theoretical maximum of 199 for most severely affected individuals. UPDRS scores can be divided into four subscales for evaluating mentation, behavior, and mood , activities of daily living , motor examination  and complications of therapy . Cognitive functioning was assessed using the Montreal Cognitive Assessment (MoCA), which ranges from 30 for no impairments to a theoretical minimum of 0 for most severely affected individuals. In addition, for the HBS cohort, Mini-Mental State Examination (MMSE) scores, which range from 30 to 0  were also available.In this study, CSF samples from two independent cross- sectional cohorts were analyzed. The first cohort consisted of 94 CSF samples from the Harvard Biomarkers Study (HBS) biobank and the second cohort was a subset of biobanked CSF samples from the Michael J. Fox Foundation for Parkinson’s Research (MJFF)-funded ,40 μL of CSF samples were aliquoted in 96-well plates and processed with an automated set-up on an Agilent Bravo liquid handling platform.Cohort-specific libraries were generated by pooling of 24 randomly selected samples of each cohort and separating the peptides of this pooled sample into 24 fractions each by high pH (pH 10) reversed-phase chromatography on the “spider fractionator”.To determine coefficients of variation, five aliquots of a pooled CSF sample on one plate were subjected to sample preparation (intra-plate) and three aliquots of the same pool were subjected to sample preparation on two different plates (inter-plate).,LC-MS/MS analysis was performed on a Q Exactive HF-X Orbitrap mass spectrometer with a nano-electrospray ion source coupled to an EASY-nLC 1200 HPLC . Peptides were separated at 60°C on 50 cm columns with an inner diameter of 75 μm packed in-house with ReproSil-Pur C18-AQ 1.9 μm resin (Dr.Maisch GmbH). Mobile phases A and B were 99.9/0.1% water/FA (v/v) and 80/20/0.1% ACN/water/FA (v/v/v). MS data for single-shot CSF samples were acquired using the MaxQuant Live software and a data-independent acquisition (DIA) mode with phase-constrained spectrum deconvolution.All fractionated samples including EV fractions were acquired using a top 12 data-dependent acquisition (DDA) mode. Full MS scans were acquired in the range of m/z 300–1,650 at a resolution of 60,000 at m/z 200. The automatic gain control (AGC) target was set to 3e6. MS/MS scans were acquired at a resolution of 15,000 at m/z 200.The MS data of the fractionated pools and the single-shot CSF samples were combined into two cohort-specific hybrid libraries using Spectronaut version 14.8.201029.47784 (Biognosys AG). For all experiments except the machine learning with OmicLearn, the two cohorts were quantified separately. All searches were performed against the human SwissProt reference proteome of canonical and isoform sequences with 42,431 entries downloaded in July 2019. Searches used carbamidomethylation as fixed modification and acetylation of the protein N-terminus and oxidation of methionines as variable modifications. The Trypsin/P proteolytic cleavage rule was used, permitting a maximum of 2 missed cleavages and a minimum peptide length of 7 amino acids. The Q-value thresholds for library generation and DIA analyses were both set to 0.01. For individual protein correlations with clinical parameters and the machine learning, the Q-value data filtering setting in Spectronaut was set to “Qvalue”. For all other analyses, the setting was set to “Qvalue percentile” with a cutoff of 25%, to use only those peptides for the protein quantification that passed the Q-value threshold in at least 25% of all analyzed samples. The various runs were normalized to each other by using median intensities of common peptides.LRRK2 status (only in PD+ vs. PD-), and PD status (only LRRK2+ vs. LRRK2-) were set as confounding factors. The FDR  was calculated using Benjamini-Hochberg correction. GO annotations were matched to the proteome data based on Uniprot identifiers. Annotation term enrichment was performed with Fisher exact test in Perseus separately for each cohort. Annotation terms were filtered for terms with an FDR of 5% after Benjamini-Hochberg correction in each cohort. Calculation of Pearson correlation scores and associated p values of protein intensities to UPDRS scores and other clinical parameters was performed in Perseus.The Perseus software package versions 1.6.0.7 and 1.6.1.3For machine learning, OmicLearn (v1.0.0) was utilized for performing the data analysis, model execution, and generating the plots and charts.


