Red blood cells as conditional recovery-phase persistence amplifiers via lipid-domain–gated vesiculation, clearance-limited EV residence, near-wall NO timing noise, and impaired SMPDL3B-mediated termination.
Author: Michael Daniels · Framework: GLA v2.6 · Date: January 21st 2026 · Interpretive synthesis only. RBCs and EVs are treated as conditional upstream persistence amplifiers during recovery, not disease initiators. Nitric oxide (NO) pathology is framed as timing/localization noise (not NO deficiency). SMPDL3B loss is framed as termination/disengagement failure (not immune triggering). This document is not clinical guidance or a treatment recommendation.
This page defines the RBC–EV–Lipid–SMPDL3B Persistence Amplifier in GLA v2.6: a recovery-phase control loop that can amplify and stabilize persistence after exertion without initiating disease.
In this framing, post-exertional malaise (PEM) is generated when physiological stress exposes a fragile execution surface and recovery fails to terminate cleanly. The v2.6 update specifies a parallel persistence branch in which red blood cells (RBCs), lipid-domain mechanics, and liver–spleen clearance-limited extracellular vesicle (EV) persistence increase recovery-phase instability and prolong “not-resolved” interface states.
v2.5: RBC deformation, hemoglobin release, and microclot interactions were treated as secondary consequences that contribute to symptoms but do not actively shape persistence.
v2.6: Under circulatory stress (shear variability · hypoxia · redox load), lipid-domain instability lowers vesiculation thresholds and drives adaptive release of RBC-derived EVs (RBC-EVs). When first-pass hepatic and splenic clearance is delayed, EVs persist near vascular control surfaces, distort NO timing/localization, and prevent re-anchoring of termination regulators (notably SMPDL3B), reinforcing a recovery-phase “not-resolved” state.
Within GLA v2.6, persistence during post-exertional malaise is shaped by a parallel recovery-phase persistence amplifier that operates downstream of exertion-time skeletal-muscle stress and after recovery-phase control has already failed. Red blood cells (RBCs), their lipid-domain architecture, RBC-derived extracellular vesicles (RBC-EVs), and the membrane-anchored termination regulator SMPDL3B do not initiate PEM or ME/CFS.
Instead, this module defines how these elements collectively form a persistence control surface that modulates clearance-to-decay, near-wall NO timing, and termination fidelity during recovery. When first-pass clearance through the liver–spleen axis is efficient, recovery completes and signaling terminates. When clearance is delayed, persistence pressure rises and exertional stress is more likely to lock into delayed PEM.
Post-exertional malaise (PEM) is the defining feature of myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS), characterized by delayed symptom worsening that emerges hours to days after physiological stress rather than during exertion itself. This temporal dissociation places a strong constraint on mechanism: exertion functions as a stress test, while the pathological failure that defines PEM unfolds during recovery.
Within the Gut–Liver–Autonomic (GLA) framework, PEM is modeled as a recovery-phase control failure, not as exertion-time injury, immune hyperactivation, or primary energy depletion. Earlier GLA versions focused on how shear-exposed skeletal muscle, endothelial timing errors, and impaired signal termination generate delayed instability during recovery. GLA v2.6 builds on this foundation by specifying an additional, parallel persistence amplifier that determines whether recovery completes or locks into prolonged PEM.
This document defines that amplifier: the RBC–EV–Lipid–SMPDL3B persistence loop. In this model, red blood cells (RBCs), their lipid-domain architecture, RBC-derived extracellular vesicles (RBC-EVs), and the membrane-anchored termination regulator SMPDL3B do not initiate disease. Instead, they act as conditional recovery-phase persistence amplifiers once recovery-phase control has already failed elsewhere in the system.
Under circulatory stress—particularly shear variability, hypoxia, and redox load—RBC membranes experience increased curvature and recovery demand. Lipid-domain instability lowers vesiculation thresholds, leading to adaptive, domain-localized release of RBC-EVs. This process is initially protective. Pathology arises only when first-pass clearance through the liver–spleen axis is delayed. In such states, EVs persist near vascular control surfaces, distort nitric oxide (NO) timing and localization, and interfere with re-anchoring of SMPDL3B, quietly reinforcing a “not-resolved” biochemical state during recovery.
