C4d is a degradation fragment of the complement protein C4b, generated when the classical complement pathway is activated. In ABO-compatible kidney transplantation, peritubular capillary C4d deposition follows the binding of anti-HLA donor-specific antibodies and is recognized as a histological hallmark of antibody-mediated injury and poor prognosis. In ABO-incompatible grafts, however, the picture is strikingly different. C4d appears in the peritubular capillaries of more than half of recipients, yet most of these grafts function well for years. This paradox has been attributed to endothelial accommodation, a state in which membrane-bound complement regulatory proteins such as CD46, CD55, and CD59 restrain complement-mediated injury on the graft endothelium despite ongoing antibody binding. The clinical significance of C4d deposition in this setting has remained unclear, and most previous studies evaluated it at only a single time point.

The Tokyo team, led by Yu Kijima and colleagues, took a different approach. Because a single determination may not reflect a sustained process, they defined C4d positivity by its persistence across two sequential post-transplant protocol biopsies, obtained at 3 months and 1 year after surgery. Between 2015 and 2022, their center performed 254 ABO-incompatible kidney transplantations, and 69 recipients with paired protocol biopsies formed the study population. Fifty-four recipients, or 78.3 percent, were classified as persistently C4d-positive, meaning C4d deposition was detectable in both biopsies, while 15 recipients were classified as C4d-negative or nonpersistent. The researchers then compared pre-transplant background characteristics, cellular infiltration, chronic Banff lesions according to the Banff 2019 classification, graft function, and graft survival between the two groups.

The immunosuppressive regimen itself is worth noting for its technical sophistication. Patients received a triple-drug protocol consisting of tacrolimus, mycophenolate mofetil, and methylprednisolone, starting seven days before transplantation, with tacrolimus doses adjusted to trough levels of 8 to 12 nanograms per milliliter during the first month. Induction with the anti-IL-2 receptor antibody basiliximab was performed in all recipients, and all patients received low-dose rituximab for B-cell depletion, which has replaced splenectomy at the center since 2004. Before surgery, recipients underwent three or four sessions of double-filtration plasmapheresis to remove anti-A and anti-B antibodies until titers fell to 1:32 or below. Recipients who received intravenous immunoglobulin at induction were excluded from the analysis because IVIG may inactivate activated complement and thereby influence C4d deposition.

The biopsy protocol was equally rigorous. Renal allograft biopsies were obtained at time zero as a back-table biopsy, at 3 months, and at 1 year, with an adequate specimen defined as one containing seven or more glomeruli and at least one arterial cross-section. C4d staining was performed by indirect immunofluorescence on cryostat sections using a mouse monoclonal anti-human C4d antibody, scored from 0 to 3 according to Banff criteria ranging from no staining to diffuse staining of more than half of the peritubular capillaries. The persistent C4d-positive group included recipients with deposition at both time points, even when the staining score was a reproducible low-grade 1 at both visits. As a sensitivity analysis, the team also repeated all comparisons using a stricter, Banff-concordant definition requiring a C4d score of at least 2 at both time points, which yielded 47 positive and 22 negative recipients and did not change the overall pattern of findings.

The results were largely reassuring. Baseline anti-A and anti-B antibody titers, ABO donor-recipient combinations, donor age, and donor relationship did not differ between the groups, although donor sex did, with a higher proportion of male donors in the C4d-positive group, a finding the authors attribute cautiously to chance given the number of comparisons performed. Microvascular inflammation, defined as a combined glomerulitis plus peritubular capillaritis score of 2 or more, was observed in 6 of 54 persistently C4d-positive recipients, or 11.1 percent, versus 1 of 15, or 6.7 percent, in the negative group. Chronic changes, defined as an interstitial fibrosis score of at least 1, appeared in 27 of 54, or 50.0 percent, versus 6 of 15, or 40.0 percent. None of these differences reached statistical significance, and cellular infiltration scores for interstitial inflammation, tubulitis, glomerulitis, and peritubular capillaritis were similarly comparable at both time points.

Graft function and survival told the same story at the group level. Serum creatinine at 1 year did not differ significantly between the groups, nor did estimated glomerular filtration rate, which measured a median of 45.5 versus 43.6 milliliters per minute per 1.73 square meters. Kaplan-Meier analysis showed no significant difference in graft survival, with a log-rank P value of 0.191, and this conclusion held in a landmark analysis with the time origin at the 1-year biopsy, designed to address the guarantee-time bias inherent in requiring a 1-year biopsy for group assignment. Biopsy-proven rejection was infrequent in both groups: nine episodes were identified among the 69 recipients, seven in the C4d-positive group and two in the negative group, including cases of borderline change, active and chronic active antibody-mediated rejection, and chronic active T-cell-mediated rejection.

Yet one finding refuses to be dismissed. All four graft losses in the analyzed cohort occurred in the persistent C4d-positive group, occurring at 1,849, 1,926, 2,231, and 3,208 days after transplantation. Three of the four were ultimately attributable to chronic active antibody-mediated rejection, and the fourth to BK virus nephropathy. Critically, these cases were not homogeneous. One recipient had biopsy-confirmed chronic active antibody-mediated rejection with a microvascular inflammation score of 3 at the very 1-year protocol biopsy used to define C4d persistence, meaning early persistent C4d was accompanied by concurrent histological injury. In the remaining two rejection cases and the BK virus case, no microvascular inflammation or overt rejection was present on either the 3-month or 1-year biopsies, and the chronic active antibody-mediated rejection was diagnosed later, at 1.9 years post-transplant in one case and at an undetermined later time in the other. This heterogeneity suggests that persistent C4d at 3 months and 1 year does not reliably distinguish recipients who will remain stable from those who will later develop overt humoral injury.

