Homotypic fibrillization of TMEM106B across diverse neurodegenerative diseases.

Misfolding and aggregation of disease-specific proteins, resulting in the formation of filamentous cellular inclusions, is a hallmark of neurodegenerative disease with characteristic filament structures, or conformers, defining each proteinopathy. Here we show that a previously unsolved amyloid fibril composed of a 135 amino acid C-terminal fragment of TMEM106B is a common finding in distinct human neurodegenerative diseases, including cases characterized by abnormal aggregation of TDP-43, tau, or α-synuclein protein. A combination of cryoelectron microscopy and mass spectrometry was used to solve the structures of TMEM106B fibrils at a resolution of 2.7 Å from postmortem human brain tissue afflicted with frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP, n = 8), progressive supranuclear palsy (PSP, n = 2), or dementia with Lewy bodies (DLB, n = 1). The commonality of abundant amyloid fibrils composed of TMEM106B, a lysosomal/endosomal protein, to a broad range of debilitating human disorders indicates a shared fibrillization pathway that may initiate or accelerate neurodegeneration.

Truncated TDP-43 proteoforms diagnostic of frontotemporal dementia with TDP-43 pathology.

INTRODUCTION: Biomarkers of TDP-43 pathology are needed to distinguish frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP) from phenotypically related disorders. While normal physiological TDP-43 is not a promising biomarker, low-resolution techniques have suggested truncated forms of TDP-43 may be specific to TDP-43 pathology. To advance biomarker efforts for FTLD-TDP, we employed a high-resolution structural technique to characterize TDP-43 post-translational modifications in FTLD-TDP. METHODS: High-resolution mass spectrometry was used to characterize TDP-43 proteoforms in brain tissue from FTLD-TDP, non-TDP-43 dementias and neuropathologically unaffected cases. Findings were then verified in a larger cohort of FTLD-TDP and non-TDP-43 dementias via targeted quantitative mass spectrometry. RESULTS: In the discovery phase, truncated TDP-43 identified FTLD-TDP with 85% sensitivity and 100% specificity. The verification phase revealed similar findings, with 83% sensitivity and 89% specificity. DISCUSSION: The concentration of truncated TDP-43 proteoforms-in particular, in vivo generated C-terminal fragments-have high diagnostic accuracy for FTLD-TDP. HIGHLIGHTS: Discovery: Truncated TDP-43 differentiates FTLD-TDP from related dementias. Verification: Truncated TDP-43 concentration has high accuracy for FTLD-TDP. TDP-43 proteoforms <28 kDa have highest discriminatory power for TDP-43 pathology.

People With Human Immunodeficiency Virus Receiving Suppressive Antiretroviral Therapy Show Typical Antibody Durability After Dual Coronavirus Disease 2019 Vaccination and Strong Third Dose Responses.

BACKGROUND: Longer-term humoral responses to 2-dose coronavirus disease 2019 (COVID-19) vaccines remain incompletely characterized in people living with human immunodeficiency virus (HIV) (PLWH), as do initial responses to a third dose. METHODS: We measured antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain, angiotensin-converting enzyme 2 (ACE2) displacement, and viral neutralization against wild-type and Omicron strains up to 6 months after 2-dose vaccination, and 1 month after the third dose, in 99 PLWH receiving suppressive antiretroviral therapy and 152 controls. RESULTS: Although humoral responses naturally decline after 2-dose vaccination, we found no evidence of lower antibody concentrations or faster rates of antibody decline in PLWH compared with controls after accounting for sociodemographic, health, and vaccine-related factors. We also found no evidence of poorer viral neutralization in PLWH after 2 doses, nor evidence that a low nadir CD4+ T-cell count compromised responses. Post-third-dose humoral responses substantially exceeded post-second-dose levels, though Omicron-specific responses were consistently weaker than responses against wild-type virus. Nevertheless, post-third-dose responses in PLWH were comparable to or higher than controls. An mRNA-1273 third dose was the strongest consistent correlate of higher post-third-dose responses. CONCLUSION: PLWH receiving suppressive antiretroviral therapy mount strong antibody responses after 2- and 3-dose COVID-19 vaccination. Results underscore the immune benefits of third doses in light of Omicron.