A central clarification in GLA v2.6 is that NO pathology reflects timing and localization noise, not NO deficiency, and that EV persistence reflects clearance limitation rather than excessive production. When clearance-to-decay is slowed, even low EV burdens can exert disproportionate effects on near-wall signaling and termination fidelity, without requiring cytokine escalation or overt inflammation.
This update also refines the role of bile-acid signaling. Whereas earlier GLA versions treated circadian bile-acid pathways as major loop-maintaining drivers of persistence, GLA v2.6 reclassifies bile acids as a secondary entrainment and bias layer that modulates clearance versus tolerance after the primary persistence loop is established. Bile acids influence the probability and timing of resolution but do not generate exertion specificity, delayed PEM, or recovery-phase collapse on their own.
Together, these refinements tighten causality, reduce reliance on speculative initiation models, and improve sequencing logic by clearly separating initiation, recovery failure, and persistence amplification. The sections that follow examine how recovery termination fails, how persistence is amplified through clearance-limited RBC-EV dynamics, and why this produces delayed, cumulative, and state-dependent PEM rather than acute injury or inflammatory disease.
Execution → Recovery → Persistence (GLA v2.6)
OrientationFigure 1. GLA v2.6 recovery-phase architecture (orientation). Exertion acts as a stress test at the skeletal-muscle execution surface. PEM emerges when recovery fails to terminate, and persistence is amplified by a parallel RBC–EV loop whose impact is gated by clearance and expressed as near-wall NO timing noise and impaired SMPDL3B re-anchoring. This figure is conceptual (not molecular) and is intended to prevent reversal of causality (persistence ≠ initiation).
This section describes mechanisms that amplify and stabilize persistence after recovery-phase control has already failed. It does not describe the primary site where recovery failure is initiated, nor does it propose disease initiation mechanisms.
Detailed Interpretation — cited text (Name, Year)
Living erythrocytes exhibit stable, sub-micrometric plasma-membrane lipid domains enriched in cholesterol or sphingomyelin, with reproducible spatial organization. Cholesterol-enriched domains preferentially localize to regions of high membrane curvature during deformation (for example, the rim during stretch), whereas sphingomyelin-enriched domains localize to low-curvature, concave regions associated with post-deformation recovery. (Leonard, 2017)
During aging or sustained stress, vesiculation occurs preferentially at these lipid domains, indicating that they function as mechanical fragility sites rather than random points of membrane failure. Experimental disruption of lipid-domain organization reduces high-curvature features and impairs deformability, confirming a mechanical—not signaling—role in stress handling and membrane off-loading. (Leonard, 2017; Bosman, 2008)
SMPDL3B integration.
SMPDL3B is GPI-anchored within these same lipid-raft domains. Lipid-domain integrity therefore simultaneously determines (i) where membrane
off-loading is energetically favored and (ii) whether SMPDL3B can remain anchored to enforce clean signal termination after stress.
(Leonard, 2017)
GLA interpretation.
Lipids act as execution-surface mechanics. They define where and when membrane shedding occurs, setting both the vesiculation threshold and
the anchoring context for SMPDL3B, without initiating pathology.
During deformation, cholesterol-enriched domains concentrate bending energy and stabilize high curvature, acting as stress concentrators and defining likely budding sites. During recovery, sphingomyelin-enriched domains transiently expand in concert with Ca²⁺ efflux and volume restoration, linking them to recovery-phase control rather than deformation initiation. (Leonard, 2017)
Disruption of sphingomyelin-rich domains impairs Ca²⁺ handling and volume recovery, increasing susceptibility to membrane shedding when recovery bandwidth is exceeded. (Dinkla, 2012)
SMPDL3B integration.
Recovery-phase sphingomyelin-domain dynamics are required to re-establish SMPDL3B anchoring following stress. When recovery-phase lipid
reorganization fails, SMPDL3B re-anchoring becomes incomplete, reducing termination precision even in the absence of overt immune activation.
(Leonard, 2017; Dinkla, 2012)
GLA interpretation.
Cholesterol domains bias deformation-phase stress localization, whereas sphingomyelin domains bias recovery-phase control and termination
fidelity. Failure emerges when recovery-phase capacity cannot resolve deformation-encoded stress and cannot re-anchor SMPDL3B.
