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Background: The interaction of CD40L and its receptor CD40 on activated T cells and B cells respectively control pro-inflammatory activation in the pathophysiology of autoimmunity and transplant rejection. Previous studies have implicated signaling pathways involving CD40L (interchangeably referred to as CD154), as well as adaptive and innate immune cell activation, in the induction of neuroinflammation in neurodegenerative diseases. This study aimed to assess the safety, tolerability, and impact on pro-inflammatory biomarker profiles of an anti CD40L antibody, tegoprubart, in individuals with amyotrophic lateral sclerosis (ALS)., Methods and Findings: In this multicenter dose-escalating open-label Phase 2A study, 54 participants with a diagnosis of ALS received 6 infusions of tegoprubart administered intravenously every 2 weeks. The study was comprised of 4 dose cohorts: 1 mg/kg, 2 mg/kg, 4 mg/kg, and 8 mg/kg. The primary endpoint of the study was safety and tolerability. Exploratory endpoints assessed the pharmacokinetics of tegoprubart as well as anti-drug antibody (ADA) responses, changes in disease progression utilizing the Revised ALS Functional Rating Scale (ALSFRS-R), CD154 target engagement, changes in pro-inflammatory biomarkers, and neurofilament light chain (NFL). Seventy subjects were screened, and 54 subjects were enrolled in the study. Forty-nine of 54 subjects completed the study (90.7%) receiving all 6 infusions of tegoprubart and completing their final follow-up visit. The most common treatment emergent adverse events (TEAEs) overall (>10%) were fatigue (25.9%), falls (22.2%), headaches (20.4%), and muscle spasms (11.1%). Mean tegoprubart plasma concentrations increased proportionally with increasing dose with a half-life of approximately 24 days. ADA titers were low and circulating levels of tegoprubart were as predicted for all cohorts. Tegoprubart demonstrated dose dependent target engagement associated and a reduction in 18 pro-inflammatory biomarkers in circulation., Conclusions: Tegoprubart appeared to be safe and well tolerated in adults with ALS demonstrating dose-dependent reduction in pro-inflammatory chemokines and cytokines associated with ALS. These results warrant further clinical studies with sufficient power and duration to assess clinical outcomes as a potential treatment for adults with ALS., Trial Registration: Clintrials.gov ID:NCT04322149., Competing Interests: SPE is an employee and Director at Eledon Pharmaceuticals. MR has Honorariums: UCB Pharmaceuticals, Argenx Pharmaceuticals, Alexion Pharmaceuticals. And Grant support: UCB Pharmaceuticals, Argenx Pharmaceuticals, Alexion Pharmaceuticals, Catalyst Pharmaceuticals, Cartesian Therapeutics, Inc., Viela Bio,Momenta Pharmaceuticals, Inc, Seelos Therapeutics, Inc., PTC THERAPEUTICS, INC., Amylyx Pharmaceuticals Inc., Healey Center, NMD Pharma, Janssen Research and Development, Apellis Pharma, MedicinNova Inc, Millennium Pharma, Orion Pharma, Biohaven Pharma, Seikagaku Corporation, Mallinckrodt ARD, Inc, Cytokinetics, Inc, Grifols., (Copyright: © 2024 Perrin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction/aims: Expanded access (EA) is a Food and Drug Administration-regulated pathway to provide access to investigational products (IPs) to individuals with serious diseases who are ineligible for clinical trials. The aim of this report is to share the design and operations of a multicenter, multidrug EA program for amyotrophic lateral sclerosis (ALS) across nine US centers., Methods: A central coordination center was established to design and conduct the program. Templated documents and processes were developed to streamline study design, regulatory submissions, and clinical operations across protocols. The program included three protocols and provided access to IPs that were being tested in respective regimens of the HEALEY ALS Platform Trial (verdiperstat, CNM-Au8, and pridopidine). Clinical and safety data were collected in all EA protocols (EAPs). The program cohorts comprised participants who were not eligible for the platform trial, including participants at advanced stages of disease progression and with long disease duration., Results: A total of 85 participants were screened across the 3 EAPs from July 2021 to September 2022. The screen failure rate was 3.5%. Enrollment for the regimens of the platform trial was completed as planned and results informed the duration of the corresponding EAP. The verdiperstat EAP was concluded in December 2022. Mean duration of participation in the verdiperstat EAP was 5.8 ± 4.1 months. The CNM-Au8 and pridopidine EAPs are ongoing., Discussion: Multicenter EAPs conducted in parallel to randomized clinical trials for ALS can successfully enroll participants who do not qualify for clinical trials., (© 2024 The Author(s). Muscle & Nerve published by Wiley Periodicals LLC.)

Amyotrophic lateral sclerosis (ALS) is an idiopathic, fatal, and fast-progressive neurodegenerative disease characterized by the degeneration of motor neurons. ALS patients often experience an initial misdiagnosis or a diagnostic delay due to the current unavailability of an efficient biomarker. Since impaired speech is typical in ALS, we hypothesized that functional differences between healthy and ALS participants during speech tasks can be explained by cortical pattern changes, thereby leading to the identification of a neural biomarker for ALS. In this pilot study, we collected magnetoencephalography (MEG) recordings from three early-diagnosed patients with ALS and three healthy controls during imagined (covert) and overt speech tasks. First, we computed sensor correlations, which showed greater correlations for speakers with ALS than healthy controls. Second, we compared the power of the MEG signals in canonical bands between the two groups, which showed greater dissimilarity in the beta band for ALS participants. Third, we assessed differences in functional connectivity, which showed greater beta band connectivity for ALS than healthy controls. Finally, we performed single-trial classification, which resulted in highest performance with beta band features (∼ 98%). These findings were consistent across trials, phrases, and participants for both imagined and overt speech tasks. Our preliminary results indicate that speech-evoked beta oscillations could be a potential neural biomarker for diagnosing ALS. To our knowledge, this is the first demonstration of the detection of ALS from single-trial neural signals., Competing Interests: 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. The handling editor RW declared a shared affiliation with the author AB-F at the time of review., (Copyright © 2024 Dash, Teplansky, Ferrari, Babajani-Feremi, Calley, Heitzman, Austin and Wang.)