Reduced Magnitude and Durability of Humoral Immune Responses to COVID-19 mRNA Vaccines Among Older Adults.

BACKGROUND: The magnitude and durability of immune responses to coronavirus disease 2019 (COVID-19) mRNA vaccines remain incompletely characterized in the elderly. METHODS: Anti-spike receptor-binding domain (RBD) antibodies, angiotensin-converting enzyme 2 (ACE2) competition, and virus neutralizing activities were assessed in plasma from 151 health care workers and older adults (range, 24-98 years of age) 1 month following the first vaccine dose, and 1 and 3 months following the second dose. RESULTS: Older adults exhibited significantly weaker responses than younger health care workers for all humoral measures evaluated and at all time points tested, except for ACE2 competition activity after 1 vaccine dose. Moreover, older age remained independently associated with weaker responses even after correction for sociodemographic factors, chronic health condition burden, and vaccine-related variables. By 3 months after the second dose, all humoral responses had declined significantly in all participants, and remained significantly lower among older adults, who also displayed reduced binding antibodies and ACE2 competition activity towards the Delta variant. CONCLUSIONS: Humoral responses to COVID-19 mRNA vaccines are significantly weaker in older adults, and antibody-mediated activities in plasma decline universally over time. Older adults may thus remain at elevated risk of infection despite vaccination.

Older Adults Mount Less Durable Humoral Responses to Two Doses of COVID-19 mRNA Vaccine but Strong Initial Responses to a Third Dose.

BACKGROUND: Third coronavirus disease 2019 (COVID-19) vaccine doses are broadly recommended, but immunogenicity data remain limited, particularly in older adults. METHODS: We measured circulating antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain, ACE2 displacement, and virus neutralization against ancestral and omicron (BA.1) strains from prevaccine up to 1 month following the third dose, in 151 adults aged 24-98 years who received COVID-19 mRNA vaccines. RESULTS: Following 2 vaccine doses, humoral immunity was weaker, less functional, and less durable in older adults, where a higher number of chronic health conditions was a key correlate of weaker responses and poorer durability. One month after the third dose, antibody concentrations and function exceeded post-second-dose levels, and responses in older adults were comparable in magnitude to those in younger adults at this time. Humoral responses against omicron were universally weaker than against the ancestral strain after both the second and third doses. Nevertheless, after 3 doses, anti-omicron responses in older adults reached equivalence to those in younger adults. One month after 3 vaccine doses, the number of chronic health conditions, but not age, was the strongest consistent correlate of weaker humoral responses. CONCLUSIONS: Results underscore the immune benefits of third COVID-19 vaccine doses, particularly in older adults.

Establishing pre-analytical requirements and maximizing peptide recovery in the analytical phase for mass spectrometric quantification of amyloid-ß peptides 1-42 and 1-40 in CSF.