Extracellular sphingomyelinase (SMase)—elevated in inflammatory or metabolic stress states—converts sphingomyelin to ceramide, producing rigid ceramide-enriched platforms that disrupt membrane–cytoskeleton coupling, increase Ca²⁺ dysregulation, and markedly enhance domain-localized vesiculation. (Dinkla, 2012; Lang, 2010)
Even low levels of SMase activity are sufficient to induce shape instability and vesicle release, with effects amplified in aged or repeatedly stressed erythrocytes. Vesiculation precedes removal and initially functions as protective off-loading; pathology arises only when clearance is delayed. (Bosman, 2008; Dinkla, 2012)
SMPDL3B integration.
Ceramide-biased membranes reduce the anchoring stability of GPI-anchored proteins, including SMPDL3B, shortening termination windows and biasing
the surface toward prolonged permissiveness. (Lang, 2010)
GLA interpretation.
Ceramide functions as a threshold shifter. It accelerates off-loading once control is strained and destabilizes SMPDL3B anchoring, increasing EV
output and persistence pressure without initiating disease.
Once released, EV fate is governed by recognition and routing, not by production rate. A substantial fraction of EVs rapidly associate with platelets and erythrocytes (“hitchhiking”) and are normally cleared as aggregates by macrophages during first-pass hepatic and secondary splenic filtration. (Pavlova, 2025/2026; Willekens, 2005)
Clearance is state-dependent. Altered flow, shear variability, and surface-identity changes (lipid composition, phosphatidylserine exposure, glycan patterns, and protein corona features) can markedly prolong EV half-life and broaden biodistribution, increasing near-wall residence time. Uptake or transient association does not guarantee resolution; effective clearance-to-decay is the rate-limiting step. (Greuter & Shah, 2016; Pavlova, 2025/2026; Tournoy, 2026)
SMPDL3B integration.
• Rapid clearance → lipid domains reset → SMPDL3B re-anchors → termination precision restored.
• Delayed clearance → persistent near-wall stress → SMPDL3B remains functionally low → termination fails quietly.
(Tournoy, 2026; Subramanian & Chavakis, 2023)
GLA interpretation.
EV persistence reflects clearance bandwidth. EV production becomes pathological only when clearance is delayed and SMPDL3B recovery is prevented.
Why clearance slows without inflammation (GLA v2.6)
In ME/CFS, autonomic instability and/or low blood volume can produce maldistributed flow and increased shear variability rather than a simple reduction in oxygen delivery. When endothelial timing control is fragile, this creates microvascular intermittency and near-wall flow disturbances. In the liver, sinusoidal endothelial cells rely on shear-regulated nitric-oxide signaling to maintain low resistance and preserve efficient first-pass filtration (Greuter & Shah, 2016).
If hepatic microcirculation becomes intermittent or congested, sinusoidal autoregulation becomes less precise and the encounter probability between circulating particles and Kupffer-cell capture surfaces falls. Under these conditions, EV clearance-to-decay slows despite normal production, increasing near-wall residence and making persistence amplification more likely during recovery (Willekens, 2005; Jiang, 2021).
Flow → sinusoidal intermittency → clearance gate → persistence amplification (GLA v2.6)
BridgeFigure 3. Autonomic instability / hypovolemia bridge (GLA v2.6). Autonomic instability and/or low blood volume can increase flow maldistribution and shear variability. When hepatic sinusoidal flow becomes intermittent, the encounter probability between circulating particles and Kupffer-cell capture surfaces falls, slowing clearance-to-decay despite normal production. Prolonged near-wall EV residence increases NO timing/localization noise (not NO deficiency), impairs SMPDL3B re-anchoring, and sustains a “not-resolved” interface state, lengthening PEM duration. This figure is conceptual and preserves the core guardrail: persistence amplification ≠ disease initiation.
