Over the past few years, several fluid biomarker candidates have been proposed for frontotemporal dementia (FTD). We have previously identified CSF proteins that could separate individuals with genetic FTD from controls. However, it is unknown whether alterations in these CSF protein levels are associated with neurodegenerative processes. The aim of this study was to explore how these CSF biomarker candidates correlate with symptom severity as well as cortical and subcortical atrophy. The levels of fourteen proteins were measured in CSF from 202 individuals, 131 mutation carriers with mutations in C9orf72, GRN, or MAPT, and 71 controls, in a cross-sectional subset from the GENFI cohort. The association between the levels of these proteins and CDR plus NACC FTLD-NM sum-of-boxes, cortical thickness, and subcortical volumes were estimated in the mutation carriers. Elevated CSF levels of five out of fourteen proteins were associated with an increased CDR score in the mutation carriers. Additionally, elevated levels of three of these proteins, NEFM, PTPRN2 and SERPINA3, were associated with reduced cortical thickness and/or subcortical volume among all mutation carriers. Some mutation-specific associations were also observed, with SPP1 and CTSS being associated with CDR and atrophy only in MAPT mutation carriers, while NPTX2 was specific for GRN mutation carriers. As indicated by the association to brain atrophy, the proposed fluid biomarker candidates continue to show promise and additional studies will further elucidate their relationship to cortical atrophy in genetic FTD, and their potential as biomarkers for diagnosis, prognosis, and disease staging.

BACKGROUND:Systemic autoimmune rheumatic diseases (SARDs) are a heterogeneous group of autoimmune conditions characterized by immune system dysregulation leading to chronic inflammation and tissue damage. The overlapping clinical manifestations make differential diagnosis challenging, highlighting the need for novel biomarkers to facilitate early diagnosis, stratification, and personalized treatment. METHODS:Five SARDs including idiopathic inflammatory myopathies (n = 210), rheumatoid arthritis (n = 84), systemic sclerosis (n = 100), Sjögren disease (n = 99), and systemic lupus erythematosus (n = 99), as well as healthy controls (n = 400) and controls with acute infectious diseases (n = 218) were selected for plasma protein profiling using Olink Explore 1536. Differential abundance analysis and machine learning were used to identify proteins with both known and novel association to SARDs. RESULTS:The five SARDs share hundreds of proteins with consistently altered abundance compared to both healthy and infectious controls, reflecting common underlying molecular dysregulation. Despite the overlap, we identify multiple proteins with higher abundance specific to individual SARDs. Machine learning further enables accurate classification of the five SARDs, identifying a panel of 48 proteins with high discriminatory performance, several of which are also supported by differential abundance analysis. CONCLUSIONS:Altogether, this explorative cross-sectional study demonstrates the importance of a pan-disease approach, including also infectious and healthy controls, to identify robust and disease-informative protein panels for improved classification of SARDs. Protein levels from this study are available open access through the Human Protein Atlas, facilitating further plasma proteome research on autoimmune disease.

Background Cognitive impairment is a recognised feature of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Despite advances in understanding cognitive impairment in ALS, no fluid biomarkers reliably predict these changes. Prior research in Alzheimer disease (AD) has demonstrated that CSF protein ratios enhance biomarker accuracy by mitigating inter-individual variability, improving diagnostic precision. In AD, ratios involving synaptic markers have shown stronger associations with cognitive outcomes than single proteins, motivating evaluation of a similar ratio-based approach in ALS. Methods Building on findings from the AD field, we analysed 47 CSF proteins, suggested to be associated to neurodegeneration, in 66 patients with ALS and explored protein ratios to evaluate their utility in detecting cognitive impairment, hypothesising shared mechanisms between neurodegenerative diseases. Elastic net regression identified the most predictive protein pairs associated with cognitive impairment, assessed with the Edinburgh Cognitive and Behavioural ALS Screen (ECAS). Results Elastic net identified seven single proteins (NEFM, NPTX2, GAP43, IGFBP4, IGFBP7, SPP1, CDH8) and eight protein pairs associated with ECAS total score. Ratios were generally more informative than individual proteins, with PTPRN2/GAP43 showing the strongest association with ECAS scores, indicating an enhanced ability to capture cognitive changes. Several of the proteins in the most predictive pairs have previously been implicated to associate to cognitive impairment in AD. Conclusion Our findings indicate that protein ratios outperform single-protein analyses in detecting associations with cognitive impairment, aligning with advancements in AD research. By extending the concept of CSF protein ratios from AD to ALS, this study highlights shared pathological mechanisms and suggests that similar proteins are linked to cognitive dysfunction in both diseases.