OBJECTIVES: Amyloid-ß (Aß) peptides in cerebrospinal fluid (CSF), including Aß42 (residues 1-42) and Aß40 (residues 1-40), are utilized as biomarkers in the diagnostic workup of Alzheimer's disease. Careful consideration has been given to the pre-analytical and analytical factors associated with measurement of these peptides via immunoassays; however, far less information is available for mass spectrometric methods. As such, we performed a comprehensive evaluation of pre-analytical and analytical factors specific to Aß quantification using mass spectrometry. METHODS: Using our quantitative mass spectrometry assay for Aß42 and Aß40 in CSF, we investigated the potential for interference from hemolysate, bilirubin, lipids, and anti-Aß-antibodies. We also optimized the composition of the calibrator surrogate matrix and Aß recovery during and after solid phase extraction (SPE). RESULTS: There was no interreference observed with total protein up to 12 g/L, hemolysate up to 10% (v/v), bilirubin up to 0.5% (v/v), intralipid up to 1% (v/v), or anti-Aß-antibodies at expected therapeutic concentrations. For hemolysate, bilirubin and lipids, visual CSF contamination thresholds were established. In the analytical phase, Aß recovery was increased by ∼50% via SPE solvent modifications and by over 150% via modification of the SPE collection plate, which also extended analyte stability in the autosampler. CONCLUSIONS: Attention to mass spectrometric-specific pre-analytical and analytical considerations improved analytical sensitivity and reproducibility, as well as, established CSF specimen acceptance and rejection criteria for use by the clinical laboratory.

Clinical reporting following the quantification of cerebrospinal fluid biomarkers in Alzheimer's disease: An international overview.

INTRODUCTION: The current practice of quantifying cerebrospinal fluid (CSF) biomarkers as an aid in the diagnosis of Alzheimer's disease (AD) varies from center to center. For a same biochemical profile, interpretation and reporting of results may differ, which can lead to misunderstandings and raises questions about the commutability of tests. METHODS: We obtained a description of (pre-)analytical protocols and sample reports from 40 centers worldwide. A consensus approach allowed us to propose harmonized comments corresponding to the different CSF biomarker profiles observed in patients. RESULTS: The (pre-)analytical procedures were similar between centers. There was considerable heterogeneity in cutoff definitions and report comments. We therefore identified and selected by consensus the most accurate and informative comments regarding the interpretation of CSF biomarkers in the context of AD diagnosis. DISCUSSION: This is the first time that harmonized reports are proposed across worldwide specialized laboratories involved in the biochemical diagnosis of AD.

Applying the Alzheimer Disease ATN Diagnostic Framework in Atypical Dementia.

Cerebrospinal fluid (CSF) biomarkers amyloid-ß and tau have been validated for the antemortem diagnosis of Alzheimer disease (AD) and are included in the AT(N) research framework for AD. Recently, an AT(N) CSF profile has been described for dementia with Lewy bodies (DLB), a disorder which is difficult to distinguish clinically from AD, particularly early in the disease course. Herein we describe a 71-year old male who presented with an atypical dementia syndrome including years of stability after an initial abrupt decline, marked visuospatial dysfunction, and relative sparing of memory. CSF biomarkers combined with the pattern of cognitive symptoms made AD unlikely and were consistent with DLB. This classification was confirmed clinically, with the emergence of classic DLB symptoms, and at postmortem pathologic examination. This case highlights the role for AD CSF biomarkers in facilitating earlier diagnosis of non-Alzheimer neurodegenerative dementias.

An Intact ACTH LC-MS/MS Assay as an Arbiter of Clinically Discordant Immunoassay Results.

BACKGROUND: Measurement of plasma adrenocorticotropic hormone (ACTH) is key in the differential diagnosis of hypothalamic-pituitary-adrenal disorders. Two-site sandwich immunoassays dominate clinical testing of ACTH in North America; however, discordant results between manufacturers have been repeatedly reported. To resolve the discrepancy, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the intended measurand, biologically active intact ACTH (iACTH). METHODS: The multiple reaction monitoring LC-MS/MS assay was designed to selectively measure full-length iACTH, as well as ACTH analogs and fragments (i.e., ACTH1-24 and ACTH18-39). Epitope assignment of the Roche Elecsys antibodies was performed by MALDI-TOF mass spectrometry. A method comparison between Roche Elecsys and Siemens Immulite ACTH immunoassays was performed and clinically concordant/discordant results identified. In a subset of these samples, the iACTH concentration was determined using the LC-MS/MS method. RESULTS: The lower limit of the measuring interval of the iACTH LC-MS/MS assay was 9 pg/mL (2 pmol/L). The assay was linear from 9 to 1938 pg/mL (2 to 427 pmol/L). Epitope mapping revealed that the Roche capture and detection antibodies bound residues 9-12 and 36-39 of ACTH, respectively. The iACTH LC-MS/MS analysis demonstrated that for discordant results between 2 immunoassays studied, only the Roche results were highly positively correlated with the iACTH concentration. CONCLUSIONS: Immunoprecipitation of biologically active ACTH molecules followed by LC-MS/MS analysis enabled selective detection of iACTH and relevant biologically active fragments in plasma. Applied to the investigation of clinically discrepant results, this method can act as an arbiter of the concentration of iACTH present.