A parallel signal emerged in the antibody data, though with important caveats. Post-transplant donor-specific antibody testing was available for 47 of the 69 recipients, and de novo donor-specific antibodies were detected in 11 of 38 evaluable recipients, or 28.9 percent, in the C4d-positive group, compared with 1 of 9, or 11.1 percent, in the negative group. This difference did not reach statistical significance, and the authors emphasize that it is exploratory at best: testing was not performed at uniform time points, measurements spanned roughly 4 months to 3 years after transplantation, and the 22 recipients without available data had significantly shorter follow-up. Whether persistent C4d deposition is unrelated to subsequent HLA sensitization or reflects low-grade humoral activity that could contribute to later sensitization simply cannot be determined from this retrospective cohort, and the absence of molecular transcript profiling further limited the ability to detect subclinical humoral activity.

The authors are admirably candid about the limits of their work. The study was retrospective and single-center, and the comparator group of only 15 patients left it substantially underpowered for low-frequency endpoints such as rejection and graft loss, meaning the null results represent a failure to detect an association rather than evidence of equivalence. The cohort was also highly selected: only 69 of 254 recipients qualified, with exclusions for insufficient pathological data, prior treated rejection, thrombotic microangiopathy, IVIG induction, retransplantation, and recurrent glomerular disease effectively removing the higher-risk population. Anti-A/B titers were available only before transplantation, so the researchers could not determine whether persistent C4d reflected ongoing antibody exposure with successful accommodation or merely residual complement footprints. Taken together, the findings are consistent with accommodation but hypothesis-generating rather than definitive, and the authors call for confirmation in larger, prospective cohorts with systematic de novo donor-specific antibody surveillance and molecular diagnostics. For now, the message for clinicians is nuanced: persistent C4d in an ABO-incompatible graft is not by itself a cause for alarm, but the small cluster of late losses in the positive group suggests that long-term immunological surveillance, rather than either reassurance or immediate treatment escalation based on early biopsies alone, may be the wisest course.

Subject of Research: Persistent C4d deposition on sequential protocol biopsies in ABO-incompatible kidney transplantation

Article Title: Clinical significance of persistent C4d deposition on sequential protocol biopsies in ABO-incompatible kidney transplantation

Article References: Kijima, Y., Ishida, H., Yagisawa, T., Hirai, T., Unagami, K., Shimizu, T., & Takagi, T. (2026). Clinical significance of persistent C4d deposition on sequential protocol biopsies in ABO-incompatible kidney transplantation. Frontiers in Nephrology, 6, Article 1940137. https://doi.org/10.3389/fneph.2026.1940137

Image Credits: AI Generated

DOI: 10.3389/fneph.2026.1940137

Keywords: ABO-incompatible kidney transplantation, C4d, accommodation, antibody-mediated rejection, protocol biopsy, microvascular inflammation, Banff classification, donor-specific antibodies, complement, graft survival, kidney allograft, transplant immunology

Duchenne muscular dystrophy is an X-linked childhood disease caused by loss of dystrophin, the protein that stabilizes muscle fibers during contraction. Delandistrogene moxeparvovec attempts to compensate by using an AAVrh74 viral vector to ferry a miniaturized micro-dystrophin gene into heart and skeletal muscle. Five FDA-approved gene therapies now rely on intravenous recombinant adeno-associated virus (rAAV), but the enormous doses needed to transduce enough tissue have produced serious adverse events and even deaths in clinical practice, making a detailed understanding of the body’s systemic reaction to these infusions a pressing safety concern.

The new study, led by researchers at the University of Florida Myology Institute, examined plasma and urine samples banked from four young boys in the NCT03375164 trial, all between four and eight years old. These participants received 2.0 × 10¹⁴ vector genomes per kilogram of rAAVrh74 carrying the micro-dystrophin transgene, and all four experienced effective muscle expression, multiyear functional improvement, and no serious adverse events. That makes them, in the researchers’ words, among the best representatives of a successful micro-dystrophin gene therapy, and therefore an ideal case study of what a favorable response looks like at the molecular level.

Using digital PCR to count viral genomes and immunoblotting to detect capsid proteins, the team found extraordinarily high levels of circulating vector one day after infusion, exceeding 10¹² vector genomes per milliliter of plasma. By day 7, plasma titers had dropped more than a thousandfold, to roughly 10⁹ vector genomes per milliliter or below. Urinary shedding was minimal and highly variable; two participants showed undetectable levels at every time point, while the highest value observed was 6 × 10⁶ vector genomes per milliliter in a single day 1 urine sample.

Critically, the circulating virus was not just inert debris. DNase pretreatment experiments showed that most of the detected genomes were protected inside intact capsids, and when day 1 plasma was added to cultured AC16 human cardiomyocytes, the particles successfully transduced the cells, proving that at least some of the vector remained biologically active a full day after infusion. This finding also addressed a recent concern in the field: laboratory studies have suggested AAV particles can spontaneously eject their genomes at body temperature, but the new data indicate genome ejection is not a prominent feature of systemic AAVrh74 therapy in humans.

The proteomic analysis told a more dramatic story. Using the SomaScan platform, which measures more than 5,000 circulating proteins with DNA-aptamer reagents, the researchers found 287 protein targets significantly altered across the treatment time course, and 99 targets changing at a stricter statistical threshold. Pathway analysis revealed that these changes were overwhelmingly concentrated in immune and infection-related programs: iron regulation, collagen biosynthesis, interleukin-6 signaling, complement and coagulation cascades, and ferroptosis all ranked among the most enriched pathways, even though none of the boys developed serious clinical complications.