Purpose: The aim of this study was to leverage data-driven approaches, including a novel articulatory consonant distinctiveness space (ACDS) approach, to better understand speech motor control in amyotrophic lateral sclerosis (ALS)., Method: Electromagnetic articulography was used to record tongue and lip movement data during the production of 10 consonants from healthy controls ( n = 15) and individuals with ALS ( n = 47). To assess phoneme distinctness, speech data were analyzed using two classification algorithms, Procrustes matching (PM) and support vector machine (SVM), and the area/volume of the ACDS. Pearson's correlation coefficient was used to examine the relationship between bulbar impairment and the ACDS. Analysis of variance was used to examine the effects of bulbar impairment on consonant distinctiveness and consonant classification accuracies in clinical subgroups., Results: There was a significant relationship between the ACDS and intelligible speaking rate (area, p = .003; volume, p = .010), and the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R) bulbar subscore (area, p = .009; volume, p = .027). Consonant classification performance followed a consistent pattern with bulbar severity, where consonants produced by speakers with more severe ALS were classified less accurately (SVM = 75.27%; PM = 74.54%) than the healthy, asymptomatic, and mild-moderate groups. In severe ALS, area of the ACDS was significantly condensed compared to both asymptomatic ( p = .004) and mild-moderate ( p = .013) groups. There was no statistically significant difference in area between the severe ALS group and healthy speakers ( p = .292)., Conclusions: Our comprehensive approach is sensitive to early oromotor changes in response due to disease progression. The preserved articulatory consonant space may capture the use of compensatory adaptations to counteract influences of neurodegeneration., Supplemental Material: https://doi.org/10.23641/asha.22044320.

Introduction/Aims . Primary lateral sclerosis (PLS) is exceedingly rare and has been an enigmatic disease. Recent progress has drastically changed this perception, with early biomarkers being investigated and potential medications for PLS emerging at the preclinical stage. The aim of this paper is to describe a study of PLS natural history and discuss the limitations and proposed solutions to the study of a rare and slowly progressive disease. Methods . The PLS Natural History Study is a 30-site, 24-month, prospective study that is supported by multiple funding sources. The study aims to enroll 50 early PLS (disease duration ≤4 years) and 50 definite PLS (disease duration 4 to 15 years) participants using modified PLS Diagnostic Criteria. Smartphone-based assessments including semi-quantitative and quantitative measures and patient-reported outcomes are utilized. In-person quantitative measures are also completed during site visits. The change in the PLS Functional Rating Scale score is the primary outcome. The study utilizes the NeuroBANK ® patient-centric data capture and management platform. The biostatistical analysis plan has been developed. Results . In one year, 28 participants have been recruited. Enrollment has been much slower than anticipated due to the COVID-19 pandemic, the rarity of PLS, and potential study competition for internal resources from ALS clinical trials. Discussion . We discuss the need for more innovative methods to enroll and study individuals with such rare diseases and propose a number of mechanisms by which more efficient enrollment could be facilitated.

Purpose: The goal of this study was to examine the efficacy of acceleration-based articulatory measures in characterizing the decline in speech motor control due to amyotrophic lateral sclerosis (ALS)., Method: Electromagnetic articulography was used to record tongue and lip movements during the production of 20 phrases. Data were collected from 50 individuals diagnosed with ALS. Articulatory kinematic variability was measured using the spatiotemporal index of both instantaneous acceleration and speed signals. Linear regression models were used to analyze the relationship between variability measures and intelligible speaking rate (a clinical measure of disease progression). A machine learning algorithm (support vector regression, SVR) was used to assess whether acceleration or speed features (e.g., mean, median, maximum) showed better performance at predicting speech severity in patients with ALS., Results: As intelligible speaking rate declined, the variability of acceleration of tongue and lip movement patterns significantly increased (p < 0.001). The variability of speed and vertical displacement did not significantly predict speech performance measures. Additionally, based on R2 and root mean square error (RMSE) values, the SVR model was able to predict speech severity more accurately from acceleration features (R2 = 0.601, RMSE = 38.453) and displacement features (R2 = 0.218, RMSE = 52.700) than from speed features (R2 = 0.554, RMSE = 40.772)., Conclusion: Results from these models highlight differences in speech motor control in participants with ALS. The variability in acceleration of tongue and lip movements increases as speech performance declines, potentially reflecting physiological deviations due to the progression of ALS. Our findings suggest that acceleration is a more sensitive indicator of speech deterioration due to ALS than displacement and speed and may contribute to improved algorithm designs for monitoring disease progression from speech signals., (© 2022 S. Karger AG, Basel.)

Purpose: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects bulbar functions including speech and voice. Voice onset time (VOT) was examined in speakers with ALS in early and late stages to explore the coordination of the articulatory and phonatory systems during speech production., Method: VOT was measured in nonword /bap/ produced by speakers with early-stage ALS ( n  = 11), late-stage ALS ( n  = 6), and healthy controls ( n  = 13), and compared with speech performance decline (a marker of disease progression) in ALS., Results: Overall comparison of the VOT values among the three groups showed a significant difference, F (2,27) = 11.71, p < .01. Speakers in late-stage ALS displayed longer voicing lead (negative VOT) than both healthy speakers and speakers in early-stage ALS. VOT was also significantly negatively correlated with speech performance (i.e., Intelligible Speaking Rate), r (15) = .74, p  < .01., Conclusions: Speakers with more severe ALS showed greater occurrence of voicing lead and longer voicing lead. Findings show voicing precedes articulatory onset with disease progression in the production of bilabial stops, which suggests that the relative timing of coordination between the supralaryngeal structures and the phonatory system is affected in the late stage of ALS.