Hemoglobin reacts with nitric oxide (NO) at diffusion-limited rates. Intact erythrocytes normally protect endothelial NO signaling through compartmentalization and near-wall cell-free zones. This protection collapses with hemolysis or Hb-containing microparticles, which scavenge NO at rates approaching those of cell-free hemoglobin—approximately three orders of magnitude faster than intact RBC-encapsulated hemoglobin. (Donadee, 2011; Helms & Kim-Shapiro, 2013)
Consequently, even low but persistent RBC-EV or microparticle burdens can introduce localized NO timing and localization noise, particularly during prolonged near-wall residence when clearance is impaired. (Donadee, 2011; Greuter & Shah, 2016)
SMPDL3B integration.
Persistent NO and redox timing noise impairs re-anchoring of SMPDL3B, locking endothelial and immune interfaces into a “not-resolved”
biochemical state without cytokine escalation. (Helms & Kim-Shapiro, 2013)
GLA interpretation.
Persistence plus proximity corrupts NO timing during recovery and prevents SMPDL3B-mediated termination, amplifying instability without altering
total NO production.
Cross-disease validation of clearance-limited persistence.
Independent work in chronic obstructive pulmonary disease (COPD) demonstrates that failure of cellular clearance (efferocytosis) can function as
an autonomous, disease-maintaining mechanism even after removal of the initiating insult, producing persistent low-grade inflammatory states
without ongoing injury. This literature is cited as conceptual validation of clearance- and termination-limited persistence, not as a
disease-specific model for ME/CFS. (Tournoy, 2026)
Closed-loop: lipid domains → EV persistence → NO timing noise → SMPDL3B termination
MechanismFigure 2. RBC–EV–Lipid–SMPDL3B persistence amplifier (GLA v2.6). Lipid-domain mechanics set vesiculation thresholds and the anchoring environment for GPI-anchored termination regulators. Ceramide shifts lower the off-loading threshold, increasing domain-localized vesiculation and RBC-EV release. EV impact is governed by routing and clearance-to-decay (often via platelet/RBC hitchhiking to liver and spleen). When clearance is delayed, near-wall EV residence introduces NO timing/localization noise and prevents clean SMPDL3B re-anchoring, reinforcing a “not resolved” interface state and amplifying delayed PEM.
In summary, post-exertional malaise (PEM) arises when physiological shear stress exposes a fragile execution surface in skeletal muscle, initiating a recovery process that fails to terminate cleanly. Exertion increases membrane coordination demand, engaging PI/PLC-linked remodeling and ER–mitochondrial calcium exchange at mitochondria-associated membranes. When calcium timing fails to resolve, recovery-phase reactive oxygen species accumulate, impair cardiolipin remodeling, and progressively reduce mitochondrial recovery bandwidth. As recovery efficiency erodes, sphingolipid resolution stalls, phosphatidylserine asymmetry is restored inconsistently, and endothelial and barrier interfaces remain biochemically flagged as unresolved.
Concurrently, lipid-raft instability reduces effective membrane anchoring of the GPI-anchored termination regulator SMPDL3B, impairing signal termination precision at endothelial and immune control surfaces and prolonging primed states without cytokine escalation. This produces a quiet persistence state in which recovery processes remain active but incompletely resolved.
In parallel, the same circulatory stressors—hypoxia, shear variability, and redox load—impose membrane stress on red blood cells (curvature demand, Ca²⁺ dysregulation, and lipid-domain reorganization), lowering vesiculation thresholds at cholesterol- and sphingomyelin-enriched membrane domains and triggering compensatory, domain-localized release of RBC-derived extracellular vesicles (RBC-EVs). These RBC-EVs—bearing phosphatidylserine, hemoglobin/heme, and oxidized lipid cargo—do not initiate pathology. Instead, they act as recovery-phase persistence amplifiers whose biological impact is gated by clearance capacity.
Under normal conditions, EVs are routed via platelet and erythrocyte “hitchhiking” to first-pass hepatic clearance with secondary splenic support, where rapid recognition and degradation resolve the signal. When clearance is delayed—due to altered flow, shear, or receptor-executed capture—prolonged near-wall EV residence amplifies localized nitric-oxide timing and localization noise (rather than NO deficiency) and prevents SMPDL3B re-anchoring. This sustains immune sentinel engagement without overt inflammation, reinforces the “not-resolved” interface state, and increases the metabolic cost of repair, reflected downstream in altered nitrogen handling.