In patients with peripheral neuropathies (PNP), neuropathic pain is present in 50% of the cases, independent of the etiology. The pathophysiology of pain is poorly understood, and inflammatory processes have been found to be involved in neuro-degeneration, -regeneration and pain. While previous studies have found a local upregulation of inflammatory mediators in patients with PNP, there is a high variability described in the cytokines present systemically in sera and cerebrospinal fluid (CSF). We hypothesized that the development of PNP and neuropathic pain is associated with enhanced systemic inflammation.To test our hypothesis, we performed a comprehensive analysis of the protein, lipid and gene expression of different pro- and anti-inflammatory markers in blood and CSF from patients with PNP and controls.While we found differences between PNP and controls in specific cytokines or lipids, such as CCL2 or oleoylcarnitine, PNP patients and controls did not present major differences in systemic inflammatory markers in general. IL-10 and CCL2 levels were related to measures of axonal damage and neuropathic pain. Lastly, we describe a strong interaction between inflammation and neurodegeneration at the nerve roots in a specific subgroup of PNP patients with blood-CSF barrier dysfunction.In patients with PNP systemic inflammatory, markers in blood or CSF do not differ from controls in general, but specific cytokines or lipids do. Our findings further highlight the importance of CSF analysis in patients with peripheral neuropathies. Polyneuropathy (PNP) is a term to describe a group of diseases with peripheral nerve dysfunction of various etiologies. Symptoms may affect the motor, sensory and/or autonomic system, and in 50% of the cases, patients experience neuropathic pain. Why some patients with PNP have pain and others not, is unknown, and even PNPs with the same etiology may be painful or painless . The avaAlthough merely 14%-20% of PNP have a definite immune-related cause –7. One oSeveral lipid compounds, such as prostaglandins (PGs) or thromboxanes, are known to be involved in inflammation and pain, and can in turn be upregulated upon inflammation or tissue injury. Moreover, high levels of PGs have been described in serum from patients with peripheral neuropathies, as well as in the spinal cord of animal models with peripheral injury , 19. In In PNP, signs and symptoms are typically length-dependent and thus more severe in the distal extremities of the body, such as feet and hands, than in the proximal regions or in the torso. Previous studies found a local upregulation of inflammatory mediators such as IL-2, IL-6, IL-8 or IL-10 in skin or nerve from patients with PNP , 25. TheWe hypothesized that PNP is associated with enhanced systemic inflammation that may correlate with the severity of the disease and the development of neuropathic pain. To test our hypothesis, we performed a comprehensive analysis of the protein, lipid, and gene expression of selected pro- and anti-inflammatory markers in blood and CSF from patients with PNP and controls. Furthermore, we measured the levels of neurofilament light-chain (NFL), as a cytoskeletal protein expressed by neurons and released upon cell damage, to assess the level of neurodegeneration present in patients with PNP , and to Between 2019 and 2021, 28 patients with PNP were prospectively recruited at the Department of Neurology, University of Würzburg, Germany, where they were seen for diagnostic work-up. In addition, 10 patients with acute headaches of unclear etiology were used as disease controls for the CSF analysis, and serum data from twelve healthy volunteers were included in the analysis to determine the inflammatory state of the headache group. Our study was approved by the Würzburg Medical Faculty Ethics Committee (# 15/19 and # 242/17) and all participants gave written informed consent prior to inclusion.Diagnoses were based on history taking and neurological examination, laboratory studies, and nerve conduction examinations. All patients underwent laboratory tests including full blood count, electrolytes, kidney and liver function tests, C-reactive protein, thyroid stimulating hormone, vitamin B12, HbA1c, oral glucose tolerance test (OGTT), screening for autoimmune antibodies , and lumbar puncture. Electrophysiological assessment with nerve conduction studies of the affected nerves including tibial motor nerve and sural sensory nerve was performed in all PNP patients.To rate the severity of the neuropathy, we used standardized scales, including the modified Toronto clinical neuropathy score (mTCNS), the overall disability sum