Background: Coformulated sodium phenylbutyrate/taurursodiol (PB/TURSO) was shown to prolong survival and slow functional decline in amyotrophic lateral sclerosis (ALS)., Objective: Determine whether PB/TURSO prolonged tracheostomy/ventilation-free survival and/or reduced first hospitalisation in participants with ALS in the CENTAUR trial., Methods: Adults with El Escorial Definite ALS ≤18 months from symptom onset were randomised to PB/ TURSO or placebo for 6 months. Those completing randomised treatment could enrol in an open-label extension (OLE) phase and receive PB/TURSO for ≤30 months. Times to the following individual or combined key events were compared in the originally randomised treatment groups over a period spanning trial start through July 2020 (longest postrandomisation follow-up, 35 months): death, tracheostomy, permanent assisted ventilation (PAV) and first hospitalisation., Results: Risk of any key event was 47% lower in those originally randomised to PB/TURSO (n=87) versus placebo (n=48, 71% of whom received delayed-start PB/TURSO in the OLE phase) (HR=0.53; 95% CI 0.35 to 0.81; p=0.003). Risks of death or tracheostomy/PAV (HR=0.51; 95% CI 0.32 to 0.84; p=0.007) and first hospitalisation (HR=0.56; 95% CI 0.34 to 0.95; p=0.03) were also decreased in those originally randomised to PB/TURSO., Conclusions: Early PB/TURSO prolonged tracheostomy/PAV-free survival and delayed first hospitalisation in ALS., Trial Registration Number: NCT03127514; NCT03488524., Competing Interests: Competing interests: SP reports grant support from Amylyx during the conduct of the study; grant support from Revalesio, Biohaven, UCB, Clene, Prilenia, and Seelos outside the submitted work; and consulting fees from Orion, Cytokinetics, and Medscape outside the submitted work. SH is owner, and SH, SPD, and NK are employees of Pentara Corporation, which was contracted by Amylyx for statistical analysis. JDB reports clinical trial support from Amylyx Pharmaceuticals, Inc, during the conduct of the study; grant support from Alexion, Biogen, Mitsubishi Tanabe Pharma America (MTPA), Anelixis Therapeutics, Brainstorm Cell Therapeutics, nQ Medical, the National Institute of Neurological Disorders and Stroke, the Muscular Dystrophy Association, ALS One, Amylyx, MT Pharma Holdings of America, The ALS Association, ALS Finding a Cure®, and RAPA Therapeutics outside the submitted work; and personal fees from Biogen, Clene Nanomedicine, MTPA, MT Pharma Holdings of America, Sawai Pharmaceutical Co, Ltd, and Janssen outside the submitted work. MAE reports payment and personal fees from Amylyx for advisory board and speakers bureau participation outside the submitted work. CK reports consulting fees from Alnylam, Takeda, CSL Behring, Argenx, Akcea/Ionis, and Sanofi; honoraria from Alnylam; and advisory board participation fees from Alnylam and Orphazyme, all outside the submitted work. JBC reports grants from AZ Therapies Inc, MTB Pharmaceuticals, and Cytokinetics Inc, lecture honorarium from Northwest Area Health Education Center, and consulting fees from the Department of Defense Congressionally Directed Medical Research Programs grant review board, all outside the submitted work. JW reports research funding from Amylyx during the conduct of the study and presentation honoraria from Amylyx outside the submitted work. SAG reports institutional funding from Amylyx and The ALS Association during the conduct of the study; institutional funding from the National Institute of Environmental Health Sciences and The ALS Association outside the submitted work; consulting fees from Biogen and ITF Pharma outside the submitted work; lecture fees from Illinois State Neurological Society Lecture and Spectrum Health outside the submitted work; and personal fees for Data Safety Monitoring Board (DSMB) participation from Watermark outside the submitted work; and is an inventor on institutional patent US10660895B2. DH reports advisory board participation for Amylyx outside the submitted work. TDH-P reports clinical trial funding from Amylyx during the conduct of the study; institutional grant support from Cytokinetics, Orion, Bio 3, MTPA, the Sean M. Healey and AMG Center for ALS & the Neurological Clinical Research Institute, UCB Pharma, Alexion, and AB Sciences outside the submitted work; institutional contract work with Samus outside the submitted work; consulting fees from Samus, Alpha Insights, and Evidera outside the submitted work; payment for educational deliverables from Platform Q Health, WebMD, MJH Holdings, IQVIA, P Value, and Vindico Medical Education outside the submitted work; and advisory board fees from MTPA, Cytokinetics, AB Bio, Alexion, Biogen, and Orphazyme outside the submitted work; and is President of the ALS Hope Foundation. CEJ reports grant support from the Muscular Dystrophy Association, The ALS Association, and the National Institutes of Health and advisory board participation for Brainstorm Cell Therapeutics, MTPA, and Anelixis, all outside the submitted work, and is President Elect of the American Academy of Neurology. CQ reports advisory board fees from Amylyx outside the submitted work. JDR reports clinical trial funding from Amylyx during the conduct of the study; licensing agreement and nonfinancial support from Ionis Pharmaceuticals; nonfinancial support from Calico, Biogen, and IBM Watson; research grant support from the National Institute of Neurological Disorders and Stroke, National Institute on Aging, Department of Defense, the Chan Zuckerberg Initiative, Microsoft, The ALS Association, the Muscular Dystrophy Association, Target ALS, F Prime, ALS Finding A Cure, Answer ALS, Robert Packard Center for ALS Research, GlaxoSmithKline, Travelers Insurance, American Airlines, Caterpillar, and the National Football League; and personal consulting fees from Expansion Therapeutics and Team Gleason, all outside the submitted work. JK reports consulting fees from Calico, Denali, Biogen, MTPA, and Amylyx outside the submitted work. LJ reports advisory board participation for Cytokinetics outside the submitted work. SL reports advisory board fees from Amylyx outside the submitted work. TMM reports licensing agreements with Ionis Pharmaceuticals and C2N, consulting fees from Cytokinetics, Ionis Pharmaceuticals, and Disarm Therapeutics, lecture honorarium from Regeneron, advisory board participation for Biogen and UCB Pharma, and receipt of reagents from Ionis Pharmaceuticals, all outside the submitted work, and is co-chair of the Northeast ALS Consortium. SNS reports funding from Amylyx during the conduct of the study; grant support from Orion Pharma and Alexion outside the submitted work; lecture (grand rounds) honorarium from New York University outside the submitted work; and payment for expert testimony from Martin Clearwater & Bell LLP; Shaub, Ahmuty, Citrin & Spratt LLP; MCIC Vermont; and Aaronson Rappaport Feinstein and Deutsch, LLP outside the submitted work. THV reports institutional funding from Amylyx during the conduct of the study; institutional grant support from Alexion, Alector, Annexon Bio, Apellis, Biogen, Cytokinetics, Healey ALS Platform Trial, MT Pharma, and Sanofi outside the submitted work; and honoraria from Amylyx, Alexion, and Cytokinetics outside the submitted work. CNF reports institutional funding from Amylyx during the conduct of the study. AS reports institutional grant support from Amylyx during the conduct of the study and from the Rare Diseases Clinical Research Network, National Institutes of Health, Healey ALS Platform Trial, Cytokinetics, and Wyck Foundation outside the submitted work; honorarium from Weill Cornell Medicine – Qatar (residency recruitment seminar) outside the submitted work; uncompensated DSMB participation for the University of Kansas outside the submitted work; and compensated independent data monitoring committee participation for Alexion outside the submitted work. NAG reports grants from Alexion, Annelixis, Annexon, Brainstorm Cell Therapeutics, Cytokinetics, Fulcrum, the Sean M. Healey and AMG Center for ALS & the Neurological Clinical Research Institute, Kezar, Medicinova, Octapharma, Orion, and Orphazyme and advisory board participation for Alexion, Argenx, AstraZeneca, CSL Behring, MT Pharma, Sanofi Genzyme, Sarepta, and UCB, all outside the submitted work. GLP reports consulting fees from MT Pharma and honoraria from MT Pharma and Cytokinetics, all outside the submitted work. GK reports provision of trial supplies from Amylyx during the conduct of the study. JMS reports grant support from the National Institutes of Health, The ALS Association, Amylyx, Biogen, Biotie Therapies (now Acorda Therapeutics), Cytokinetics Incorporated, MTPA, Alexion, Medicinova, Ionis, Alector, and Orphazyme; royalties from UpToDate; consulting fees from Amylyx, Apic Biosciences, Neurosense, Cytokinetics, Denali, GSK, MTPA, Orphazyme, Pinteon, RRD, Swanbio, Helixsmith, Novartis, Sanofi, and EMD Serono; and honoraria from Amylyx (symposium) and Oakstone (online presentation); all outside the submitted work. JC and JK are co-CEOs of and own stock in Amylyx, the trial sponsor, and report grant support from ALS Finding a Cure and The ALS Association during the conduct of the study. RET is Founding Chair of Amylyx’s Scientific Advisory Board and holds equity in Amylyx. Dr Gilbert reports stock ownership in Amylyx. PDY reports full-time employment and stock option ownership with Amylyx outside the submitted work. MEC reports consulting fees from Faze, Regeneron, AB Sciences, Avexis, Orion, Lilly, Biohaven, Mt Pharma, Revalasio, Aclipse, Anelexis, Cytokinetics, Disarm, ALS Pharma, Immunity Pharma, Wave, Sunovian, Pontifex, Denali, Transposen, Quralis, Helixsmith, and RRD and is a board member for Praxis, all outside the submitted work., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Answer ALS is a biological and clinical resource of patient-derived, induced pluripotent stem (iPS) cell lines, multi-omic data derived from iPS neurons and longitudinal clinical and smartphone data from over 1,000 patients with ALS. This resource provides population-level biological and clinical data that may be employed to identify clinical-molecular-biochemical subtypes of amyotrophic lateral sclerosis (ALS). A unique smartphone-based system was employed to collect deep clinical data, including fine motor activity, speech, breathing and linguistics/cognition. The iPS spinal neurons were blood derived from each patient and these cells underwent multi-omic analytics including whole-genome sequencing, RNA transcriptomics, ATAC-sequencing and proteomics. The intent of these data is for the generation of integrated clinical and biological signatures using bioinformatics, statistics and computational biology to establish patterns that may lead to a better understanding of the underlying mechanisms of disease, including subgroup identification. A web portal for open-source sharing of all data was developed for widespread community-based data analytics., (© 2022. The Author(s).)

