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Many vertebrate species act as both plant pollinators and seed-dispersers, thus interconnecting these processes, particularly on islands. Ecological multilayer networks are a powerful tool to explore interdependencies between processes; however, quantifying the links between species engaging in different types of interactions (i.e. inter-layer edges) remains a great challenge. Here, we empirically measured inter-layer edge weights by quantifying the role of individually marked birds as both pollinators and seed-dispersers of Galápagos plant species over an entire year. Although most species (80%) engaged in both functions, we show that only a small proportion of individuals actually linked the two processes, highlighting the need to further consider intra-specific variability in individuals' functional roles. Furthermore, we found a high variation among species in linking both processes, i.e. some species contribute more than others to the modular organization of the multilayer network. Small and abundant species are particularly important for the cohesion of pollinator seed-dispersal networks, demonstrating the interplay between species traits and neutral processes structuring natural communities.

ObjectivesTo investigate if quantitative apparent diffusion coefficient (ADC) measurements can predict genetic subtypes of non-gadolinium-enhancing gliomas, comparing whole tumour against single slice analysis.MethodsVolumetric T2-derived masks of 44 gliomas were co-registered to ADC maps with ADC mean (ADCmean) calculated. For the slice analysis, two observers placed regions of interest in the largest tumour cross-section. The ratio (ADCratio) between ADCmean in the tumour and normal appearing white matter was calculated for both methods.ResultsIsocitrate dehydrogenase (IDH) wild-type gliomas showed the lowest ADC values throughout (p < 0.001). ADCmean in the IDH-mutant 1p19q intact group was significantly higher than in the IDH-mutant 1p19q co-deleted group (p < 0.01). A volumetric ADCmean threshold of 1201 × 10−6 mm2/s identified IDH wild-type with a sensitivity of 83% and a specificity of 86%; a volumetric ADCratio cut-off value of 1.65 provided a sensitivity of 80% and a specificity of 92% (area under the curve (AUC) 0.9–0.94). A slice ADCratio threshold for observer 1 (observer 2) of 1.76 (1.83) provided a sensitivity of 80% (86%), specificity of 91% (100%) and AUC of 0.95 (0.96). The intraclass correlation coefficient was excellent (0.98).ConclusionsADC measurements can support the distinction of glioma subtypes. Volumetric and two-dimensional measurements yielded similar results in this study.Key Points• Diffusion-weighted MRI aids the identification of non-gadolinium-enhancing malignant gliomas• ADC measurements may permit non-gadolinium-enhancing glioma molecular subtyping• IDH wild-type gliomas have lower ADC values than IDH-mutant tumours• Single cross-section and volumetric ADC measurements yielded comparable results in this study

[...]the number of permanent black holes (PBH), which reflect irreversible tissue destruction where acute inflammation has settled, was significantly lower among those patients receiving laquinimod. [...]patients on laquinimod had less microscopic demyelination over time than patients receiving placebo as reflected by the magnetisation transfer ratio-derived parameters.