Abstract Background Aquaporin-4 (AQP4) is crucial for brain fluid regulation and glymphatic system function. Idiopathic normal pressure hydrocephalus (INPH) is characterized by impaired CSF flow and is treated with shunt surgery. This study investigated AQP4 levels in INPH patients to explore its role in pathophysiology and as a potential biomarker for shunt response. Methods CSF samples from 233 INPH patients and 29 controls were analysed. AQP4 levels were compared between preoperative patients and controls, before and after shunt surgery (110 patients), and between shunt responders and non-responders (204 patients). A bead-based assay was used to measure AQP4, and outcomes were assessed by postoperative changes in maximum gait velocity. Results In unadjusted analyses, preoperative AQP4 levels were lower in INPH patients than in controls; however, this difference did not remain after adjustment for pre-analytical and demographic confounders (p = 0.87). Postoperative AQP4 levels were higher (median 1646 AU IQR 1347–1976) than preoperative levels (1166 AU IQR: 976–1345; p < 0.001) and the magnitude of increase showed a modest correlation with gait improvement (rₛ = 0.22, p = 0.022). Shunt responders had lower preoperative AQP4 levels median 1089 AU, IQR 971–1277) than non-responders (median 1213 AU, IQR 1074–1361; p = 0.008). Pre-analytical factors, including storage duration and sample processing, were strong determinants of measured AQP4 levels. Conclusions CSF AQP4 levels in INPH did not differ from those in controls and were highly sensitive to pre-analytical sample handling. CSF AQP4 levels increased following shunt surgery. A potential prognostic value of CSF AQP4 is suggested but requires further investigation.

Background: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with clinical heterogeneity. Biomarker development remains dominated by neurofilament light chain (NEFL), a non-specific marker ofneuroaxonal injury that fails to capture ALS phenotypic diversity, including onset site and ALS-frontotemporalspectrum disorder (ALS-FTSD). We investigated whether plasma proteomics could reveal pathway-levelsignatures that stratify and explain ALS heterogeneity. Methods: We profiled ~5,400 plasma proteins (Olink Explore HT) in 299 patients with ALS and 50 healthycontrols using two complementary analytic frameworks: (i) differential protein abundance analysis to identifyproteins altered in ALS and across clinical subgroups, and (ii) weighted gene correlation network analysis(WGCNA) to identify coordinated protein modules and relate them to ALS diagnosis and clinical traits (site ofonset, ALS-FTSD, ALSFRS-R score, and plasma NEFL). Findings: Differential abundance analysis identified 56 proteins altered in ALS versus controls, 40 of which wereincreased. WGCNA identified 11 proteins correlation modules; ALS samples correlated most strongly with amuscle-enriched module (n=51 proteins), with 29 of the 40 upregulated proteins mapping to this module. Thismuscle-associated signal extended to clinical stratification: spinal-onset patients showed a strong positiveassociation with the muscle module, and differential abundance analysis of spinal- versus bulbar-onset ALSmapped predominantly to the same module. ALS-FTSD instead mapped to distinct modules enriched forextracellular matrix and cell-adhesion pathways, indicating a separable biological axis. The muscle-enrichedmodule correlated with higher NEFL and lower ALSFRS-R, supporting its role as a severity-linked, muscle-involvement proxy. Interpretation: Large-scale plasma proteomics reveals that ALS heterogeneity reflects distinct underlyingbiological structures. A dominant muscle-associated protein network distinguished ALS from controls andcorrelated with onset phenotype and severity, while separate networks linked to ALS-FTSD. These pathway-levelsignatures extend beyond NEFL, enabling biologically informed patient stratification.​