Resolution of Spurious Immunonephelometric IgG Subclass Measurement Discrepancies by LC-MS/MS.

BACKGROUND: The Binding Site immunonephelometric (IN) IgG subclass reagents (IgG1, IgG2, IgG3, IgG, BSIN) are used for assessment of both immunodeficiency and IgG4-related disease (IgG4-RD). In our laboratory, suspected analytic errors were noted in patients with increases in IgG4: The sum of the individual IgG subclasses was substantially greater than the measured total IgG concentrations (unlike samples with normal IgG4), and the IgG4 concentration was always less than the IgG2 concentration. METHODS: We developed a tryptic digest LC-MS/MS method to quantify IgG1, IgG2, IgG3, and IgG4 in serum. Samples with IgG4 concentrations ranging from <0.03 g/L to 32 g/L were reanalyzed by LC-MS/MS, and a subset was also reanalyzed by Siemens IN (SIN) subclass measurements. RESULTS: Multivariate linear regression identified 3 subclass tests with multiple predictors of the measured subclass concentration. For these 3 subclasses, the predominant predictors were (in terms of LC-MS/MS IgG subclass measurement coefficients) BSIN IgG1 = 0.89·IgG1 + 0.4·IgG4; BSIN IgG2 = 0.94·IgG4 + 0.89·IgG2; and SIN IgG2 = 0.72·IgG2 + 0.24·IgG4. CONCLUSIONS: There is apparent IgG4 cross-reactivity with select IN subclass measurements affecting tests from both vendors tested. These findings can be explained either by direct cross-reactivity of the IN reagents with the IgG4 subclass or unique physicochemical properties of IgG4 that permit nonspecific binding of IgG4 heavy chain to other IgG immunoglobulin heavy chains. Irrespective of the mechanism, the observed intermethod discrepancies support the use of LC-MS/MS as the preferred method for measurement of IgG subclasses when testing patients with suspected IgG4-RD.

Biomarker Development for Chronic Obstructive Pulmonary Disease. From Discovery to Clinical Implementation.

Chronic obstructive pulmonary disease (COPD) is one of the major causes of morbidity and mortality in the world. Regrettably, there are no biomarkers to objectively diagnose COPD exacerbations, which are the major drivers of hospitalization and deaths from COPD. Moreover, there are no biomarkers to guide therapeutic choices or to risk stratify patients for imminent exacerbations and no objective biomarkers of disease activity or disease progression. Although there has been a tremendous investment in COPD biomarker discovery over the past 2 decades, clinical translation and implementation have not matched these efforts. In this article, we outline the challenges of biomarker development in COPD and provide an overview of a developmental pipeline that may be able to surmount these challenges and bring novel biomarker solutions to accelerate therapeutic discoveries and to improve the care and outcomes of the millions of individuals worldwide with COPD.

Renal leukocyte chemotactic factor 2 (LECT2) amyloidosis in First Nations people in Northern British Columbia, Canada: a report of 4 cases.

Leukocyte chemotactic factor 2 (LECT2) amyloidosis is a recently identified type of amyloidosis that may represent an underdiagnosed cause of chronic kidney disease. LECT2 amyloidosis typically is reported as being renal limited and, in the United States, more prevalent in Hispanic patients. We add to the epidemiologic data of this condition by describing 4 First Nations people from Northern British Columbia, Canada, who presented with slowly progressive chronic kidney disease that was found to be due to LECT2 amyloidosis.