Background:CD19-targeting CAR T cells showed remarkable improvements in autoimmune diseases including refractory lupus erythematodes [1,2] and inflammatory myopathy [3] and first experiences in patients with dcSSc have been reported [4,5].Objectives:Here we present up to 1-year follow-up data of six patients with severe dcSSc who received CD19-targeting CAR T cell treatment. The aim of this study was to assess the effects of deep B-cell depletion using CD19 CAR T cells on the disease course of fibrotic skin and organ involvement, autoimmunity and vascular phenomena one year after CD19 CAR T cell therapy.Methods:CD19 CAR T cells were acquired as previously described [1]. Immunosuppression was stopped before CAR T cell infusion and the therapy was performed upon lymphodepletion with fludarabine (25 mg/m2 on days -5, -4, -3) and cyclophosphamide (1g/m2 on day -3) as single infusion with 1x106 CAR T cells/kg. Patients were followed up for a 12 months period with the following assessments being performed: mRSS, EUSTAR activity index, patient reported outcomes, FVC, extent of ILD on CT scans and 68Ga-04-FAPI-uptake. Consecutive skin biopsies were taken before CAR T cell therapy and in regular intervals during one year follow up.Results:Six patients (4 male, 2 female) with diffuse and progressive SSc, who had failed state of the art therapies, were treated with CD19-targeting CAR T cells. The median age was 42 (IQR 36,25 – 53) with a median disease duration of 36 months (IQR 24,75 – 45,75). At baseline, patients were positive for either anti-Scl-70 antibodies (5/6) or anti-RNAP-III antibodies (1/6). After CAR T therapy, mRSS decreased by around 30-45% within the first 3 to 4 months and remained stable or decreased even further by up to 60% during one year follow up. Raynaud’s syndrome improved and digital ulcerations were less frequent to absent. Histologically, we observed reduced counts of FAP positive fibroblasts. Moreover, the papillary skin structure tended to shift to a more physiological phenotype with reduced collagen alignment and an increased number of papillae. Serologically, ANA titers showed an up to 10-fold decrease on three months follow up and declined further during the following 9 months. Anti-RNAP-III antibodies disappeared early after CAR T application and were not detectable during all follow up appointments, anti-Scl70 persisted on immunoblot with regressive titers as analyzed by ELISA. Lung function parameters remained stable in all patients with ILD during 1 year follow up, 68GA-FAPI-04-uptake as a potential marker of fibrotic disease activity decreased within three months and remained low during the whole year follow up in the lung and in the heart.Conclusion:These data on the first six SSc patients receiving CD19-targeting CAR T cell therapy show that the procedure can lead to stabilization of SSc disease activity without additional immunosuppression during a period of up to 1 year after treatment. Histological analysis furthermore show structural changes in the skin after therapy that could indicate a shift to a more physiological phenotype. Further mechanistic studies are ongoing.REFERENCES:[1] Mackensen et al. Anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus. Nat Med. 2022 Oct;28(10):2124-2132. doi: 10.1038/s41591-022-02017-5.[2] Mougiakakos et al. CD19-Targeted CAR T Cells in Refractory Systemic Lupus Erythematosus. N Engl J Med. 2021 Aug 5;385(6):567-569. doi: 10.1056/NEJMc2107725.[3] Müller et al. CD19-targeted CAR T cells in refractory antisynthetase syndrome. Lancet. 2023 Mar 11;401(10379):815-818. doi: 10.1016/S0140-6736(23)00023-5.[4] Bergmann et al. Treatment of a patient with severe systemic sclerosis (SSc) using CD19-targeted CAR T cells. Ann Rheum Dis. 2023 Aug;82(8):1117-1120. doi: 10.1136/ard-2023-223952.[5] Merkt et al. Third-generation CD19.CAR-T cell-containing combination therapy in Scl70+ systemic sclerosis. Ann Rheum Dis. 2023 Dec 22:ard-2023-225174. doi: 10.1136/ard-2023-225174. Epub ahead of print. PMID: 38135464.Acknowledgements:NIL.Disclosure of Interests:None declared.

Background:Autoimmune diseases such as systemic lupus erythematosus (SLE), idiopathic inflammatory myositis (IIM) and systemic sclerosis (SSc) often require long-term immune suppression. In some cases, the disease can be severe, progressive and does not adequately respond to standard immunosuppressive drug treatment. We and others have previously shown that administration of autologous CD19 chimeric antigen receptor (CAR) T cells can induce remission or at least major improvement in patients with SLE, IIM and SSc. Resetting aberrant autoimmunity in these diseases through deep depletion of B cell is a potential strategy for achieving sustained drug free remission in these diseases.Objectives:To investigate the long-term safety and efficacy of autologous CD19 CAR T cell therapy in in patients with SLE, IIM and SSc.Methods:CD19 CAR-T cells were prepared from leukapheresis specimen after enrichment of T cells and transfection with MB-CART19.1 lentiviral vector (Miltenyi). Cells were expanded for 12 days and 1 million CAR T cells/kg body weight were administered as a single intravenous infusion after standard conditioning therapy with 1g/m2 cyclophosphamide and 3x 25 mg fludarabine, as described previously [1,2]. Safety parameters, including cytokine-release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), myelotoxicity and infections, were recorded up to 3 years post CAR-T cell infusion. Efficacy was assessed using the Definition of Remission in SLE (DORIS), clinical response according to 2016 ACR/EULAR total improvement score (TIS) and the European Scleroderma Trials and Research Group (EUSTAR) activity index up to three years after CAR-T cell infusion.Results:Between February 2021 and May 2023, 15 patients (8 SLE, 4 SSc, and 3 IIM) were treated with CD19 CAR-T cells. Among them were ten women and five men. Patients were mostly young (median age 36 years; range [18-60] years). Median disease duration before CAR-T was 3 years [1-20]. All patients failed on multiple previous immune suppressive treatments (median 5 [2-14]). Median follow up time after CD19-CAR-T cell infusion was 18 months [8-32] with 5 patients followed-up over more than two years and 12 patients followed-up over more than one year. Regarding safety, no higher grade CRS was observed. Grade 1 CRS occurred in 8 patients, grade 2 CRS in 1 patient. Only a single grade 1 ICANS was observed. No acute myelotoxicity (grade 3 or 4 neutropenia or leucocytopenia > 28 days post CAR-T cell treatment) occurred. One patient, however, developed late-stage grade 4 neutropenia 120 days after CD19CAR-T cell treatment, which resolved upon G-CSF treatment. With respect to infections only one case of severe infection (pneumonia) requiring hospital admission was recorded, which required antibiotic treatment. All other infections were mild and mostly manifested as upper respiratory tract infections. With respect to efficacy, all SLE patients achieved DORIS remission, all IIM patients reached ACR/EULAR major response and all SSc patients improved in the EUSTAR activity index. Immunosuppressive therapy including glucocorticoids was completely stopped in all patients.Conclusion:This case series provides first evidence for the long-term safety and efficacy of CD19 CAR-T cell treatment in three different autoimmune diseases, providing the rationale for further controlled clinical trials.REFERENCES:[1] Mougiakakos D et al., CD19-Targeted CAR T Cells in Refractory Systemic Lupus Erythematosus. N Engl J Med 2021;385:567-569.[2] Mackensen A. et al., Anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus; Nat Med 2022 Oct; 28(10):2124-2132.Acknowledgements:NIL.Disclosure of Interests:None declared.