Proteomic research enhances our understanding of health- and disease-related biological processes. Protein profiling during healthy childhood provides important insights into normal physiological development. We longitudinally measured 5416 plasma proteins at four follow-ups during childhood (4-, 8-, 16 years) and early adulthood (24 years) in 100 randomly selected subjects participating in a population-based Swedish cohort, using Olink Explore HT. In total, 3509 proteins were included in the analysis. 54% of the proteins were found to be associated with age, and we observed several protein trajectories from childhood to adulthood based on clustering. In addition to proteins involved in bone, teeth and cartilage formation, we identified differences in proteins involved in neural function, drug metabolism, and hormonal control. There were pronounced sex-related differences in protein levels, particularly at follow-ups 16 and 24, characterized by, for example, growth, response to stimuli and regulation of catabolic processes. We demonstrate dynamic age- and sex-related changes in protein levels during the first two decades of life. Our study results may serve as an important resource in understanding human physiological development, disease etiology, and for future protein biomarker research.

Background Aquaporin-4 (AQP4) is crucial for brain fluid regulation and glymphatic system function. Idiopathic normal pressure hydrocephalus (INPH), characterized by impaired CSF flow, is often treated with shunt surgery. This study investigated AQP4 levels in INPH patients to explore its role in pathophysiology and potential as a biomarker for shunt response. Methods CSF samples from 233 INPH patients and 29 controls were analysed. AQP4 levels were compared between preoperative patients and controls, before and after shunt surgery (110 patients), and between shunt responders and non-responders (204 patients). A bead-based assay was used to measure AQP4, and outcomes were assessed by postoperative changes in maximum gait velocity. Results Preoperative AQP4 levels were lower in INPH patients (1177 ± 259 AU) than in controls (1351 ± 279 AU, p = 0.003), but the difference did not persist after adjustment for confounders (p = 0.87). Postoperatively, AQP4 levels increased (1714 ± 473 vs 1186 ± 269 AU, p < 0.001), with a modest correlation between AQP4 increase and gait improvement (rₛ = 0.22, p = 0.022). Compared with non-responders, shunt responders (130 patients) had lower preoperative AQP4 levels (1144 ± 250 vs. 1238 ± 273 AU, p = 0.016) Conclusions INPH patients presented AQP4 levels comparable to those of controls. The postoperative increase in AQP4 may result from altered CSF dynamics induced by the shunt; however, the underlying mechanism remains unclear, underscoring the need for further studies to clarify this relationship. Notably, lower preoperative AQP4 levels in shunt responders suggest a potential predictive role.

PROBLEM:Female sex workers (FSWs) are at higher risk of acquiring HIV. Interestingly, some FSWs who are highly exposed remain seronegative for HIV (HESN). This natural resistance to HIV infection has been attributed to an immune quiescence (IQ) phenotype. Our study investigates how the menstrual cycle phases (follicular and luteal) impact the immune responses in Kenyan FSWs. METHODS:This is a part of the Longitudinal Assessment of Mucosal Immune Quiescence study (LAMIQ), 48 FSWs not living with HIV and not using hormonal contraception were followed for a menstrual cycle and divided into two groups based on duration of sex work: New Negative (NN) with 3 years or less and HESN with at least 7 years of involvement in sex work. We obtained blood and cervicovaginal samples and measured sex hormone, cytokine, and chemokine levels, and blood and endocervical T-cell and NK-cell phenotypes. RESULTS:We observed differences in how the immune response of NN and HESN responds to sex hormones. Indeed, the level of mucosal Annexin A3 measured was higher during the luteal phase in HESN, which was not observed in NN. HESN exhibited a higher CD39 expression on their Treg during the luteal phase, while maintaining CTLA-4 expression compared to NN. Furthermore, in HESN, NK cell activation varied across the menstrual cycle phases. They had a higher expression of NKG2D and an increase in the cluster of CD95+ HLA-DR+ NK cells during the follicular phase. This suggests stronger innate immune activation in HESN during the follicular phase of the menstrual cycle. CONCLUSION:Our data indicate that, in HESN, there is a modulation of the immune response based on the menstrual cycle, which potentially limits the availability of  HIV target cells at the female genital tract during the luteal phase of the menstrual cycle (window of susceptibility).