PEM therefore represents a recovery-phase control failure with clearance-limited persistence and impaired SMPDL3B-mediated termination, rather than exertional injury, immune hyperactivation, or primary energy depletion.
Integrated mechanistic chain (compressed; lipid-, clearance-, and SMPDL3B-explicit — GLA v2.6)
Clarification:
In this framework, low membrane-anchored SMPDL3B—whether arising from episodic shedding or chronic deficiency—reflects failure of recovery-phase
signal termination driven by lipid-domain instability and clearance-limited stress persistence, rather than primary immune activation or intrinsic
enzymatic deficiency.
These predictions follow directly from the GLA v2.6 separation of initiation, recovery failure, and persistence amplification. They are intended to be mechanistically constrained, state-dependent, and falsifiable, rather than correlational or pathway-agnostic.
For a given exertional stressor, the magnitude and duration of PEM will correlate more strongly with markers of EV clearance efficiency (e.g., hepatic or splenic uptake proxies, EV half-life, glycan or phosphatidylserine exposure patterns) than with measures of exertional intensity, total EV production, or acute mechanical stress.
Prediction: Individuals with comparable exertion but slower EV clearance will exhibit longer PEM duration and greater delayed symptom amplification.
RBC-derived EV and microparticle signals measured during PEM will show weak correlation with peak exertional metrics but strong correlation with recovery duration and symptom persistence.
Prediction: EV burden will remain elevated during prolonged PEM even when exertion has ceased and energetic demand has normalized.
Nitric-oxide signaling during PEM will exhibit altered timing, spatial heterogeneity, and near-wall disruption rather than a uniform reduction in total NO bioavailability.
Prediction: Manipulations that alter near-wall residence of Hb-containing microparticles will disproportionately affect PEM severity without measurably changing bulk NO production.
Membrane-anchored SMPDL3B levels or functional proxies will be lowest during prolonged recovery and will normalize only after EV clearance and NO timing stabilize, independent of cytokine levels or overt immune activation.
Prediction: SMPDL3B recovery will lag behind exertion and normalize only when persistence resolves, even in the absence of inflammatory markers.
Markers of sphingomyelinase activity and ceramide-enriched membrane domains will predict reduced tolerance to subsequent stress and earlier onset of PEM, without producing symptoms in isolation.
Prediction: Ceramide enrichment will shift the threshold for persistence amplification but will not generate PEM without exertion-induced recovery failure.
Interventions or physiological states that improve EV clearance or reduce near-wall EV residence will shorten PEM duration even if exertional capacity remains unchanged.
Prediction: Recovery duration will improve before exercise tolerance improves.
Age-associated reductions in membrane reserve, clearance capacity, and recovery bandwidth will amplify PEM duration and variability without changing the fundamental initiation mechanism.
Prediction: Older individuals will exhibit longer persistence phases and slower SMPDL3B recovery after comparable exertional stress.
Changes in bile-acid signaling will modulate the probability and timing of recovery resolution only after persistence is established, without altering the exertion threshold for PEM onset.
Prediction: Bile-acid interventions will affect recovery variability and duration but will not prevent PEM initiation under identical exertional stress.
Immune features associated with PEM (e.g., MAIT and γδ T-cell programs) will scale with persistence duration and recovery incompleteness rather than with exertional load or primary immune activation.
Prediction: Immune signatures will decay only after EV persistence and SMPDL3B anchoring recover, not immediately after exertion ends.
Exertion applied before recovery completion will produce disproportionately greater persistence amplification than equivalent exertion applied after full resolution.
Prediction: Repeated stress during unresolved PEM will increase EV persistence, NO timing noise, and recovery duration in a non-linear, history-dependent manner.
If hepatic and splenic clearance functions normally in ME/CFS, RBC-derived vesicles should be removed from circulation on a minutes-scale following exertion, as observed in healthy physiology. If clearance is impaired, vesicle persistence will be prolonged despite normal or near-normal production.
Prediction: In ME/CFS, direct measurement of post-exertional RBC-EV clearance kinetics will reveal delayed clearance-to-decay relative to healthy controls, consistent with clearance-limited persistence rather than excessive vesicle generation.