Proliferation of Schwann cells during peripheral nerve development is stimulated by the heregulin/neuregulin family of growth factors expressed by neurons. However, for neonatal rat Schwann cells growing in culture, heregulins produce only a weak mitogenic response. Supplementing heregulin with forskolin, an agent that elevates cyclic AMP levels, produces a dramatic increase in the proliferation of cultured Schwann cells. The mechanisms underlying this synergistic effect required for Schwann cell proliferation in vivo is not well established. Characterizing the A-kinase anchoring proteins (AKAPs) in Schwann cells might help identify substrates tethered to and phosphorylated by the cAMP-dependent protein kinase A (PKA). Using an RII overlay assay that detects AKAPs that are bound to the type II regulatory subunits of PKA, we identified AKAP150 in Schwann cells. Western blot analysis revealed that additional AKAPs, specifically AKAP95, and yotiao were also present. Disruption of PKA/AKAP interaction with Ht-31 peptide resulted in an increase in luciferase-conjugated cyclin D3 promoter activity. Transfection with sequence-specific AKAP siRNAs for AKAP150 and AKAP95 produced a marked reduction in cell proliferation. Immunoblot analysis revealed that knock down of AKAP95 protein caused a significant decrease in expression of the cell cycle regulatory proteins cyclin D2, cyclin D3 and the cell survival signal Akt/Protein Kinase B (Akt/PKB). Morphological characterization of Schwann cell AKAPs indicated the presence of nuclear (AKAP95), cytoplasm-associated (AKAP150) and perinuclear (yotiao) A-kinase anchoring proteins. These results indicate a role for AKAP95 and AKAP150 in the synergistic response of Schwann cells to treatment with heregulin and forskolin., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Purpose Kinematic measurements of speech have demonstrated some success in automatic detection of early symptoms of amyotrophic lateral sclerosis (ALS). In this study, we examined how the region of symptom onset (bulbar vs. spinal) affects the ability of data-driven models to detect ALS. Method We used a correlation structure of articulatory movements combined with a machine learning model (i.e., artificial neural network) to detect differences between people with ALS and healthy controls. The performance of this system was evaluated separately for participants with bulbar onset and spinal onset to examine how region of onset affects classification performance. We then performed a regression analysis to examine how different severity measures and region of onset affects model performance. Results The proposed model was significantly more accurate in classifying the bulbar-onset participants, achieving an area under the curve of 0.809 relative to the 0.674 achieved for spinal-onset participants. The regression analysis, however, found that differences in classifier performance across participants were better explained by their speech performance (intelligible speaking rate), and no significant differences were observed based on region of onset when intelligible speaking rate was accounted for. Conclusions Although we found a significant difference in the model's ability to detect ALS depending on the region of onset, this disparity can be primarily explained by observable differences in speech motor symptoms. Thus, when the severity of speech symptoms (e.g., intelligible speaking rate) was accounted for, symptom onset location did not affect the proposed computational model's ability to detect ALS.