Disturbed cerebral autoregulation (represented by a positive pressure reactivity index [PRx]), elevated intracranial pressure (ICP), and decreased cerebral perfusion pressure (CPP) are key treatment targets following severe traumatic brain injury (sTBI). This study investigated neuroinflammation as a potential mechanism underlying these intracranial disturbances. Plasma samples from 11 sTBI patients (from a prior Phase II drug trial) were analyzed for 174 proteins using an antibody-based suspension bead array, with intervention effects accounted for where possible. Dimensionality reduction techniques, including principal component analysis (PCA) and supervised methods, were applied to protein data, informed by physiological variables (ICP, CPP, and PRx). PCA revealed distinct protein clustering patterns related to ICP >20 mmHg and PRx > 0, with PC1 linked to patient ID, time from injury, and intervention, and PC2/PC3 significantly associated with PRx dose (p < 0.001). Markers relating to inflammation of the vascular system comprised 20% of the top 50 proteins influencing PC2, implicating complement inflammation in these processes. Notably, MASP-2 (p = 0.027) and complement factor I (p = 0.039) were significantly associated with PRx dose in a mixed-effects model. These findings suggest that vascular inflammation, particularly complement activation, may contribute to intracranial physiological disturbances in sTBI, highlighting the complement pathway as a potential target for further investigation.

Accurate diagnosis and monitoring of neurodegenerative diseases require reliable biomarkers. Cerebrospinal fluid (CSF) proteins are promising candidates for reflecting brain pathology; however, their diagnostic utility may be compromised by natural variability between individuals, weakening their association with disease. Here, we measured the levels of 69 pre-selected proteins in cerebrospinal fluid using antibody-based suspension bead array technology in a multi-disease cohort of 499 individuals with neurodegenerative disorders including Alzheimer’s disease (AD), behavioral variant frontotemporal dementia, primary progressive aphasias, amyotrophic lateral sclerosis (ALS), corticobasal syndrome, primary supranuclear palsy, along with healthy controls. We identify significant inter-individual variability in overall CSF levels of brain-derived proteins, which could not be attributed to specific disease associations. Using linear modelling, we show that adjusting for median CSF levels of brain-derived proteins increases the diagnostic accuracy of proteins previously identified as altered in CSF in the context of neurodegenerative disorders. We further demonstrate a simplified approach for the adjustment using pairs of correlated proteins with opposite alteration in the diseases. With this approach, the proteins adjust for each other and further increase the biomarker performance through additive effect. When comparing the diseases, two proteins—neurofilament medium and myelin basic protein—showed increased levels in ALS compared to other diseases, and neurogranin showed a specific increase in AD. Several other proteins showed similar trends across the studied diseases, indicating that these proteins likely reflect shared processes related to neurodegeneration. Overall, our findings suggest that accounting for inter-individual variability is crucial in future studies to improve the identification and performance of relevant biomarkers. Importantly, we highlight the need for multi-disease studies to identify disease-specific biomarkers.

Background & Aims: Primary sclerosing cholangitis (PSC) is a rare cholestatic liver disease with heterogeneous phenotypes and progression. Autoimmune traits, such as the presence of autoantibodies, are suspected to drive its heterogeneity. Methods: We performed a proteome-scale autoantibody screen of IgG and IgA isotypes using >42,100 protein fragments. This was followed by a validation of 1,153 selected autoantibodies, in serum samples from 466 patients with PSC in a longitudinal setting using the SUPRIM cohort and 214 controls. Results: We identified autoantibodies associated with clinical phenotypes, biochemical and clinical severity, comorbidities, and disease progression (e.g. alkaline phosphatase and albumin level p

Malaria presents with varying degrees of severity. To improve clinical management and prevention, it is crucial to understand the pathogenesis and host response. We analyzed 1,463 plasma proteins during and after acute Plasmodium falciparum malaria in adult travelers and linked responses to peripheral immune cells by integrating with single-cell RNA sequencing (RNA-seq) data from a subset of donors. We identified extensive perturbations in over 250 proteins with diverse origins, including many not previously analyzed in malaria patients, such as hormones, circulating receptors, and intracellular or membrane-bound proteins from affected tissues. The protein profiles clustered participants according to disease severity, enabling the identification of a compressed 11-protein signature enriched in severe malaria. Conceptually, this study advances our understanding of malaria by linking systemic proteomic changes to immune cell communication and organ-specific responses. This resource, which includes an interactive platform to explore data, opens new avenues for hypothesis generation, biomarker discovery, and therapeutic target identification.