The temporal pattern suggested an acute, self-limiting innate antiviral response. Interferon-stimulated genes such as MX1, IFIT3, IFI16, and TLR5 spiked sharply at day 7, coinciding with vector clearance, and largely returned to baseline by day 14. Macrophage markers like HMOX1, LST1, and MARCKSL1, along with neutrophil proteins including MPO, LCP1, and EZR, followed the same trajectory. Complement activation markers C3d, C1QTNF1, and C1S also peaked at day 7, while C1q components dropped at day 14, consistent with consumption during cascade activation. Notably, IL1R1 rose later, peaking at days 60 to 90 during the glucocorticoid taper, suggesting a rebound of interleukin-1 signaling as steroid suppression wore off rather than a direct effect of the virus.

Muscle biomarkers painted a nuanced picture of treatment effect. Sustained declines in TNNI3, a cardiac troponin, and MYL6B, a slow-fiber myosin light chain, hint at protection of heart and slow skeletal muscle. But a striking day 1 spike in titin, TTN, suggested transient muscle damage at the moment of infusion, and the classic damage markers fast troponin, cardiac troponin T, and creatine kinase showed no significant change at all. A newly proposed marker, the ubiquitin-conjugating enzyme UBE2S, declined steadily across the study and may reflect reduced muscle turnover as the transgene takes hold. Meanwhile, organ injury markers HAVCR1, FBP1, and IGFBP7 all peaked at days 60 to 90, and GDF15, a tissue stress signal, rose from day 7 and stayed elevated for at least three months, possibly explaining the vomiting that all participants experienced, since GDF15 is now linked to nausea in pregnancy and chemotherapy.

To test whether these immune signatures translate across species, the team injected dystrophic D2.mdx mice with a matching systemic rAAV dose and examined their livers a week later. Genes central to viral sensing and interferon signaling, including Ifit3, Ifnb, and Mavs, were significantly elevated, confirming that the antiviral response seen in human plasma also occurs in dystrophic liver tissue. The mouse work offers a tractable model for dissecting mechanisms and testing interventions, though the top human hit, MX1, only trended upward in mice, likely because inbred mouse strains carry nonfunctional Mx1 mutations.

The findings establish that robust immune activation is a natural and probably universal consequence of high-dose rAAV therapy, not merely a warning sign of impending toxicity. The key clinical question is what separates the transient, self-limiting response observed here from the sustained immune pathology seen in patients who develop hepatotoxicity, thrombotic microangiopathy, or fatal outcomes. By providing a detailed molecular reference point from a best-case scenario, the study gives clinicians a benchmark for monitoring future patients and points toward interventions, including complement control and anti-GDF15 strategies, that could make systemic gene therapy safer for the children who need it.

Subject of Research: Systemic immune and viral dynamics following intravenous rAAV micro-dystrophin gene therapy in boys with Duchenne muscular dystrophy

Article Title: Systemic responses to intravenous rAAV administration in boys with Duchenne muscular dystrophy

Article References: Harris, B. E., Seldin, B. M., Towne, S. C., Hobson, O. R., Zerpa, E., Prima, V., Sweeney, H. L., & Hammers, D. W. (2026). Systemic responses to intravenous rAAV administration in boys with Duchenne muscular dystrophy. Frontiers in Molecular Medicine, 6, Article 1953867. https://doi.org/10.3389/fmmed.2026.1953867

Image Credits: AI Generated

DOI: 10.3389/fmmed.2026.1953867

Keywords: rAAV gene therapy, Duchenne muscular dystrophy, micro-dystrophin, delandistrogene moxeparvovec, AAVrh74, SomaScan proteomics, interferon response, complement activation, digital PCR, vector dynamics, GDF15, immune response biomarkers

The study, published in Physiological Reports, enrolled twenty experienced male mixed-gas divers, nineteen of whom completed the protocol after one was excluded because of a leaking drysuit and severe cold stress. All were veterans of the sport, averaging around twenty-two years of diving experience and roughly 1,800 lifetime dives, with a median of 150 trimix dives each. Working in water at 15.5 degrees Celsius, buddy teams of two performed identical open-water dives to a mean maximum depth of about 50 metres of seawater, with total dive durations of roughly 147 minutes. Each team paired one diver breathing heliox, composed of 16 percent oxygen and 84 percent helium, with a partner breathing trimix, composed of 16 percent oxygen, 32 percent helium and 52 percent nitrogen. Both used closed-circuit rebreathers, which recycle exhaled gas, scrub carbon dioxide and deliver a warm, humidified mixture at a constant oxygen partial pressure.

The experimental design was elegant in its simplicity. Because each buddy team followed the same profile, with the same depth, bottom time, water temperature, equipment class and oxygen exposure, any differences between the groups could plausibly be attributed to the composition of the inert gas itself. Decompression was managed by the Bühlmann ZHL-16C algorithm built into each diver’s computer, with conservative gradient factor settings, and the ascent schedule was anchored to the heliox diver’s obligations, which ran longer. Oxygen toxicity exposure, tracked by the central nervous system clock, was nearly identical between groups at around 86 to 88 percent of the allowable limit. The researchers hypothesized that the higher helium content, by slashing gas density from 5.8 grams per litre in the trimix group to just 2.7 grams per litre at maximum depth, would ease the work of breathing and improve decompression kinetics, thereby reducing overall physiological stress.