Rationale: A modern, ethically appropriate replication of classic hepatic clearance kinetics—using contemporary labeling or tracking methods—would provide a decisive test of the GLA v2.6 persistence model.
Summary constraint
Across all predictions, interventions that improve recovery termination and clearance will reduce PEM duration before they improve capacity,
whereas interventions that increase throughput or stress without improving termination will worsen persistence even if short-term performance
appears unchanged.
GLA v2.6 one-line tag: Post-exertional malaise in ME/CFS reflects a recovery-phase control failure that is stabilized and prolonged by clearance-limited RBC-EV persistence and impaired SMPDL3B-mediated termination, rather than by exertional injury, immune hyperactivation, or primary energy depletion.
References are grouped by priority (Tier 0–6) to preserve causal ordering: physiology → execution mechanics → threshold shifts → clearance failure logic → flow/shear bridge → ME/CFS context → genetics/state-mapping.
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The following studies establish SMPDL3B relevance in ME/CFS as a biomarker and state-defining factor. Within GLA v2.6, these findings are interpreted through the lens of recovery-phase termination failure and persistence, not primary immune triggering or disease initiation.
Rostami-Afshari, B., Elremaly, W., Franco, A., Elbakry, M., Akoume, M. Y., Boufaied, I.,
Moezzi, A., Leveau, C., Rompré, P., Godbout, C., Mella, O., Fluge, Ø., & Moreau, A. (2025).
SMPDL3B: a novel biomarker and therapeutic target in myalgic encephalomyelitis.
Journal of Translational Medicine, 23(1), 748.
Erratum: Journal of Translational Medicine, 23(1), 911.
doi:10.1186/s12967-025-06829-0
https://pmc.ncbi.nlm.nih.gov/articles/PMC12236014/
Rostami-Afshari, B., Elremaly, W., McGregor, N. R., Huang, K. J. K., Armstrong, C. W.,
Franco, A., Godbout, C., Elbakry, M., Abdelli, R., & Moreau, A. (2025).
Circulating levels of SMPDL3B define metabolic endophenotypes and subclinical kidney
alterations in myalgic encephalomyelitis.
International Journal of Molecular Sciences, 26(18), 8882.
doi:10.3390/ijms26188882
https://pubmed.ncbi.nlm.nih.gov/41009450/
GLA v2.6 interpretation note: These studies demonstrate that SMPDL3B levels stratify ME/CFS patients and track systemic alterations. In GLA v2.6, reduced circulating or membrane-associated SMPDL3B is interpreted as a marker of impaired recovery-phase signal termination and persistence (across shedding and deficient phenotypes), rather than evidence of primary immune activation, enzymatic insufficiency, or disease initiation.
Xiong, R., Aiken, E., Caldwell, R., et al. (2025). AI-driven multi-omics modeling of myalgic encephalomyelitis/chronic fatigue syndrome. Nature Medicine, 31, 2991–3001. https://doi.org/10.1038/s41591-025-03788-3 (Used for state signatures; predictive ≠ causal.)
Huang, K., Muneeb, M., Thomas, N., Schneider-Futschik, E. K., Gooley, P. R., Ascher, D. B., & Armstrong, C. W. (2026). Exploring a genetic basis for the metabolic perturbations in ME/CFS using UK Biobank. iScience, 29(1), Article 110577. https://www.cell.com/iscience/fulltext/S2589-0042(25)02577-5
PrecisionLife Ltd. (2025). Combinatorial analysis identifies genetic networks associated with ME/CFS. medRxiv (preprint). https://www.medrxiv.org/content/10.64898/2025.12.01.25341362v1
Scope note: Tier 6 references support susceptibility and state-mapping context; they are not used as evidence for clearance kinetics or recovery-phase persistence mechanisms.
The documents listed below define the conceptual and methodological framework used to interpret genetic signals and physiological mechanisms in this paper. Collectively, they establish layer boundaries, phenotype discipline, and phase dependence within the Gut–Liver–Autonomic (GLA) system architecture.
These materials are provided for transparency and interpretive context only. They are not cited as evidentiary sources and should be read as evolving systems-biology models used to organize and constrain interpretation, rather than as claims of mechanism or causation.
Core GLA framework documents
SMPDL3B phenotype frameworks
Control layers & system modulators