Objective: To evaluate safety, dose response, and preliminary efficacy of reldesemtiv over 12 weeks in patients with amyotrophic lateral sclerosis (ALS). Methods: Patients (≤2 years since diagnosis) with slow upright vital capacity (SVC) of ≥60% were randomized 1:1:1:1 to reldesemtiv 150, 300, or 450 mg twice daily (bid) or placebo; active treatment was 12 weeks with 4-week follow-up. Primary endpoint was change in percent predicted SVC at 12 weeks; secondary measures included ALS Functional Rating Scale-Revised (ALSFRS-R) and muscle strength mega-score. Results: Patients ( N  = 458) were enrolled; 85% completed 12-week treatment. The primary analysis failed to reach statistical significance ( p  = 0.11); secondary endpoints showed no statistically significant effects (ALSFRS-R, p  = 0.09; muscle strength mega-score, p  = 0.31). Post hoc analyses pooling all active reldesemtiv -treated patients compared against placebo showed trends toward benefit in all endpoints (progression rate for SVC, ALSFRS-R, and muscle strength mega-score (nominal p values of 0.10, 0.01 and 0.20 respectively)). Reldesemtiv was well tolerated, with nausea and fatigue being the most common side effects. A dose-dependent decrease in estimated glomerular filtration rate was noted, and transaminase elevations were seen in approximately 5% of patients. Both hepatic and renal abnormalities trended toward resolution after study drug discontinuation. Conclusions: Although the primary efficacy analysis did not demonstrate statistical significance, there were trends favoring reldesemtiv for all three endpoints, with effect sizes generally regarded as clinically important. Tolerability was good; modest hepatic and renal abnormalities were reversible. The impact of reldesemtiv on patients with ALS should be assessed in a pivotal Phase 3 trial. (ClinicalTrials.gov Identifier: NCT03160898).

An orally administered, fixed-dose coformulation of sodium phenylbutyrate-taurursodiol (PB-TURSO) significantly slowed functional decline in a randomized, placebo-controlled, phase 2 trial in ALS (CENTAUR). Herein we report results of a long-term survival analysis of participants in CENTAUR. In CENTAUR, adults with ALS were randomized 2:1 to PB-TURSO or placebo. Participants completing the 6-month (24-week) randomized phase were eligible to receive PB-TURSO in the open-label extension. An all-cause mortality analysis (35-month maximum follow-up post-randomization) incorporated all randomized participants. Participants and site investigators were blinded to treatment assignments through the duration of follow-up of this analysis. Vital status was obtained for 135 of 137 participants originally randomized in CENTAUR. Median overall survival was 25.0 months among participants originally randomized to PB-TURSO and 18.5 months among those originally randomized to placebo (hazard ratio, 0.56; 95% confidence interval, 0.34-0.92; P = .023). Initiation of PB-TURSO treatment at baseline resulted in a 6.5-month longer median survival as compared with placebo. Combined with results from CENTAUR, these results suggest that PB-TURSO has both functional and survival benefits in ALS., (© 2020 The Authors. Muscle & Nerve published by Wiley Periodicals LLC.)

Importance: Cryptogenic sensory polyneuropathy (CSPN) is a common generalized slowly progressive neuropathy, second in prevalence only to diabetic neuropathy. Most patients with CSPN have significant pain. Many medications have been tried for pain reduction in CSPN, including antiepileptics, antidepressants, and sodium channel blockers. There are no comparative studies that identify the most effective medication for pain reduction in CSPN., Objective: To determine which medication (pregabalin, duloxetine, nortriptyline, or mexiletine) is most effective for reducing neuropathic pain and best tolerated in patients with CSPN., Design, Setting, and Participants: From December 1, 2014, through October 20, 2017, a bayesian adaptive, open-label randomized clinical comparative effectiveness study of pain in 402 participants with CSPN was conducted at 40 neurology care clinics. The trial included response adaptive randomization. Participants were patients with CSPN who were 30 years or older, with a pain score of 4 or greater on a numerical rating scale (range, 0-10, with higher scores indicating a higher level of pain). Participant allocation to 1 of 4 drug groups used the utility function and treatment's sample size for response adaptation randomization. At each interim analysis, a decision was made to continue enrolling (up to 400 participants) or stop the whole trial for success (80% power). Patient engagement was maintained throughout the trial, which helped guide the study and identify ways to communicate and disseminate information. Analysis was performed from December 11, 2015, to January 19, 2018., Interventions: Participants were randomized to receive nortriptyline (n = 134), duloxetine (n = 126), pregabalin (n = 73), or mexiletine (n = 69)., Main Outcomes and Measures: The primary outcome was a utility function that was a composite of the efficacy (participant reported pain reduction of ≥50% from baseline to week 12) and quit (participants who discontinued medication) rates., Results: Among the 402 participants (213 men [53.0%]; mean [SD] age, 60.1 [13.4] years; 343 White [85.3%]), the utility function of nortriptyline was 0.81 (95% bayesian credible interval [CrI], 0.69-0.93; 34 of 134 [25.4%] efficacious; and 51 of 134 [38.1%] quit), of duloxetine was 0.80 (95% CrI, 0.68-0.92; 29 of 126 [23.0%] efficacious; and 47 of 126 [37.3%] quit), pregabalin was 0.69 (95% CrI, 0.55-0.84; 11 of 73 [15.1%] efficacious; and 31 of 73 [42.5%] quit), and mexiletine was 0.58 (95% CrI, 0.42-0.75; 14 of 69 [20.3%] efficacious; and 40 of 69 [58.0%] quit). The probability each medication yielded the highest utility was 0.52 for nortriptyline, 0.43 for duloxetine, 0.05 for pregabalin, and 0.00 for mexiletine., Conclusions and Relevance: This study found that, although there was no clearly superior medication, nortriptyline and duloxetine outperformed pregabalin and mexiletine when pain reduction and undesirable adverse effects are combined to a single end point., Trial Registration: ClinicalTrials.gov Identifier: NCT02260388.

Background: Sodium phenylbutyrate and taurursodiol have been found to reduce neuronal death in experimental models. The efficacy and safety of a combination of the two compounds in persons with amyotrophic lateral sclerosis (ALS) are not known., Methods: In this multicenter, randomized, double-blind trial, we enrolled participants with definite ALS who had had an onset of symptoms within the previous 18 months. Participants were randomly assigned in a 2:1 ratio to receive sodium phenylbutyrate-taurursodiol (3 g of sodium phenylbutyrate and 1 g of taurursodiol, administered once a day for 3 weeks and then twice a day) or placebo. The primary outcome was the rate of decline in the total score on the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R; range, 0 to 48, with higher scores indicating better function) through 24 weeks. Secondary outcomes were the rates of decline in isometric muscle strength, plasma phosphorylated axonal neurofilament H subunit levels, and the slow vital capacity; the time to death, tracheostomy, or permanent ventilation; and the time to death, tracheostomy, permanent ventilation, or hospitalization., Results: A total of 177 persons with ALS were screened for eligibility, and 137 were randomly assigned to receive sodium phenylbutyrate-taurursodiol (89 participants) or placebo (48 participants). In a modified intention-to-treat analysis, the mean rate of change in the ALSFRS-R score was -1.24 points per month with the active drug and -1.66 points per month with placebo (difference, 0.42 points per month; 95% confidence interval, 0.03 to 0.81; P = 0.03). Secondary outcomes did not differ significantly between the two groups. Adverse events with the active drug were mainly gastrointestinal., Conclusions: Sodium phenylbutyrate-taurursodiol resulted in slower functional decline than placebo as measured by the ALSFRS-R score over a period of 24 weeks. Secondary outcomes were not significantly different between the two groups. Longer and larger trials are necessary to evaluate the efficacy and safety of sodium phenylbutyrate-taurursodiol in persons with ALS. (Funded by Amylyx Pharmaceuticals and others; CENTAUR ClinicalTrials.gov number, NCT03127514.)., (Copyright © 2020 Massachusetts Medical Society.)