score (ODSS) and the Medical Research Council-sumscore (MRC-sumscore). Depression was assessed with the “Allgemeine Depressionsskala” (ADS) . Pain wa® Serum-Gel Tubes  and Tempus™ Blood RNA Tubes . For the collection of sera, whole blood was left to clot for 30 min at room temperature and later centrifuged for 10 min at 1200 g. Supernatant was aliquoted and stored at -20°C until further analysis. Tempus™ Blood RNA Tubes were immediately shaken for 30 s to lyse the cells and stabilize the RNA and stored at -80°C until extraction.From each patient, venous blood was drawn in the morning between 8 a.m. and 9 a.m. into S-MonovetteCSF samples were obtained after a lumbar puncture at the L4/5 level. The amount of CSF taken varied between 10-15 ml. Samples of 2 ml were aliquoted and subsequently stored at -20 °C.RNA was extracted from Tempus™ Blood RNA Tubes following the manufacturer’s protocol , 39 from2, 20 μL deoxyribonucleoside triphosphate, 6.25 μL multiscribe reverse transcriptase and 2 μL RNase inhibitor were added per sample. Lastly, reaction was performed under these conditions: annealing , reverse transcription , and enzyme inactivation .For cDNA synthesis from mRNA, TaqMan Reverse Transcription reagents  were used. 250 ng mRNA of each sample were pre-incubated with 5 µl random hexamer at 85°C for 3 min. Next, 10 μL 10× PCR buffer, 22 μL MgClFor miRNA, reverse transcription was carried out with the miRCURY LNA RT Kit . 10 ng of RNA were mixed with 2 μL of 5x reaction buffer, 5 μL of nuclease free water and 1 μL of enzyme mix, per sample. Reaction was performed using the following program: reverse transcription  and enzyme deactivation .Reactions were carried out on a PRISM 7700 Cycler  and transcribed cDNA was stored at -20°C until further analysis.Real time qPCR of mRNA and miRNA targets was performed to analyze gene expression using the StepOnePlus Real-Time PCR System . For mRNA, RT-qPCR was carried out with TaqMan qRT-PCR reagents  and pre-designed assays. For target normalization, different endogenous controls were used: ribosomal protein L13a (RPL13A), actin beta (ACTb), Hydroxymethylbilane Synthase (HMBS) and TATA-Box binding protein (TBP) were tested. TBP was the most stable across groups, and thus selected as suitable endogenous control. For each reaction, 3.5 µl cDNA (8.75 ng cDNA) was mixed with 0.5 µl nuclease free water, 5 µl Fast Advanced Mastermix and 0.5 µl TBP primer and 0.5 µl target primer , the ribosomal RNA 5s and hsa-miR-16 were tested as endogenous controls. Due to differences found in the expression of 5s between groups, hsa-miR-16 was selected as suitable endogenous control, based on its comparability and standard deviation across groups and samples. Each miRNA was run adding 5 µl 2× miRCURY SYBR Green Master Mix with 1 µl ROX per 50 µl and 1 µl primer  and pre-designed miRCURY LNA miR PCR assays  were used. Based on previous experience from our group , 13 and Each mRNA and miRNA was amplified in triplicates and relative quantitation (RQ) values were obtained by the StepOnePlus™ Software v2.3  using interplate calibrators through the 2-ΔΔCt method.Cytokine levels were measured in serum and CSF using the Ella™ technology . Ella™ is a fully automated cartridge-based system that allows you to perform multiple sample, multi-analyte immunoassays with the specificity of a traditional single-plex ELISA (enzyme-linked immunosorbent assay). Samples were thawed on ice and more than two freeze-thaw cycles were avoided. Samples were diluted 1:1 in the appropriate sample diluent from each kit and 50 µl were added to each sample well of the Simple Plex™ cartridge, after 1 ml of washing buffer was added to their corresponding wells. Each sample was measured in triplicates, and the levels of each cytokine were displayed in pg/ml.Serum and CSF samples were quantitatively analyzed for their levels of the endogenous metabolites carnitine (CAR), palmitoylcarnitine (PC) and oleoylcarnitine (OC) by LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry).All samples were thawed on ice for the first time for this analysis, in order to avoid freeze-thaw cycles, and processed within two years upon collection. Serum samples were analyzed at a neat dilution and after applying a 1:10 dilution step in 70% (v/v) ethanol. For sample preparation, 50 µl of serum or CSF samples were mixed with 150 µl internal standard solution (100 ng/ml acetylcarnitine-d3 in acetonitrile). Samples were centrifuged  and the supernatant was used for analysis.