The bubble data told an unexpected story. Using two-dimensional echocardiography at 45 and 90 minutes after surfacing, with an operator blinded to the gas mixture counting bubbles frame by frame across ten cardiac cycles, the team found that vascular gas emboli were detected in every one of the nine heliox divers but in only five of the ten trimix divers. At the 45-minute mark, heliox divers carried a median of 12 bubbles per ten cardiac cycles, compared with zero in the trimix group, a statistically significant difference that vanished by 90 minutes. The timing fits the physics: helium’s low solubility coefficient means it both forms bubbles faster during ascent and washes out faster once the diver is back on the surface. Previous work has suggested that bubble peaks occur around 45 minutes after helium-rich dives, whereas nitrogen-based mixtures tend to peak later, so the true divergence between the groups may have been even sharper than the two sampling points captured.

Crucially, no diver in either group developed symptoms of decompression sickness, and the absolute bubble grades remained low, with few divers reaching the high grades associated with elevated risk. The authors caution that the trimix divers, whose computers prescribed shorter decompression than the shared ascent schedule actually delivered, effectively enjoyed a longer in-water decompression than their algorithms demanded, which could itself have suppressed bubble formation. Commercial dive computers, the researchers note, impose a decompression penalty for helium that is not physiologically well grounded, and comparisons by the US Navy Experimental Diving Unit have even hinted at increased risk with trimix under some conditions. Both groups, in other words, likely dove conservatively, and a more aggressive profile might have amplified the differences.

The lung function results were largely reassuring. Whole-body plethysmography performed within 150 minutes of surfacing showed no significant changes in lung volumes or flow rates, with the exception of a slight decrease in mid-expiratory flow at 50 percent of vital capacity confined to the heliox group, possibly reflecting cold-induced small airway reactivity driven by helium’s high thermal conductivity. Lung diffusion capacity for carbon monoxide declined modestly after the dive, by about 6 to 7 percent, a well-recognized and clinically insignificant finding linked to oxygen exposure and bubble formation, and it did not differ between gases. Lung ultrasound B-lines, transient markers of extravascular lung water, showed a temporal trend in the heliox group but no statistically significant post-dive increase at specific time points. One trimix diver experienced a brief post-dive cough; otherwise respiratory complaints were absent.

The biological readouts added a layer of intrigue. Both groups lost roughly two litres of fluid during their dives, yet hematocrit remained stable, indicating compensatory fluid shifts that preserved intravascular volume. Heart rate variability, a window into cardiac autonomic regulation, was remarkably stable, which the authors attribute to the exceptional experience of their participants and the moderate decompression stress. In the blood, both groups showed a neutrophil-driven leukocytosis and a rise in the neutrophil-derived inflammatory mediator MRP8/14, hallmarks of a dive-induced inflammatory response. But the heliox divers stood out in two respects: their platelet counts rose significantly, the opposite of the platelet consumption typically reported after air dives, and their total hemolytic complement activity and C3c levels dropped, a pattern consistent with complement activation at the gas-liquid interface of circulating bubbles.

These findings challenge the long-standing assumption that helium is inherently gentler on the body. Experimental work outside the hyperbaric setting has suggested helium may have organ-protective, anti-inflammatory properties, reducing platelet activation and vascular injury in animal models. Yet recent human data, and now this field study, hint that helium exposure can promote a more pro-inflammatory profile, possibly by increasing membrane microparticle release under prolonged exposure. The paradox, the authors propose, is that the higher transient bubble burden in heliox divers may have driven a more pronounced thrombo-inflammatory activation that overwhelmed any protective effect of the gas itself, a hypothesis supported by the complement changes and the CCL2 chemokine decline seen only in the heliox group.

The study has honest limitations. The sample was small, gas assignment was not randomized, and each diver performed only one condition, leaving room for unmeasured confounding, particularly given the notorious inter-individual variability in bubble production. No air-diluent control was tested, and bubble measurements at only two time points may have missed the true peak. Still, the real-world conditions, matched buddy teams and blinded bubble counting give the findings unusual ecological validity. For the growing community of rebreather divers pushing into deeper water, the message is nuanced rather than alarming: within conservative limits, both helium-based mixtures were tolerated with minimal subclinical stress, but more helium is not automatically better. The relationship between helium, bubble dynamics, inflammation and the actual risk of decompression sickness remains unresolved, and the authors call for further research to untangle whether the extra bubbles of a heliox dive carry any meaningful clinical cost.

Subject of Research: Comparative physiological effects of heliox and trimix breathing gases on pulmonary function and decompression stress in closed-circuit rebreather diving

Article Title: Heliox versus trimix in closed‐circuit rebreather diving: A comparative study of pulmonary and decompression stress in male divers

Article References: Gouin, E., Orsat, J., Dugrenot, E., Brenner, R. J., Leman, G., Bettacchioli, E., Theron, M., Giroux‐Metges, M.-A., L'Her, E., & Guerrero, F. (2026). Heliox versus trimix in closed‐circuit rebreather diving: A comparative study of pulmonary and decompression stress in male divers. Physiological Reports, 14(19), Article e71129. https://doi.org/10.14814/phy2.71129

Image Credits: AI Generated

DOI: 10.14814/phy2.71129

Keywords: heliox, trimix, closed-circuit rebreather, decompression sickness, vascular gas emboli, diving physiology, helium, inflammation, pulmonary function, complement activation, technical diving, gas density

The numbers behind the argument are striking. Antiphospholipid syndrome, the archetypal autoimmune clotting disorder, affects roughly 40 to 50 per 100,000 people in western populations, with a four-to-one female predominance, and carries an annual incidence of one to two cases per 100,000. Around 20 to 30 percent of patients with systemic lupus erythematosus carry antiphospholipid antibodies, and about 40 percent of those go on to develop secondary antiphospholipid syndrome. Patients who have both lupus and antiphospholipid antibodies face significantly higher risks of clotting and of recurrence than either condition alone. Rheumatoid arthritis, giant cell arteritis, Takayasu’s arteritis, polyarteritis nodosa and ANCA-associated vasculitis all add to the burden, as do immune-driven syndromes such as heparin-induced thrombocytopenia and the vaccine-induced clotting disorder VITT that emerged during the COVID-19 pandemic.