Objective: To determine the associations between plasma creatinine (PCr), plasma uric acid (PUA), and urinary oxidative stress (OS) biomarkers with the ALSFRS-R at baseline and survival in a large epidemiological cohort study (ALS COSMOS) with a well-phenotyped patient population ( N  = 355). Methods: Fasting plasma and first void urine samples were obtained. PCr, PUA, urinary 8-oxo-deoxy guanosine (8-oxodG), and 15-F 2t -isoprostane (IsoP) were analyzed at baseline, near the midpoint of follow-up, and at the final blood draw (before death or withdrawal from study). We estimated associations between these biomarkers and the ALSFRS-R at baseline and survival. Results: At baseline, PCr correlated with ALSFRS-R (Spearman r  = 0.30), percent (%) FVC ( r  = 0.20), PUA ( r  = 0.37), and 8-oxodG ( r = -0.13, all p  < 0.05). Baseline PCr significantly predicted survival (adjusted hazard ratio 0.28, p  < 0.001). Time to death from baseline was shortest for those in the lowest two PCr quartiles relative to the highest two quartiles. PCr and ALSFRS-R values were significantly correlated at all three time points (baseline: r  = 0.29, midpoint: r  = 0.23, final: r  = 0.38, all p  < 0.001). PCr and PUA significantly declined over time, whereas OS biomarkers significantly increased over time. Conclusions: To date, PCr predicted survival the best, compared to PUA, 8-oxodG, and IsoP. Although PCr represents the degree of muscle mass, it may also represent complex biochemical changes in ALS. Because the field has no reliable prognostic biomarkers, the importance of PCr warrants further investigation through clinical studies in ALS.

Introduction: Our research aim was to develop a novel clinimetric scale sensitive enough to detect disease progression in primary lateral sclerosis (PLS)., Methods: A prototype of the PLS Functional Rating Scale (PLSFRS) was generated. Seventy-seven participants with PLS were enrolled and evaluated at 21 sites that comprised the PLSFRS study group. Participants were assessed using the PLSFRS, Neuro-Quality of Life (QoL), Schwab-England Activities of Daily Living (ADL), and the Clinical Global Impression of Change scales. Participants completed telephone assessments at 12, 24, and 48 weeks after enrollment., Results: The PLSFRS demonstrated internal consistency as well as intrarater, interrater, telephone test-retest reliability, and construct validity. Significant changes in disease progression were detected at 6 and 12 months; changes measured by the PLSFRS vs the ALSFRS-R were significantly higher., Discussion: The PLSFRS is a valid tool to assess the natural history of PLS in a shorter study period., (© 2019 Wiley Periodicals, Inc.)

[This corrects the article DOI: 10.1155/2018/5969137.]., (Copyright © 2019 Susan Woolley et al.)

Decoding speech directly from the brain has the potential for the development of the next generation, more efficient brain computer interfaces (BCIs) to assist in the communication of patients with locked-in syndrome (fully paralyzed but aware). In this study, we have explored the spectral and temporal features of the magnetoencephalography (MEG) signals and trained those features with convolutional neural networks (CNN) for the classification of neural signals corresponding to phrases. Experimental results demonstrated the effectiveness of CNNs in decoding speech during perception, imagination, and production tasks. Furthermore, to overcome the long training time issue of CNNs, we leveraged principal component analysis (PCA) for spatial dimension reduction of MEG data and transfer learning for model initialization. Both PCA and transfer learning were found to be highly beneficial for faster model training. The best configuration (50 principal coefficients + transfer learning) led to more than 10 times faster training than the original setting while the speech decoding accuracy remained at a similarly high level.

Introduction: Universally established comprehensive clinical bulbar scales objectively assessing disease progression in amyotrophic lateral sclerosis (ALS) are currently lacking. The goal of this working group project is to design a best practice set of provisional bulbar ALS guidelines, available for immediate implementation within all ALS clinics., Methods: ALS specialists across multiple related disciplines participated in a series of clinical bulbar symposia, intending to identify and summarize the currently accepted best practices for the assessment and management of bulbar dysfunction in ALS Results: Summary group recommendations for individual speech, Augmentative and Alternative Communication (AAC), and swallowing sections were achieved, focusing on the optimal proposed level of care within each domain., Discussion: We have identified specific clinical recommendations for each of the 3 domains of bulbar functioning, available for incorporation within all ALS clinics. Future directions will be to establish a formal set of bulbar guidelines through a methodological and evidence-based approach. Muscle Nerve 59:531-531, 2019., (© 2019 Wiley Periodicals, Inc.)

Purpose : This research aimed to automatically predict intelligible speaking rate for individuals with Amyotrophic Lateral Sclerosis (ALS) based on speech acoustic and articulatory samples. Method : Twelve participants with ALS and two normal subjects produced a total of 1831 phrases. NDI Wave system was used to collect tongue and lip movement and acoustic data synchronously. A machine learning algorithm (i.e. support vector machine) was used to predict intelligible speaking rate (speech intelligibility × speaking rate) from acoustic and articulatory features of the recorded samples. Result : Acoustic, lip movement, and tongue movement information separately, yielded a R 2 of 0.652, 0.660, and 0.678 and a Root Mean Squared Error (RMSE) of 41.096, 41.166, and 39.855 words per minute (WPM) between the predicted and actual values, respectively. Combining acoustic, lip and tongue information we obtained the highest R 2 (0.712) and the lowest RMSE (37.562 WPM). Conclusion : The results revealed that our proposed analyses predicted the intelligible speaking rate of the participant with reasonably high accuracy by extracting the acoustic and/or articulatory features from one short speech sample. With further development, the analyses may be well-suited for clinical applications that require automatic speech severity prediction.