Clinical value of Alzheimer's disease biomarker testing.

INTRODUCTION: In the Investigating the Impact of Alzheimer's Disease Diagnostics in British Columbia (IMPACT-AD BC) study, we aimed to understand how Alzheimer's disease (AD) cerebrospinal fluid (CSF) biomarker testing-used in medical care-impacted medical decision-making (medical utility), personal decision-making (personal utility), and health system economics. METHODS: The study was designed as an observational, longitudinal cohort study. A total of 149 patients were enrolled between February 2019 and July 2021. Patients referred to memory clinics were approached to participate if their dementia specialist ordered AD CSF biomarker testing as part of their routine medical care, and the clinical scenario met the appropriate use criteria for lumbar puncture and AD CSF biomarker testing. For the medical utility pillar, detailed clinical management plans were collected via physician questionnaires pre- and post-biomarker disclosure. RESULTS: Patients with completed management questionnaires (n = 142) had a median age of 64 (interquartile range: 59-69) years, 48% were female, and 60% had CSF biomarker profiles on the AD continuum. Clinical management changed in 89.4% of cases. AD biomarker testing was associated with decreased need for other diagnostic procedures, including brain imaging (-52.0%) and detailed neuropsychological assessments (-63.2%), increased referrals and counseling (57.0%), and guided AD-related drug prescriptions (+88.4% and -50.0% in biomarker-positive and -negative cases, respectively). DISCUSSION: AD biomarker testing was associated with significant and positive changes in clinical management, including decreased health care resource use, therapy optimization, and increased patient and family member counseling. While certain changes in management were linked to the AD biomarker profile (e.g., referral to clinical trials), the majority of changes were independent of baseline clinical presentation and level of cognitive impairment, demonstrating a broad value for AD biomarker testing in individuals meeting the appropriate use criteria for testing.

Personal value of Alzheimer's disease biomarker testing and result disclosure from the patient and care partner perspective.

INTRODUCTION: We described patients' and care partners' experiences with Alzheimer's disease (AD) cerebrospinal fluid (CSF) biomarker testing and result disclosure in routine care. METHODS: IMPACT-AD BC is an observational study of clinic patients who underwent AD CSF biomarker testing as part of their routine medical care (n = 142). In the personal utility arm of the study, semi-structured phone interviews were conducted with a subset of patients (n = 34), and separately with their care partners (n = 31). Post-disclosure interviews were conducted ∼1 month and ∼6 months after biomarker result disclosure and investigated the patients' decision-making process around testing, impact of receiving results, wellness and lifestyle changes, and future planning. RESULTS: A majority of patients (90%) rated their decision to undergo testing as "easy." Post-disclosure, the majority (82%) reported overall positive feelings from having greater certainty and the ability to plan ahead, and results spurred them to adopt/continue healthy behaviors such as exercise (84%) and cognitive activities (54%). Care partners expressed relief from having more diagnostic certainty, increased appreciation of future caregiving responsibilities, and a desire to connect with support resources. DISCUSSION: Perspectives of persons with lived experience in dementia provide new insight into the value of biomarker testing and should be included as part of evidence-guided considerations for pre-test counseling and result disclosure. Moreover, study findings identify an interval when patients and care partners are highly receptive to positive lifestyle and medical interventions.

Effects of COVID-19 mRNA vaccination on HIV viremia and reservoir size.