What makes these conditions clinically treacherous is that, unlike inherited clotting tendencies, they can strike any part of the circulation. In antiphospholipid syndrome, venous thromboembolism accounts for 39 percent of first events, but stroke follows at nearly 20 percent, with transient ischaemic attacks and heart attacks behind it. Clots also form in unusual places: the cerebral venous sinuses, the splanchnic veins, even the retina. In a five-year international cohort, the single most frequent recurrent event was stroke, a sobering signal that standard anticoagulation incompletely protects these patients. Microvascular thrombosis and pregnancy complications complete a picture that disproportionately afflicts younger adults, especially women, precisely the people conventional cardiovascular screening tends to overlook.

At the mechanistic heart of the problem lies immunothrombosis, the ancient host-defence programme that uses clot formation to wall off circulating pathogens. When autoantibodies hijack this machinery, the result is pathological thrombo-inflammation. The best-characterised culprit is the family of antiphospholipid antibodies, particularly those directed against beta-2 glycoprotein I. These antibodies bind phospholipid-binding proteins on endothelial cells, monocytes and platelets, triggering tissue factor expression, thrombin generation and platelet aggregation, and creating a sustained hypercoagulable state that spans arteries, veins and the microcirculation.

Platelets themselves turn out to be immune sentinels, and the review places the platelet Fc gamma receptor IIa at the centre of the story. This receptor, the only Fc receptor on platelets, binds the tail of IgG immune complexes. When pathogenic antibodies cross-link it, a signalling cascade through Src family kinases, Syk and phospholipase C gamma 2 rapidly activates the platelet, spurring granule release, integrin activation and aggregation. Activated platelets shed phosphatidylserine-rich microparticles that serve as catalytic surfaces for thrombin generation and express P-selectin, recruiting neutrophils and monocytes. In antiphospholipid syndrome, heparin-induced thrombocytopenia and VITT alike, Fc gamma receptor IIa signalling is the common final pathway converting adaptive immune mischief into arterial thrombosis, a fact already exploited therapeutically through high-dose intravenous immunoglobulin.

Neutrophils add a second layer of amplification. Activated neutrophils cast out neutrophil extracellular traps, web-like lattices of DNA, histones and granular enzymes that normally ensnare microbes but in autoimmunity provide scaffolds for fibrin deposition, activate platelets and injure the endothelium. In lupus and antiphospholipid syndrome, impaired clearance of these traps sustains vascular inflammation. Meanwhile complement fragments C3a and C5a stoke cytokine release and thrombin generation, while the membrane attack complex punches holes in endothelial cells; experimental work shows complement activation is essential to clotting in antiphospholipid syndrome, making complement inhibition a promising strategy for severe disease.

The endothelium itself is transformed from a guardian into an accomplice. Healthy vessels secrete nitric oxide and prostacyclin and display anticoagulant proteins, but inflammatory cytokines such as tumour necrosis factor-alpha, interleukin-1 and interferon-alpha flip the switch: adhesion molecules appear, von Willebrand factor floods out, natural anticoagulants are downregulated, and plasminogen activator inhibitor-1 throttles fibrinolysis. Oxidative stress strips away nitric oxide bioavailability. In lupus and rheumatoid arthritis this same endothelial dysfunction accelerates atherosclerosis, so patients develop premature arterial disease years or decades ahead of schedule. Traditional risk factors then multiply the damage: smoking boosts autoantibody production, obesity feeds chronic low-grade inflammation, and long-term glucocorticoid therapy worsens blood pressure, lipids and insulin resistance.

Paradoxically, several of these conditions cause clotting despite depleting platelets. In immune thrombocytopenia, antibodies against platelet glycoproteins drive the count down, yet patients still clot more often, through microparticle formation, enhanced thrombin generation and endothelial dysfunction. In autoimmune haemolytic anaemia, antibodies destroy red cells, and the liberated iron, procoagulant microparticles, complement activation and nitric oxide depletion together conspire toward thrombosis. Even antibodies against the natural anticoagulants protein C and protein S have been described, producing catastrophic syndromes such as purpura fulminans and symmetrical peripheral gangrene. The lesson, the authors stress, is that thrombosis in strange contexts or without traditional risk factors should prompt a search for an immune cause.

Diagnosis and risk stratification remain stubbornly difficult because conventional tools are calibrated to ordinary populations. Framingham and SCORE2 scores systematically underestimate vascular risk in inflammatory disease, which is why several rheumatology societies recommend adjustment factors. Laboratory assessment centres on antiphospholipid antibody testing, where persistence on repeat testing after at least twelve weeks confirms clinical significance, and so-called triple positivity marks particularly high recurrence risk. Anticoagulation strategy is similarly nuanced: vitamin K antagonists remain the standard for thrombotic antiphospholipid syndrome because randomised trials showed rivaroxaban and other direct oral anticoagulants carry unacceptably high arterial recurrence rates in triple-positive patients, though carefully selected low-risk patients intolerant of warfarin may still qualify. Hydroxychloroquine, with its platelet-calming and endothelial-protecting effects, statins, aggressive inflammation control and rigorous management of blood pressure, lipids and glucose all form part of the preventive package, alongside minimising cumulative steroid exposure.