The human blood proteome provides a holistic readout of health states through the assessment of thousands of circulating proteins. In this study, we present a pan-disease resource to enable the study of diverse disease phenotypes within a harmonized proteomics dataset. By profiling protein concentrations across 59 diseases and healthy cohorts, we identified proteins associated with age, sex, and body mass index, as well as disease-specific signatures. This study highlights shared and distinct protein patterns across conditions, demonstrating the power of a unified proteomics approach to uncover biological insights. The dataset, covering 8262 individuals and up to 5416 proteins, serves as an online resource for exploring disease-specific protein profiles and advancing precision medicine research.

Background Amyloid and tau aggregates are considered to cause neurodegeneration and consequently cognitive decline in individuals with Alzheimer’s disease (AD). Here, we explore the potential of cerebrospinal fluid (CSF) proteins to reflect AD pathology and cognitive decline, aiming to identify potential biomarkers for monitoring outcomes of disease-modifying therapies targeting these aggregates. Method We used a multiplex antibody-based suspension bead array to measure the levels of 49 proteins in CSF from the Swedish GEDOC memory clinic cohort at the Karolinska University Hospital. The cohort comprised 148 amyloid- and tau-negative individuals (A-T-) and 65 amyloid- and tau-positive individuals (A+T+). An independent sample set of 26 A-T- and 26 A+T+ individuals from the Amsterdam Dementia Cohort was used for validation. The measured proteins were clustered based on their correlation to CSF amyloid beta peptides, tau and NfL levels. Further, we used support vector machine modelling to identify protein pairs, matched based on their cluster origin, that reflect AD pathology and cognitive decline with improved performance compared to single proteins. Results The protein-clustering revealed 11 proteins strongly correlated to t-tau and p-tau (tau-associated group), including mainly synaptic proteins previously found elevated in AD such as NRGN, GAP43 and SNCB. Another 16 proteins showed predominant correlation with Aβ42 (amyloid-associated group), including PTPRN2, NCAN and CHL1. Support vector machine modelling revealed that proteins from the two groups combined in pairs discriminated A-T- from A+T+ individuals with higher accuracy compared to single proteins, as well as compared to protein pairs composed of proteins originating from the same group. Moreover, combining the proteins from different groups in ratios (tau-associated protein/amyloid-associated protein) significantly increased their correlation to cognitive decline measured with cognitive scores. The results were validated in an independent cohort. Conclusions Combining brain-derived proteins in pairs largely enhanced their capacity to discriminate between AD pathology-affected and unaffected individuals and increased their correlation to cognitive decline, potentially due to adjustment of inter-individual variability. With these results, we highlight the potential of protein pairs to monitor neurodegeneration and thereby possibly the efficacy of AD disease-modifying therapies.