OBJECTIVE: The immunogenic nature of coronavirus disease 2019 (COVID-19) mRNA vaccines led to some initial concern that these could stimulate the HIV reservoir. We analyzed changes in plasma HIV loads (pVL) and reservoir size following COVID-19 mRNA vaccination in 62 people with HIV (PWH) receiving antiretroviral therapy (ART), and analyzed province-wide trends in pVL before and after the mass vaccination campaign. DESIGN: Longitudinal observational cohort and province-wide analysis. METHODS: Sixty-two participants were sampled prevaccination, and one month after their first and second COVID-19 immunizations. Vaccine-induced anti-SARS-CoV-2-Spike antibodies in serum were measured using the Roche Elecsys Anti-S assay. HIV reservoirs were quantified using the intact proviral DNA assay; pVL were measured using the cobas 6800 (lower limit of quantification: 20 copies/ml). The province-wide analysis included all 290 401 pVL performed in British Columbia, Canada between 2012 and 2022. RESULTS: Prevaccination, the median intact reservoir size was 77 [interquartile range (IQR): 20-204] HIV copies/million CD4 + T-cells, compared to 74 (IQR: 27-212) and 65 (IQR: 22-174) postfirst and -second dose, respectively (all comparisons P > 0.07). Prevaccination, 82% of participants had pVL <20 copies/ml (max: 110 copies/ml), compared to 79% postfirst dose (max: 183 copies/ml) and 85% postsecond dose (max: 79 copies/ml) ( P  > 0.4). There was no evidence that the magnitude of the vaccine-elicited anti-SARS-CoV-2-Spike immune response influenced pVL nor changes in reservoir size ( P  > 0.6). We found no evidence linking the COVID-19 mass vaccination campaign to population-level increases in detectable pVL frequency among all PWH in the province, nor among those who maintained pVL suppression on ART. CONCLUSION: We found no evidence that COVID-19 mRNA vaccines induced changes in HIV reservoir size nor plasma viremia.

T-Cell Responses to COVID-19 Vaccines and Breakthrough Infection in People Living with HIV Receiving Antiretroviral Therapy.

People living with HIV (PLWH) can exhibit impaired immune responses to vaccines. Accumulating evidence indicates that PLWH, particularly those receiving antiretroviral therapy, mount strong antibody responses to COVID-19 vaccines, but fewer studies have examined cellular immune responses to the vaccinations. Here, we used an activation-induced marker (AIM) assay to quantify SARS-CoV-2 spike-specific CD4+ and CD8+ T cells generated by two and three doses of COVID-19 vaccines in 50 PLWH receiving antiretroviral therapy, compared to 87 control participants without HIV. In a subset of PLWH, T-cell responses were also assessed after post-vaccine breakthrough infections and/or receipt of a fourth vaccine dose. All participants remained SARS-CoV-2 infection-naive until at least one month after their third vaccine dose. SARS-CoV-2 infection was determined by seroconversion to a Nucleocapsid (N) antigen, which occurred in 21 PLWH and 38 control participants after the third vaccine dose. Multivariable regression analyses were used to investigate the relationships between sociodemographic, health- and vaccine-related variables, vaccine-induced T-cell responses, and breakthrough infection risk. We observed that a third vaccine dose boosted spike-specific CD4+ and CD8+ T-cell frequencies significantly above those measured after the second dose (all p < 0.0001). Median T-cell frequencies did not differ between PLWH and controls after the second dose (p > 0.1), but CD8+ T-cell responses were modestly lower in PLWH after the third dose (p = 0.02), an observation that remained significant after adjusting for sociodemographic, health- and vaccine-related variables (p = 0.045). In PLWH who experienced a breakthrough infection, median T-cell frequencies increased even higher than those observed after three vaccine doses (p < 0.03), and CD8+ T-cell responses in this group remained higher even after a fourth vaccine dose (p = 0.03). In multivariable analyses, the only factor associated with an increased breakthrough infection risk was younger age, which is consistent with the rapid increase in SARS-CoV-2 seropositivity that was seen among younger adults in Canada after the initial appearance of the Omicron variant. These results indicate that PLWH receiving antiretroviral therapy mount strong T-cell responses to COVID-19 vaccines that can be enhanced by booster doses or breakthrough infection.