The review closes with a policy prescription that amounts to a cultural shift. The authors call for European health systems to designate autoimmune diseases as cardiovascular risk-enhancing conditions, embedding cardiovascular prevention within routine rheumatology and haematology care rather than leaving it to cardiology alone. Multidisciplinary pathways linking primary care, rheumatology, haematology, cardiology and obstetrics, supported by harmonised registries and autoimmune-specific risk prediction tools, would allow earlier detection of high-risk patients through blood pressure, lipid and glucose monitoring, vascular imaging and structured screening of carriers of antiphospholipid antibodies. The economic stakes are substantial: venous thromboembolism costs the United States an estimated seven to ten billion dollars annually, and stroke cost 32 European countries roughly 31 billion euros in 2017, with the autoimmune contribution disproportionately large because patients are young and comorbidities multiply. Emerging therapies, from CD19-directed CAR-T cells to bispecific antibodies that deplete the plasma cells making pathogenic antibodies, hint that in the future the source of the problem, not just its downstream clot, might finally be silenced.

Subject of Research: Mechanisms, epidemiology and prevention of autoimmune-associated thrombosis

Article Title: Autoimmune-associated thrombosis: mechanisms, population burden, and prevention strategies

Article References: Arachchillage, D. J., Preece, M. V., & Laffan, M. (2026). Autoimmune-associated thrombosis: mechanisms, population burden, and prevention strategies. The Lancet Regional Health – Europe, 70, Article 101850. https://doi.org/10.1016/j.lanepe.2026.101850

Image Credits: AI Generated

DOI: 10.1016/j.lanepe.2026.101850

Keywords: autoimmune disease, thrombosis, antiphospholipid syndrome, systemic lupus erythematosus, immunothrombosis, endothelial dysfunction, neutrophil extracellular traps, complement activation, VITT, heparin-induced thrombocytopenia, cardiovascular risk, anticoagulation

The liver has long been considered the most difficult organ to transplant across species. Unlike the heart or kidney, whose failures can be bridged by dialysis or mechanical support, the liver performs hundreds of interlocking tasks: synthesising clotting factors and albumin, clearing toxins and ammonia, producing bile, and orchestrating immune surveillance through its unique sinusoidal vasculature. Previous pig-to-human attempts relied on auxiliary transplantation, in which the pig liver was added alongside the recipient’s own liver rather than replacing it, providing partial metabolic support without fully testing the xenograft’s capacity. Earlier work, including a 2024 case in which a gene-edited pig liver supported a patient for ten days, and a 2025 report of gene-modified pig-to-human liver xenotransplantation by the same Chinese group, demonstrated feasibility but left open the question of whether an orthotopic graft, placed where the human liver once sat, could sustain life-sustaining function on its own.

Central to the new study was the donor animal itself. The team used a pig engineered with six genetic modifications, or 6-GE, designed to blunt the three-pronged human immune attack on pig tissue. Three genes were knocked out: GGTA1, CMAH and B4GALNT2, which encode enzymes that decorate pig cell surfaces with carbohydrate antigens, including the galactose-alpha-1,3-galactose epitope, N-glycolylneuraminic acid and the Sd(a)-like antigen, against which humans carry preformed natural antibodies. Three human genes were added: CD46 and CD55, which regulate complement activation at the cell surface, and thrombomodulin (THBD), an anticoagulant protein that helps convert protein C to its activated form and dampens the inflammatory coagulation cascade. Polymerase chain reaction and Sanger sequencing confirmed the edits at the genomic level, and screening found no evidence of porcine endogenous retrovirus transmission or microchimerism in the recipient’s tissues during the observation window.

The recipient was a brain-dead individual, a model that allows researchers to evaluate xenograft function under genuine human physiology while respecting the ethical framework of organ donation. Brain death, however, is a double-edged sword: the dying brain triggers a storm of systemic inflammation and endothelial injury that can itself destabilise haemodynamics and complicate interpretation of graft outcomes. The surgical team performed the orthotopic transplant, removing the native liver and connecting the pig organ to the recipient’s portal vein, hepatic artery, hepatic veins and biliary system. Preoperative ultrasound confirmed that the diameters of the donor and recipient portal veins and hepatic arteries were compatible, a practical prerequisite that is far from trivial given the size and anatomical variability of both human and porcine vasculature.

Over 11 days of continuous monitoring, the xenograft demonstrated that a pig liver can execute core hepatic functions in a human host. The graft produced albumin continuously and generated bile with normal composition, findings that indicate functioning hepatocytes and an intact biliary epithelium, even though the absolute quantities remained below what a healthy human liver would deliver. Metabolomic analyses, deposited in a public database under accession OMIX017282, tracked the biochemical interplay between graft and host. Systemic haemodynamic stability was maintained throughout, meaning the recipient did not require escalating vasopressor support attributable to graft failure, and the pig liver did not provoke the catastrophic collapse that has historically accompanied cross-species organ transplants.

Yet the study is equally notable for what went wrong, and the authors are candid about it. Imaging and functional assessment revealed suboptimal hepatic perfusion arising from microthrombosis, the formation of microscopic clots within the graft’s sinusoidal vessels, which the researchers believe contributed substantially to the late-phase decline in hepatic function. Coagulation abnormalities emerged as progressive thrombocytopenia, a falling platelet count, alongside reduced activity of clotting factors. This pattern echoes the thrombocytopenia and coagulation dysregulation seen in pig-to-baboon liver transplants and in the landmark pig-to-human heart transplants, and it points to a fundamental incompatibility between pig endothelium and the human coagulation system that current genetic engineering has only partially resolved. The liver’s consumption of platelets and clotting factors, combined with impaired thrombomodulin-mediated protein C activation in the face of endothelial stress, appears to drive a vicious cycle of microvascular clotting and bleeding risk.