Importance: Aquaporin-4 (AQP4) plays a critical role in the glymphatic system, responsible for clearing brain solutes like Abeta peptides. Exploring AQP4 as an Alzheimer's disease (AD) biomarker might aid in the understanding of AD neuropathology and monitor the effects of novel drug candidates on the glymphatic system. Objective: To determine the potential of CSF AQP4 as an early stage AD biomarker using a newly established immunoassay. Design: A discovery cohort (n = 157) (2010-2022), composed by AD patients, other neurodegenerative conditions and controls (CON), was used to assess the diagnostic performance of CSF AQP4. Subsequently, AQP4 concentration across the clinical AD spectrum was analyzed in two independent validation cohorts (n = 176) (2016-2023). Stratified randomization based on diagnosis and blinded analyses were performed. Setting Multicenter study: Ulm University Hospital (discovery), University of Perugia (validation cohort I), University Hospital of Torino (validation cohort II). Participants: Discovery cohort: 38 CON, 40 AD, 21 primary progressive aphasia, 20 behavioural variant frontotemporal dementia, 17 amyotrophic lateral sclerosis (ALS), and 21 Lewy body disease (LBD). Validation cohorts: 55 CON, 14 preclinical AD, 51 AD with mild cognitive impairment (AD-MCI), 39 AD dementia (ADD) and 17 mild cognitive impairment with non-AD pathology (non-AD MCI). The discovery cohort was selected through random sampling, while validation cohort I and II followed a consecutive sampling method. Exposures: CSF AQP4 Main Outcome (s) and Measure (s): AQP4 CSF biomarker detection Results: A total of 333 participants were included in this study. In the discovery cohort, the median (IQR) age was 69 (61-75) years and 46.5% of the cohort were women. CSF AQP4 concentration was increased in AD patients compared to CON (p < 0.001), ALS (p = 0.015), and LBD (p = 0.012) patients. CSF AQP4 in AD patients were further analyzed in validation cohort I (median (IQR) age, 74 (71-77) years; 62.0% women), and II (median (IQR) age, 71 (65-75) years; 58.5% women). When analyzing the different stages of the AD continuum in validation cohort I, AD-MCI (p = 0.011) and ADD (p = 0.002) patients had significantly higher AQP4 concentrations than CON. Similar results were obtained in cohort II, where AQP4 levels were higher in AD-MCI (p < 0.001) and ADD (p = 0.028) patients compared to controls. The AQP4 accuracy (area under the receiver operating characteristic curve [AUC]) to distinguish AD patients from CON was 0.81 (95% CI: 0.71 to 0.90, p <0.001) in the discovery cohort, 0.70 (95% CI: 0.60 to 0.81, p<0.001) in validation cohort I and 0.82 (95% CI 0.71 to 0.94, p <0.001) in II. Moreover, patients with AD-MCI could be distinguished from non-AD MCI with an AUC of 0.79 (95% CI: 0.65 to 0.93, p = 0.002). Conclusions and Relevance: Three independent cohorts consistently showed elevated AQP4 levels in AD (including AD-MCI and ADD) compared to CON and other neurodegenerative conditions, suggesting specificity to AD pathology. These findings contribute to understanding AD neuropathology and propose AQP4 as a potential early biomarker of AD. Further investigations are needed to proof AQP4 as a fluid blood brain barrier damage marker.### Competing Interest StatementMs. Gomez de San Jose has no disclosures to report Dr. Halbgebauer has no disclosures to report Prof. Steinacker has no disclosures to report Dr. Anderl-Straub has no disclosures to report Dr. Abu-Rumeileh has no disclosures to report Dr. Barba has no disclosures to report Dr. Oeckl has no disclosures to report Dr. Bellomo has no disclosures to report Dr. Gaetani participated on advisory boards for, and received writing honoraria and travel grants from Almirall, Biogen, Euroimmun, Fujirebio, Lilly, Merck, Mylan, Novartis, Roche, Sanofi, Siemens Healthineers and Teva. Dr. Toja has no disclosures to report Ms. Mravinacova has no disclosures to report Dr. Bergstrom has no disclosures to report Dr. Manberg has no disclosures to report Dr. Grassini has no disclosures to report Prof. Rainero has no disclosures to report Prof. Nilsson has no disclosures to report Prof. Parnetti has no disclosures to report Prof. Otto has no disclosures to report ### Funding StatementMO, LP, PN and GSJN are supported by the Marie Skłodowska-Curie grant agreement No. 860197 - MIRIADE project (European Union's Horizon 2020 research and innovation program) GB is supported by the Postdoctoral Fellowship for Basic Scientists grant of the Parkinson's Foundation (Award ID: PF-PRF-934916). LP and LG are funded by the European Union-Next Generation EU - PNRR M6C2 - Investimento 2.1 Valorizzazione e potenziamento della ricerca biomedica del SSN (PNRR-MAD-2022-12376035). LB is supported by the Medical Faculty of Martin-Luther University Halle Wittenberg (Junior Clinician Scientist Programm No. JCS24/02). SAB received research support from the Medical Faculty of Martin-Luther University Halle-Wittenberg (Clinician Scientist-Programm No. CS22/06). ### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:The Ethics Comittee of the University of Ulm gave ethical approval for this work. The Ethics Comittee of the University of Perugia gave ethical approval for this work. The Ethics Comittee of the University of Turin gave ethical approval for this work.I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors

The effect of varying brain ventricular volume on the cerebrospinal fluid (CSF) proteome has been discussed as possible confounding factors in comparative protein level analyses. However, the relationship between CSF volume and protein levels remains largely unexplored. Moreover, the few existing studies provide conflicting findings, indicating the need for further research. Here, we explored the association between levels of 88 pre-selected CSF proteins and ventricular volume derived from magnetic resonance imaging (MRI) measurements in 157 cognitively healthy 70-year-olds from the H70 Gothenburg Birth Cohort Studies, including individuals with and without pathological levels of Alzheimer’s disease (AD) CSF markers (n = 123 and 34, respectively). Both left and right lateral, the inferior horn as well as the third and the fourth ventricular volumes were measured. Different antibody-based methods were employed for the protein measurements, with most being analyzed using a multiplex bead-based microarray technology. Furthermore, the associations between the protein levels and cortical thickness, fractional anisotropy, and mean diffusivity were assessed. CSF levels of many brain-derived proteins correlated with ventricular volumes in A-T- individuals, with lower levels in individuals with larger ventricles. The strongest negative correlations with total ventricular volume were observed for neurocan (NCAN) and neurosecretory protein VGF (rho = -0.34 for both). Significant negative correlations were observed also for amyloid beta (Ab) 38, Ab40, total tau (t-tau), and phosphorylated tau (p-tau), with correlation ranging between − 0.34 and − 0.28, while no association was observed between ventricular volumes and Ab42 or neurofilament light chain (NfL). Proteins with negative correlations to ventricular volumes further demonstrated negative correlations to mean diffusivity and positive correlation to fractional anisotropy. However, only weak or no correlations were observed between the CSF protein levels and cortical thickness. A + T + individuals demonstrated higher CSF protein levels compared to A-T- individuals with the most significant differences observed for neurogranin (NRGN) and synuclein beta (SNCB). Our findings suggest that the levels of many brain-derived proteins in CSF may be subjected to dilution effects depending on the size of the brain ventricles in healthy individuals without AD pathology. This phenomenon could potentially contribute to the inter-individual variations observed in CSF proteomic studies.

Objective: To evaluate the prognostic potential of neurofilament medium chain (NfM) in CSF from patients with ALS and explore its relationship with the extensively studied neurofilament light chain (NfL) and phosphorylated heavy chain (pNfH). Method: CSF levels of NfL, NfM, and pNfH were analyzed in 235 samples from patients with ALS, ALS mimics, and healthy controls in a well-characterized cohort from Karolinska ALS Clinical Research Center in Stockholm, Sweden. NfM levels were analyzed using an antibody-based suspension bead-array and NfL and pNfH levels were measured using ELISA. Clinical data, including ALS Revised Functional Rating Scale (ALSFRS-R), and survival outcomes were utilized for disease progression estimations. Result: Increased NfM levels were observed in patients with ALS compared with mimics and healthy controls. Similarly, higher NfM levels were found in fast compared with slow progressing patients for baseline and longitudinal progression when evaluating both total and subscores of ALSFRS-R. These findings were consistent with the results observed for NfL and pNfH. All three proteins, used individually as well as in combination, showed comparable performance when classifying fast vs slow progressing patients (AUCs 0.78-0.85). For all neurofilaments, higher survival probability was observed for patients with low CSF levels. Conclusion: Based on this cross-sectional study, the prognostic value provided by NfM aligns with the more established markers, NfL and pNfH. Additional investigations with independent cohorts and longitudinal studies are needed to further assess the potential added value of NfM.