Objective: To evaluate longitudinal cognitive/behavioral change over 12 months in participants enrolled in the ALS Multicenter Cohort Study of Oxidative Stress (ALS COSMOS)., Methods: We analyzed data from 294 ALS participants, 134 of whom were studied serially. Change over time was evaluated controlling for age, sex, symptom duration, education, race, and ethnicity. Using multiple regression, we evaluated associations among decline in ALS Functional Rating Scale-Revised (ALSFRS-R) scores, forced vital capacity (FVC), and cognitive/behavioral changes. Change in cognitive/behavioral subgroups was assessed using one-way analyses of covariance., Results: Participants with follow-up data had fewer baseline behavior problems compared to patients without follow-up data. We found significant worsening of behavior (ALS Cognitive Behavioral Screen (ALS CBS) behavioral scale, p < 0.001; Frontal Behavioral Inventory-ALS (FBI-ALS) disinhibition subscale, p = 0.044). Item analysis suggested change in frustration tolerance, insight, mental rigidity, and interests ( p < 0.05). Changes in ALSFRS-R correlated with the ALS CBS. Worsening disinhibition (FBI-ALS) did not correlate with ALSFRS-R, FVC, or disease duration., Conclusion: We did not detect cognitive change. Behavioral change was detected, and increased disinhibition was found among patients with abnormal baseline behavioral scores. Disinhibition changes did not correlate with disease duration or progression. Baseline behavioral problems were associated with advanced, rapidly progressive disease and study attrition.

Objective: The aim of this Symposium was to develop a consensus based, bulbar assessment protocol for implementation within NEALS clinics., Methods: A one-day symposium, held in April 2017, was organized into Speech and Swallowing sections to establish summary recommendations for the assessment of bulbar dysfunction within each group., Results: Summary recommendations included speech referrals and AAC evaluations at initial visit, CNS-BFS, maximum sustained phonation, and speaking rate. Dysarthria evaluation included the speech subsystem involvement of respiration, phonation, resonance, and articulation. Specific recommendations for swallowing were established for each of the following domains: dietary/oral intake, airway defense physiologic capacity, swallow safety screen, patient-reported swallow-related outcomes, oral sensorimotor exam, and pulmonary function. Practice parameters focused upon patient education and unresolved questions included the use of videofluoscopy, monitoring diet progression, and swallow safety screening., Conclusions: The working goal is to establish a clinical bulbar protocol, designed to be incorporated within ALS clinics and ultimately to formulate a best practice set of bulbar ALS guidelines, available for implementation throughout the international ALS community.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurological disease that affects the speech motor functions, resulting in dysarthria, a motor speech disorder. Speech and articulation deterioration is an indicator of the disease progression of ALS; timely monitoring of the disease progression is critical for clinical management of these patients. This paper investigated machine prediction of intelligible speaking rate of nine individuals with ALS based on a small number of speech acoustic and articulatory samples. Two feature selection techniques - decision tree and gradient boosting - were used with support vector regression for predicting the intelligible speaking rate. Experimental results demonstrated the feasibility of predicting intelligible speaking rate from only a small number of speech samples. Furthermore, adding articulatory features to acoustic features improved prediction performance, when decision tree was used as the feature selection technique.

Objectives: To characterize the prevalence of cognitive and behavioral symptoms using a cognitive/behavioral screening battery in a large prospective multicenter study of amyotrophic lateral sclerosis (ALS)., Methods: Two hundred seventy-four patients with ALS completed 2 validated cognitive screening tests and 2 validated behavioral interviews with accompanying caregivers. We examined the associations between cognitive and behavioral performance, demographic and clinical data, and C9orf72 mutation data., Results: Based on the ALS Cognitive Behavioral Screen cognitive score, 6.5% of the sample scored below the cutoff score for frontotemporal lobar dementia, 54.2% scored in a range consistent with ALS with mild cognitive impairment, and 39.2% scored in the normal range. The ALS Cognitive Behavioral Screen behavioral subscale identified 16.5% of the sample scoring below the dementia cutoff score, with an additional 14.1% scoring in the ALS behavioral impairment range, and 69.4% scoring in the normal range., Conclusions: This investigation revealed high levels of cognitive and behavioral impairment in patients with ALS within 18 months of symptom onset, comparable to prior investigations. This investigation illustrates the successful use and scientific value of adding a cognitive-behavioral screening tool in studies of motor neuron diseases, to provide neurologists with an efficient method to measure these common deficits and to understand how they relate to key clinical variables, when extensive neuropsychological examinations are unavailable. These tools, developed specifically for patients with motor impairment, may be particularly useful in patient populations with multiple sclerosis and Parkinson disease, who are known to have comorbid cognitive decline., (© 2016 American Academy of Neurology.)

Abstract In a multicenter study of newly diagnosed ALS patients without a reported family history of ALS, we are prospectively investigating whether markers of oxidative stress (OS) are associated with disease progression. Methods utilize an extensive structured telephone interview ascertaining environmental, lifestyle, dietary and psychological risk factors associated with OS. Detailed assessments were performed at baseline and at 3-6 month intervals during the ensuing 30 months. Our biorepository includes DNA, plasma, urine, and skin. Three hundred and fifty-five patients were recruited. Subjects were enrolled over a 36-month period at 16 sites. To meet the target number of subjects, the recruitment period was prolonged and additional sites were included. Results showed that demographic and disease characteristics were similar between 477 eligible/non-enrolled and enrolled patients, the only difference being type of health insurance among enrolled patients. Sites were divided into three groups by the number of enrolled subjects. Comparing these three groups, the Columbia site had fewer 'definite ALS' diagnoses. This is the first prospective, interdisciplinary, in-depth, multicenter epidemiological investigation of OS related to ALS progression and has been accomplished by an aggressive recruitment process. The baseline demographic and disease features of the study sample are now fully characterized.

Substantial disparities in TIV utilization rates among ALS patients have been observed, with rates in Japan far exceeding rates in the United States. Our objective was to elicit national preferences and their determinants. We predicted more Japanese than American patients would desire TIV, as would sicker patients, those already using non-invasive interventions, and those with more positive mood and outlook. Patients were enrolled in five U.S. states and six Japanese regions. Eligible patients completed surveys during clinic visits (U.S.) or at home (Japan). Survey responses were in multiple-choice format and took about 15 min to complete. One hundred and fifty-six Americans and 66 Japanese patients participated. Contrary to expectations, Japanese patients were more likely to oppose TIV, as were those on 24-h NIV and patients who knew someone using TIV. Most Japanese and American patients with advanced respiratory impairment were undecided or opposed to TIV, while nearly 20% in both countries were in favor. Finally, patients who favored TIV or who were undecided had more energy, greater wish to live, and more sense of control over ALS management. In conclusion, factors other than patient preferences, such as neurologist preferences, caregiver attitudes and perhaps lack of advance planning, may influence probability of TIV utilization.