®, Sciex) in multiple reaction monitoring (MRM) mode. Measurements were carried out in the positive electrospray ionization (ESI) mode. The LC-MS/MS system was operated with the Software Analyst®, Version 1.7.1 (Sciex).Chromatographic separation was performed with a UHPLC System  using a Waters Acquity UHPL CSH™ Fluoro-Phenyl  column. The injection volume was 2 µl and the flow rate was set to 0.3 mL/min. For the chromatographic separation a gradient was run using 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Detection was performed by means of a triple quadrupole mass spectrometer  or PBS buffer pH 7.4 diluted 1:10 in 30% acetonitrile were used to mimic serum or CSF, respectively).Correlation coefficients of 0.9996  and 0.9992 (PC) were obtained. Accuracies of the calibration samples were found within +/-15% or within +/-20% for the lowest calibration level, respectively. Mean accuracies for all QC samples were found between 87.07% and 109.77% with CVs < 15%, indicating an acceptable method performance for all matrices investigated.Further analytical information can be found in the https://smart.servier.com/, under the Creative common Attribution 3.0 Unported Licence.Statistical analysis was performed in SPSS 27 , where the Shapiro-Wilk test was used to determine the normal distribution of the data. For parametric data, a T-test was used for comparison between two groups and a Pearson test was performed for correlations. In non-parametric data, the Mann–Whitney U Test was applied for comparison of two groups, and the Spearman test was used for correlations. Data results were plotted in GraphPad Prism 9  for visualization. Graphical images were incorporated from Smart Servier Medical Art, Baseline characteristics of the study group and the diagnostic subgroups are summarized in Patients were classified as painless when they presented an NRS = 0 , and as painful with an NRS ≥ 4 . From the full cohort of PNP patients, at the time of inclusion fifteen patients had been treated for their neuropathy with either an immunosuppressive/immunomodulatory drug (six patients) and/or pain treatment (twelve patients), while thirteen received no treatment.Further laboratory and electrophysiological data are given in Patients that presented with acute headaches of unclear etiology (AH) and had a lumbar puncture to exclude meningitis or a subarachnoid bleed were used as controls for the CSF data. Only patients that had less than four leukocytes/µl in their CSF and normal protein values were included. After full clinical work-up, five of these patients had some signs of inflammation . Thus, the AH group was divided into two groups , and only patients with UH were further used as controls for inflammatory markers . Patients with systemic inflammation had a higher expression of IL-10 in comparison to those with normal CRP levels (≤0.5 mg/dl) (p < 0.001)  and divided the patients in subgroups according to the B-CSF barrier integrity: Normal B-CSF barrier integrity (QAlb < 0.007) and mild (0.007 < QAlb < 0.01), moderate (0.01 < QAlb < 0.02) and severe (QAlb > 0.02) B-CSF barrier dysfunction (Alb) correlated with the severity of the clinical phenotype  among subgroups and between PNP and AH (NFL measurements in serum and CSF in the different subgroups of twenty-eight PNP patients and ten AH (IL-6 and QCCL2 (cytokine ratio in CSF/in serum), than those with a normal B-CSF barrier integrity  had higher levels of carnitine in CSF and a lower serum/CSF ratio than those with normal protein levels (p < 0.001)  and in UH (p < 0.001)  and the severity of the neuropathy may indicate that patients with a moderate to severe B-CSF barrier dysfunction also present more severe neuropathy and thus, more neurodegeneration, explaining the higher levels of NFL in these patients. Furthermore, while a break of the B-CSF barrier would allow the exchange of proteins between serum and CSF, the levels of NFL were consistently higher in CSF than in sera, thus indicating an intrathecal production. Since we found higher NFL levels in serum related with a decreased SNAP, and in accordance with previous studies . The patients/participants provided their written informed consent to participate in this study.PG-F, NÜ and CS contributed to conception and design of the study. AR performed the gene expression analysis. KH and NC performed the protein analysis. KS and AN performed the lipid analysis. KH, NC, A-KR, HR, NÜ and CS contributed to recruitment of patients and collection of clinical data. A-KR and HR provided samples from healthy controls. PG-F organized the database and performed the statistical analysis. PG-F wrote the first draft of the manuscript. KS and AN wrote a section of the manuscript. All authors contributed to the article and approved the submitted version.