The immunological portrait drawn from histopathology was in some respects encouraging. Examination of biopsy and autopsy tissue found scant infiltration by T cells and B cells and minimal deposition of immunoglobulin G, suggesting that the adaptive immune response, the arm of immunity responsible for chronic cellular rejection, was largely held in check by the genetic modifications and the targeted immunosuppressive regimen. Instead, the dominant pathology was innate: prominent activation of innate immune cells and evidence of immunoglobulin M-mediated complement activation, the rapid, antibody-triggered cascade that has historically destroyed unmodified pig organs within minutes. That the classical hyperacute rejection was avoided, while a slower innate-driven injury emerged, marks a shift in the nature of the problem rather than its elimination.

The authors argue that these findings define the research agenda for the next phase of clinical translation. Endothelial damage, microthrombosis and coagulopathy now stand out as the critical challenges, and addressing them will likely require additional genetic modifications, such as further human anticoagulant and complement-regulatory transgenes, together with refined immunosuppressive protocols tailored to the liver’s distinctive immunology. The study also underscores the value of the brain-dead recipient model, which generates clinically relevant data without exposing a living patient to an unproven therapy, even as it cautions that the inflammatory milieu of brain death may exaggerate some forms of injury. Metabolomics, serial histology and comprehensive coagulation profiling of the kind reported here give transplant teams a granular, time-resolved picture of how a xenograft fails, information that no animal model can fully replicate.

For the field at large, the experiment represents a careful step forward rather than a breakthrough cure. Global shortages of donor livers leave millions of patients with end-stage liver disease facing limited options, and pig organs, which can be gene-edited, pathogen-screened and produced on demand, remain the most credible long-term solution. The demonstration that a six-gene-edited pig liver can sustain albumin and bile production and haemodynamic stability for 11 days in a human body, while the precise mechanisms of its gradual functional decline are documented at molecular resolution, converts xenogeneic liver transplantation from a speculative concept into an engineering problem with identifiable targets. Whether the next iteration of gene-edited donors and immunosuppressive strategies can extend graft survival from days to months, and ultimately to the years a clinical therapy would demand, will be decided by the coagulation and innate immune pathways this study has now brought into sharp focus.

Subject of Research: Orthotopic xenotransplantation of a six-gene-edited pig liver into a brain-dead human recipient

Article Title: Orthotopic liver xenotransplantation from gene-modified pig to decedent human

Article References: Tao, K., Yang, Z., Zhang, X., Zhang, H., Lin, Z., Yue, S., Yang, Y., Song, W., Wang, D., Liu, Z., Li, H., Chen, Y., Zhou, J., Ding, R., Sun, S., Yu, M., Li, J., Duan, W., Wang, Z., … Dou, K. (2026). Orthotopic liver xenotransplantation from gene-modified pig to decedent human. Nature Biomedical Engineering. https://doi.org/10.1038/s41551-026-01797-2

Image Credits: AI Generated

DOI: 10.1038/s41551-026-01797-2

Keywords: xenotransplantation, pig liver transplant, gene-edited pigs, orthotopic liver transplantation, brain-dead recipient, microthrombosis, coagulopathy, complement activation, thrombocytopenia, albumin production, bile production, organ shortage

The study, led by María Payá, Gemma Serrano-Heras, and Tomás Segura of the General University Hospital of Albacete and collaborators across Spain, compared cerebral thrombi from six pre-Omicron COVID-19 stroke patients with those from six matched non-infected controls treated at the same institutions. All patients had experienced acute ischemic stroke due to large vessel occlusion and underwent endovascular thrombectomy, during which the retrieved clot material was carefully preserved for research. Although the cohort was small, the breadth of analytical techniques applied to each thrombus was unusual: whole-slide digital imaging quantified immune cells, platelets, and red blood cells; transmission electron microscopy revealed clot architecture at nanometer resolution; liquid chromatography–tandem mass spectrometry with SWATH acquisition profiled more than 1,200 proteins; and total reflection X-ray fluorescence measured fifteen chemical elements.

Clinically, the contrast between the two groups was sobering. Twenty-four hours after treatment, COVID-19 patients had significantly worse neurological scores than controls, and at ninety days their functional outcomes, measured by the modified Rankin Scale, were markedly poorer. Cryptogenic stroke, in which no clear cause is identified, was the most common subtype in both groups. Routine blood counts showed no overt anemia or thrombocytopenia, but COVID-19 patients tended toward higher monocyte counts, elevated hemolysis indices, increased ferritin, and lactate dehydrogenase levels above the normal range, subtle laboratory signals hinting at red blood cell injury and iron release that had gone largely unrecognized at stroke presentation.

Under the microscope, the COVID-19 clots looked fundamentally different. Immunostaining for citrullinated histone H3 and myeloperoxidase showed similar burdens of neutrophil extracellular traps and neutrophils in both groups, but COVID-19 thrombi contained a trend toward more macrophages displaying a diffuse, scattered CD68 staining pattern suggestive of a hyperactivated state. Platelet content, quantified by CD61 staining, was reduced by roughly a third in the infected clots. Histology revealed an extracellular fibrillar matrix that was irregular and less densely packed, and electron microscopy confirmed a disordered ultrastructure: instead of the well-organized peripheral fibrin meshwork enclosing packed cellular regions seen in control clots, COVID-19 thrombi showed fibrillar material strewn chaotically through the section, along with numerous membrane protrusions resembling the filopodia of highly activated platelets.