To promote clinical and patient oriented research, as part of the Second International ALS Conference in Tarrytown, NY, USA, seven pairs of clinicians and scientists were asked to lead discussions with meeting attendees on six major topics (one of which was discussed by two groups); each one the focus of a 90-min Breakout Session. Approximately 25 meeting attendees participated in each session. The Breakout Sessions considered six major themes: 1) Approaches to encourage clinicians to engage in more clinical research to discover the pathogenesis and cause of ALS; 2) Exploring avenues to build more effective partnerships between basic scientists and ALS physicians; 3) Increasing patient interest and commitment to participating in non-trial clinical research; 4) Brainstorming about factors that are most critical to the discovery of the pathogenesis and cause of ALS; 5) Finding ways to incorporate clinical research projects into clinical trials; and 6) Developing state-of-the-art epidemiological studies to solve the mystery of ALS. In this paper, we present the reports from each of the Breakout Sessions; and we provide a wrap-up of the entire conference.

Embryonic sensory neurons express membrane-anchored growth factors that stimulate proliferation and differentiation of Schwann cells. The most important of these are members of the neuregulin-1 (Nrg-1) family that activate the erbB2/erbB3 receptor kinase on Schwann cells. Nrg-1 growth factors display a complex pattern of alternative mRNA splicing. We investigated the expression of the Nrg-1 type I in rat embryo dorsal root ganglion (DRG) neurons. Nrg-1 type I mRNA was abundantly expressed in DRG neurons; molecular cloning identified three distinct isoforms. The most prominent structural difference produced by alternative splicing was truncation of the C-terminal cytoplasmic domain. In sensory neurons and other cells, Nrg-1 type I proteins with the full-length 374-amino-acid cytoplasmic domain were expressed on the cell surface. In contrast, an isoform with a partially truncated cytoplasmic domain was retained in an intracellular compartment. Deletion studies demonstrated the presence of a cryptic intracellular retention signal that was exposed in the truncated cytoplasmic domain. Cell surface Nrg-1 type I molecules were subject to protease-dependent release of the biologically active ectodomain. As a consequence of their intracellular localization, the Nrg-1 type I isoform with a truncated cytoplasmic domain was not subject to membrane shedding. Nrg-1 type I ectodomain release was accelerated by factors present in Schwann cell-conditioned medium. In cells with active Nrg-1 type I ectodomain, shedding products corresponding to the cytoplasmic domain were not detected, because of rapid gamma-secretase- and proteasome-dependent degradation. These results demonstrate that sensory neurons express alternatively spliced neuregulin polypeptides with distinct subcellular localizations and processing., (Copyright 2006 Wiley-Liss, Inc.)

Four 13762NF rat mammary adenocarcinoma clones were examined for their survival response to heating under conditions that induced transient thermal resistance (thermotolerance). Clones MTC and MTF7 were isolated from the subcutaneous locally growing tumor, whereas clones MTLn2 and MTLn3 were derived from spontaneous lung metastases. There was heterogeneity among these clones in thermotolerance induced by either fractionated 45 degrees C or continuous 42 degrees C heating, but the order of sensitivity was not necessarily the same. The clones developed thermal resistance at different rates and to different degrees within the same time intervals. There was heterogeneity between clones isolated from within either the primary site or metastatic lesions. However, clones derived from metastatic foci did not intrinsically acquire more or less thermotolerance to fractionated 45 degrees C or continuous 42 degrees C heating than did clones from the primary tumor. Further, there was no apparent relationship between any phenotypic properties that conferred more or less thermotolerance in vitro and any phenotypic properties that conferred enhanced metastatic success of these same clones by spontaneous (subcutaneous) or experimental (intravenous) routes in vivo. These tumor clones also differ in their karyotype, metastatic potential, cell surface features, sensitivity to x-irradiation and drugs, and ability to repair sublethal radiation damage. These results provide further credence to the concept that inherent heterogeneity within tumors may be as important in therapeutic success as other known modifiers of outcome such as site and treatment heterogeneity.

Exposure of clone MTC rat 13672NF mammary adenocarcinoma cells to continuous heating at 42 degrees C or to heating for 20 min at 45 degrees C followed by incubation at 37 degrees C induced or enhanced synthesis of several heat-stress proteins (hsp) detectable by [3H]leucine pulse labeling, gel electrophoresis, and fluorography. Hsp synthesis occurred during development and expression of thermal resistance. For example, continuous heating of MTC cells at 42 degrees C after a rapid temperature transient (about 4 min) produced thermal resistance within 4 to 5 hr of initiation of heating. Hsp synthesis was observed within 2 hr of the cells reaching 42 degrees C and continued throughout 8 hr of heating; four major hsp appeared at nominal molecular weights of 112, 90, 70, and 22 kDa. A slow temperature transient from 37 degrees to 42 degrees C over a 3-hr period increased thermal resistance by a factor of about 2 relative to a rapid transient or "shock" to 42 degrees C. However, hsp synthesis was not significant during the slow heat transient and was either reduced (at 70 and 22 kDa) or not increased (at 112 and 90 kDa) compared with the rates of hsp synthesis at the same time intervals after the rapid transient to 42 degrees C. In these mammary tumor cells, no differences in the number of hsp were detected during heating at 42 degrees or after 45 degrees C heating. Other proteins did not appear to change their relative rates of synthesis, with the exception of a clear decrease in proteins with nominal molecular weights of histones H2A, H2B, H3, and H4. Hsp synthesis was not triggered by cold shock to 23 degrees or 0 degree C, or by radiation of from 2.5 to 10 Gy. Thus hypothermic stress did not enhance hsp synthesis nor were hsp nonspecifically associated with eventual cell lethality. In these experiments, the synthesis of hsp was generally correlated with the development of thermal resistance. Determining the quantity, function, and location of stress proteins may identify targets for thermal killing and resistance. However, one paradoxical observation remains to be resolved. Under the slow temperature transient conditions, the tumor cells attained more thermal resistance with generally reduced rates of hsp synthesis. These results have two possible implications: (a) one or more hsp were not directly related to thermal resistance, or (b) hsp were involved in thermal resistance but their relative rate of synthesis depended on the severity of the heat transient. The latter would imply that the cells made other regulatory or metabolic adjustments and either utilized the hsp more effectively or required less additional hsp.