Intrathecal protein synthesis (ITS) occurs in various central nervous system disorders, but few quantitative studies have focused on ITS for neurosyphilis (NS) in southwestern China. We made a study to quantitatively assess the ITS in patients with NS and to investigate the association between ITS and the stages of NS.Ig/Qalb), Ig extended index (Ig_EI), and intrathecally synthesized fraction (IgIF) using the hyperbolic function. The role of QTPPA/QIgG as an antibody index (AI = Q specific Ig/QIgG) was also explored.CSF–serum specimen pairs from 142 patients (66 NS and 76 non-NS/syphilis) were collected for routine CSF and serum tests. The NS group was divided into slight and severe subgroups according to the NS stages. Three formulas for the quantitative determination of the intrathecal synthesis were calculated to characterize the specimens, including the Ig index (Qp < 0.05) were found to be significantly elevated in the NS group. Intrathecal Ig synthesis can be identified using all three formulas in the NS group. The pattern of Ig intrathecal synthesis was IgIF-G (48.62%) > IgIF-A = IgIF-M (p < 0.05), with the dominant intrathecal fraction being IgG , which was also verified by QIgG> Qalb> QIgM = QIgA. In the slight NS group, the intrathecal fractions of IgM (>0 in 4 out of 20 cases) and IgG (>0 in 16 out of 20) were lower than the intrathecal fractions of IgM (>0 in 19 out of 35 cases) and IgG (>0 in 33 out of 38) in the severe group (p < 0.05). The area under the curve (AUC) of the CSF_TPPA antibody index was 0.867 , with an optimal cutoff point of 0.81, providing a sensitivity of 88.91% and specificity of 84.62%.Sero_TRUST titres , sero_TPPA titres , total protein (MTP), and CSF_Igs (Although the intrathecal synthesis pattern is IgG dominant in patients with NS, brain-derived IgM and IgA can also be found. Moreover, intrathecal IgM and IgG were associated with a parenchymatous type of neurosyphilis. Syphilis-specific antibodies are a new potential tool for NS diagnosis. Neurosyphilis (NS), syphilis of the central nervous system (CNS), has attracted a lot of attention in recent years , 2. NS hpallidum particle agglutination assay) titers, CSF_CXCL13, and serological trust titers have the potential to diagnose or predict neurosyphilis in recent years , it is difficult to discriminate the source of TTs and/or nTT antibodies, as immunoglobulins (Ig) in CSF might be brain-derived or blood-derived. Rieber and other researchers have developed formulas for assessing the intrathecal synthesis of immunoglobin quantitatively  CSF abnormalities: elevated CSF protein (>450 mg/L) and/or leukocyte count (>10 cells/μl) and (2) clinical symptoms or signs consistent with neurosyphilis without other known causes for these clinical abnormalities or a reactive CSF_TRUST. We divided the NS group into slight and severe subgroups according to the stages of neurosyphilis. Here, asymptomatic neurosyphilis, meningeal syphilis, meningovascular syphilis, and ocular syphilis were divided into the slight group, while general paresis and tabes dorsalis were classified into the severe group. Patients with syphilis without a confirmed diagnosis of neurosyphilis were defined as the non-neurosyphilis (N-NS) group.In this study, we included 162 syphilis patients with CSF examinations between December 2019 and July 2021 at West China Hospital. The patients' basic characteristics were collected such as sex, age, syphilis stage, and HIV coinfection. The stages of syphilis were based on clinician diagnosis. Their residual CSF-serum specimen pairs were collected and stored at −80°C for subsequent analysis. CSF samples with macroscopic blood contamination or from duplicate patients were excluded. A total of 142 patients (66 patients with NS and 76 patients with N-NS) were ultimately included, whose information is illustrated in Elecsys Syphilis assays , and FTA-ABS , were performed according to the manufacturer's instructions. CSF IgA and IgM were measured by Siemens Healthcare Diagnostics on a BN II platform , while CSF IgG was measured by immunoturbidimetry . Serum Igs were measured by immunoturbidimetry . However, some values were missing, which may have been caused by a lower limit of detection in CSF .Some related clinical and routine laboratory parameters, including routine CSF results and serum results, were collected from the hospital information system (HIS) and laboratory information system (LIS). The syphilis serological tests, including TRUST , TPPA  tests, alb) was used to evaluate the function of BCB. The IgG index  is considered to be the most commonly used measure to evaluate the function of BCB and can be used in patients with normal BCB function (Ig/ a) based on the linear relationship between ln (QIg) and ln . However, the groundwork of ITS laid by Reiber et al. is a hyperbolic curve between QIg and Qalb (QTPPA/QIgG), providing indirect evidence for T. pallidum invasion in the CNS, to reflect the local pathogen-specific antibody response.The albumin quotient  analysis was performed on MedCalc Statistical Software version 15.2.2 to determine the performance of the Treponema-specific antibody index, and the