BackgroundThe cervicovaginal epithelial barrier is crucial for defending the female reproductive tract against sexually transmitted infections. Hormones, specifically estradiol and progesterone, along with their respective receptor expressions, play an important role in modulating this barrier. However, the influence of estradiol and progesterone on gene and protein expression in the ectocervical mucosa of naturally cycling women is not well understood.MethodsMucosal and blood samples were collected from Kenyan female sex workers at high risk of sexually transmitted infections. All samples were obtained at two time points, separated by two weeks, aiming for the follicular and luteal phases of the menstrual cycle. Ectocervical tissue biopsies were analyzed by RNA-sequencing and in situ immunofluorescence staining, cervicovaginal lavage samples (CVL) were evaluated using protein profiling, and plasma samples were analyzed for hormone levels.ResultsUnsupervised clustering of RNA-sequencing data was performed using Weighted gene co-expression network analysis (WGCNA). In the follicular phase, estradiol levels positively correlated with a gene module representing epithelial structure and function, and negatively correlated with a gene module representing cell cycle regulation. These correlations were confirmed using regression analysis including adjustment for bacterial vaginosis status. Using WGCNA, no gene module correlated with progesterone levels in the follicular phase. In the luteal phase, no gene module correlated with either estradiol or progesterone levels. Protein profiling on CVL revealed that higher levels of estradiol during the follicular phase correlated with increased expression of epithelial barrier integrity markers, including DSG1. This contrasted to the limited correlations of protein expression with estradiol levels in the luteal phase. In situ imaging analysis confirmed that higher estradiol levels during the follicular phase correlated with increased DSG1 expression.ConclusionWe demonstrate that estradiol levels positively correlate with specific markers of ectocervical epithelial structure and function, particularly DSG1, during the follicular phase of the menstrual cycle. Neither progesterone levels during the follicular phase nor estradiol and progesterone levels during the luteal phase correlated with any specific sets of gene markers. These findings align with the expression of estradiol and progesterone receptors in the ectocervical epithelium during these menstrual phases.

Background Plasma biomarkers reflecting the pathology of frontotemporal dementia would add significant value to clinical practice, to the design and implementation of treatment trials as well as our understanding of disease mechanisms. The aim of this study was to explore the levels of multiple plasma proteins in individuals from families with genetic frontotemporal dementia. Methods Blood samples from 693 participants in the GENetic Frontotemporal Dementia Initiative study were analysed using a multiplexed antibody array targeting 158 proteins. Results We found 13 elevated proteins in symptomatic mutation carriers, when comparing plasma levels from people diagnosed with genetic FTD to healthy non-mutation controls and 10 proteins that were elevated compared to presymptomatic mutation carriers. Conclusion We identified plasma proteins with altered levels in symptomatic mutation carriers compared to non-carrier controls as well as to presymptomatic mutation carriers. Further investigations are needed to elucidate their potential as fluid biomarkers of the disease process.

A novel panel of 14 proteins measured in the CSF could separate individuals with genetic frontotemporal dementia (FTD) from controls, with most significant findings observed for neurofilament medium (NEFM), neuronal pentraxin 2 (NPTX2), neurosecretory protein VGF (VGF) and aquaporin 4 (AQP4) [1]. However, it is currently unknown whether these altered protein levels in the CSF reflect neurodegenerative changes in the brain. The aim of this study was to explore the cross-sectional associations between the previously identified CSF biomarker candidates and cortical thickness in presymptomatic and symptomatic mutation carriers, and whether those associations differ by FTD mutation type. We have analyzed T1 MRI scans alongside concurrent CSF samples from 202 individuals from the GENFI cohort, belonging to families that carry FTD mutations in either C9orf72 , GRN or MAPT genes. The study sample included symptomatic mutation carriers, presymptomatic mutation carriers and non-carrier controls. Cortical thickness was estimated with FreeSurfer and CSF protein levels were measured via a multiplexed antibody-based suspension bead array. The correlations between regional cortical thickness and protein levels were calculated via linear regression. Altered levels of NEFM, AQP4, APOA1, PTPRN2, CTSS, SERPINA3, C4, AMPH and CD14 were all correlated with increased atrophy of at least one cortical region. Some effects were mutation specific, but NEFM, AQP4 and APOA1 correlated with atrophy in all mutation groups. We also observed mutation specific effects for 10 of the proteins. CTSS levels were only correlated with cortical atrophy in C9orf72 mutation carriers while NPTX2, VGF and PTPRN2 correlated with atrophy in GRN mutation carriers. In MAPT mutation carriers, 6 different proteins correlated with atrophy in the right temporal pole. The proposed fluid biomarker candidates continue to show promise and further longitudinal studies will contribute to elucidate their relationship to cortical atrophy and their prognostic value in genetic FTD. [1] Bergström et al. Mol Neurodegener. 2021; 16(1):79