The most striking discovery emerged from elemental and proteomic analysis. The COVID-19 clots had a distinctive dark-red, brownish coloration that persisted even in protein extracts, a visual clue typically pointing to heme pigments. Total reflection X-ray fluorescence confirmed markedly elevated iron concentrations: more than 80 percent of the COVID-19 thrombi exceeded nine sulfur-normalized units of iron, compared with fewer than 30 percent of controls, while all other measured elements were unchanged. Crucially, the excess iron could not be explained by a simple excess of red blood cells, because glycophorin A immunostaining showed comparable erythrocyte content in both groups.

Proteomics then revealed where that iron was coming from. Of 720 proteins shared between the two groups, 48 were significantly differentially expressed, and among the 28 upregulated in COVID-19 clots were the alpha, beta, gamma, and delta hemoglobin subunits, haptoglobin, biliverdin reductase B, and carbonic anhydrase 1, a signature of free hemoglobin and heme degradation. Because erythrocyte structural proteins such as spectrin, ankyrin-1, and band 3 were not differentially expressed, the hemoglobin within the COVID-19 thrombi appears to have been largely liberated from its cellular packaging, circulating in a cell-free form before being swept into the growing clot. The researchers also found proteins found exclusively in COVID-19 clots: classical complement components including C1QB, C1QC, and C1S, acute-phase reactants such as C-reactive protein and serum amyloid proteins, ubiquitin–proteasome components, and immunoglobulin chains, together painting a picture of intense immune-inflammatory activation and oxidative stress inside the infected thrombi.

How does free hemoglobin promote clotting? The authors draw on a substantial literature showing that cell-free and oxidized hemoglobin are potent procoagulant molecules. Free hemoglobin scavenges nitric oxide, promoting vasoconstriction, and it directly activates platelets by binding glycoprotein Ib alpha, while enhancing von Willebrand factor–mediated platelet adhesion and inhibiting ADAMTS13, the enzyme that normally regulates von Willebrand factor. Heme, its breakdown product, activates endothelial cells through TLR4, stimulates tissue factor expression, and triggers complement deposition on endothelial surfaces. Iron overload further damages endothelial mitochondria through reactive oxygen species. In this framework, the reduced platelet content seen in COVID-19 clots likely reflects platelet consumption and hyperactivation during widespread microthrombus formation rather than a quiet coagulation system, with the surviving platelets showing the morphological stress of filopodia formation observed by electron microscopy.

The source of the free hemoglobin remains an open question. The authors propose that SARS-CoV-2 infection may drive subclinical or compensated hemolysis through cytokine-mediated erythroid injury, complement attack on red blood cells, or direct viral effects on erythrocyte membrane integrity, mechanisms that previous proteomic and lipidomic studies have independently suggested. They caution that their observational design cannot prove causation, that the pre-Omicron viral strains studied may differ from current variants, and that the small sample size and selection of only fully analyzable thrombi impose limitations. Notably, the proteomic signature tracked infection status rather than stroke etiology, suggesting that the hemoglobin-iron enrichment is a distinctive feature of COVID-19 clots independent of the underlying embolic source.

Still, the implications are considerable. Retrieved thrombi are thought to preserve at least part of the molecular milieu present at the moment of clot formation, and the convergence of hemolysis markers, complement activation, antioxidant responses, and platelet depletion in these clots offers a coherent mechanistic account of COVID-19 immunothrombosis that goes beyond the traditional triad of endothelial injury, immune activation, and coagulopathy. If cell-free hemoglobin and iron indeed help drive the prothrombotic state, they become candidate therapeutic targets: haptoglobin infusion, heme-scavenging strategies, or iron-chelating approaches could, in principle, blunt thrombotic risk during severe viral infection. The authors call for larger multicenter studies to directly quantify circulating cell-free hemoglobin and iron-handling biomarkers in COVID-19 patients and to test whether targeting these pathways can reduce ischemic complications, including stroke, and improve the response of these unusually stubborn clots to thrombolysis and mechanical removal.

Subject of Research: Cell-free hemoglobin and iron accumulation in cerebral thrombi as drivers of the SARS-CoV-2-induced prothrombotic state in COVID-19 stroke patients

Article Title: High levels of cell-free hemoglobin and iron in cerebral thrombi of pre-Omicron COVID-19 stroke patients: novel drivers of SARS-CoV-2–induced prothrombotic state

Article References: Payá, M., Alcahut-Rodríguez, C., Barbella-Aponte, R. A., Hernández-Fernández, F., Molina-Nuevo, J. D., Barroso-García, G., Castro-Robles, B., López-López, S., Arias-Salazar, L., Ayo-Martín, Ó., García-García, J., Yélamos-Sanz, B., García-Flores, N., Arandilla, A. G., Moreno-Luna, R., Aliena-Valero, A., Vielba-Gómez, I., Tembl, J. I., Salom, J. B., … Segura, T. (2026). High levels of cell-free hemoglobin and iron in cerebral thrombi of pre-Omicron COVID-19 stroke patients: novel drivers of SARS-CoV-2–induced prothrombotic state. Acta Neuropathologica, 152(1), Article 26. https://doi.org/10.1007/s00401-026-03074-7

Image Credits: AI Generated

DOI: 10.1007/s00401-026-03074-7

Keywords: COVID-19, stroke, cell-free hemoglobin, iron, cerebral thrombi, hypercoagulability, immunothrombosis, proteomics, complement activation, platelets, hemolysis, SARS-CoV-2