optimal cutoff was determined corresponding to the maximal Youden's index (sensitivity + specificity−100%). Missing values were not taken into the statistical calculations mentioned. A p < 0.05 was considered statistically significant.The data were expressed as X ± SD or median (Range) according to its distribution. The unpaired This study was approved by (2020-920) the Ethics Committee of West China Hospital of Sichuan University.p > 0.05). However, more male patients were seen in the NS group (p < 0.05). Higher sero_TRUST titres  and sero_TPPA titres  were observed in the NS group (p < 0.001). No statistically significant differences were found in sero_Igs between the two groups (p > 0.05). Compared with CSF protein levels in the N-NS group, the total protein (MTP) and CSF_Igs were elevated in the NS group, but the ALB level was not elevated.In total, 66 patients with NS and 76 patients with N-NS were enrolled . Nearly alb indicated BCB dysfunction (p < 0.05). Intrathecal Igs can be found in the NS groups using all three formulas. However, more patients were found to have intrathecal Ig synthesis using the Ig index than those patients using the Ig extent index and IgIFs. In the N-NS group, the three intrathecal Ig syntheses were not so dissimilar among the three formulas. The molecular diffusion/CSF flow theory provides the sensitive identification of the intrathecal fraction of pathological protein (p < 0.05), with the dominant intrathecal fraction being IgG  in the NS group, which was also verified by QIgG > Qalb > QIgM= QIgA. The Ig quotient diagram with hyperbolic curves is presented in Compared with data in the N-NS group, increased concentrations of the three CSF_Ig classes were seen in the NS group . In the  protein , 24. Whep < 0.05), while there was no statistically significant difference in the intrathecal fractions of IgA between the two groups. Moreover, intrathecal IgM and IgG were associated with general paresis and tabes dorsalis, late stages, or parenchymatous types of neurosyphilis.Intrathecal IgM analysis is not considered a sign of the acute phase of a disease, as there is no Ig isotype switch in the brain . MoreoveTPPA/QIgG) was 0.867 , with an optimal cutoff of ≥0.81, a sensitivity of 88.91% and a specificity of 84.62% , which evaluates the intrathecal synthesis of treponemal antibody, has not yet been validated for the diagnosis. Here, the AUC of the CSF_TPPA index antibody index . Compared with CSF protein levels in the N-NS group, the MTP and CSF_Igs were elevated in the NS group (p < 0.05), but the ALB level was not elevated since all CSF albumin is blood-derived (p > 0.05), interfering with the elevation of immunoglobulins and other brain-derived proteins. Intrathecal synthesis of immunoglobulins occurs in many CNS disorders , suggesting the existence of brain-derived immunoglobulins. The discordant constituent ratio or pattern of ITS by different quantitative determinations is caused by different conditions of application of the three formulas in the NS group. The use of the Ig index assumes a linear relationship between the QIg and the Qalb. Logarithmic transformations of QIg and Qalb did not result in a linear response in the NS group, indicating that Ig_EI may also not be suitable. Compared with the Ig index and Ig-EI, the hyperbolic function showed high sensitivity and a low rate of false-positive findings for central nervous inflammatory diseases , a quotient of CSF/serum albumin  and immunoglobulin quotients (QIg), is useful to confirm a suspicion of infection in the CNS . Here, tiagnosis . The higiagnosis . FurtherP < 0.05), and intrathecal IgM and IgG were associated with general paresis and tabes dorsalis, late stages or parenchymatous types of neurosyphilis. The AI for specific T. pallidum antibodies is another new potential tool for NS diagnosis. However, there are several limitations in this study, including the small sample size, the possibility of misclassification of grouping error, and some missing data due to sample volume or detection limitation of assays. Also, we ignored the effect of HIV infection on intrathecal immunoglobulin synthesis and blood–cerebrospinal fluid barrier dysfunction due to only two cases of coinfection with HIV.In conclusion, the NS Ig ITS pattern was IgIF-G (48.62%) > IgIF-A = IgIF-M . These samples were analyzed anonymously. For researchers, all personal or private information was blind. Written or oral informed consents can be exempted according to rules of the Ethics Committee of West China Hospital of Sichuan University. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.XYH: conceptualization, formal analysis, investigation, writing-original draft, and writing—review and editing. SSY: methodology, validation, and review. LXL and LL: investigation and review. YX: project administration. DDL: formal analysis, conceptualization, writing—review and editing, and supervision. All authors contributed to the article and approved the submitted version.The study was funded by Key R&D projects of the Sichuan Provincial Department of Science and Technology (2022YFS0309).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.