Background:Treatment with autologous CD19 chimeric antigen receptor (CAR) T cells induces deep depletion of B cells in humans. This potential may allow resetting aberrant B-cell immunity in patients with autoimmune diseases such as systemic lupus erythematosus (SLE), idiopathic inflammatory myositis (IIM) and systemic sclerosis (SSc).Objectives:To investigate the effects of CD19 CAR-T cell treatment on B cell immunity in patients with SLE, IIM and SSc.Methods:Spectral analyzer-based characterization of B cell subsets in 8 SLE, 2 IIM and 2 SSc patients at baseline, early reconstitution (EaR) 4 months and established reconstitution (EsR) one year after CD19 CAR-T cell therapy. High-throughput single-cell mRNA sequencing of B cells with an Illumina NovaSeq platform in 5 SLE patients before and one year after CD19 CAR-T cell therapy. VH repertoire analysis of B cells based on VDJ library preparation and Illumina MiSeq sequencing in each one patient with SLE, IIM and SSc one year after CD19 CAR-T cell therapy and comparison with VH repertoire of two healthy controls.Results:Characterization of B cell subsets revealed that reconstituted B cells at EaR and EsR showed a naïve phenotype, while CD19+CD27+ memory B cells were drastically reduced. A limited increase in CD19+CD27+ memory B cells occurred between EaR and EsR, which was predominantly seen in pre-switched IgD+ CD27+ B cells. CD27+CD38+ plasmablasts and SLE-associated activated CD11c+ memory B cells disappeared in SLE patients after B cell reconstitution. Immature CD38+ B cells, indicating reconstitution of B cells from the bone marrow boosted at EaR and declined thereafter. Similar results were also seen in at EaR an EsR of B cells in IIM and SSc. Single cell sequencing-based analysis of heavy chains in SLE patients showed virtual disappearance of IGHG1, IGHG2, IGHG3, IGHG4, IGHA1 and IGHA2 chains, while the expression of IGHM and IGHD increased, resembling a non-class switched B cell receptor phenotype. The expression of distinct chains associated with SLE and autoimmunity, such as immunoglobulin kappa variable (IGKV) 4-1, immunoglobulin heavy variable (IGHV) 4-59 chain and immunoglobulin lambda variable (IGLV) 3-21 chain was downregulated. In addition, a broad and balanced VH – JH usage was found in SLE, IIM and SSc patients treated with CAR-T cells without any noticeable clonal expansions, very similar to a normal B cell repertoire of naïve B cells from healthy controls. Analysis of the frequency of somatic mutations in the VH genes revealed that all IgM sequences had a very low mutation frequency similar to a repertoire from sorted naïve cells from healthy controls.Conclusion:Altogether the appearance of a naïve non-class-switched B cell system, the disappearance of circulating plasmablasts, the down regulation of specific disease- associated heavy and light chains and the broad and balanced VH – JH usage support the concept that CD19 CAR-T cell therapy may have induced a reset of pathologic B cell autoimmunity in patients with systemic autoimmune disease.REFERENCES:[1] Mougiakakos D et al., CD19-Targeted CAR T Cells in Refractory Systemic Lupus Erythematosus. N Engl J Med 2021;385:567-569.[2] Mackensen A. et al., Anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus; Nat Med 2022 Oct; 28(10):2124-2132.Acknowledgements:NIL.Disclosure of Interests:None declared.

Background:Bi-specific T-cell engagers (BiTEs) kill B cells by engaging T cells. BITEs are highly effective in acute lymphoblastic leukemia but have not yet been tested in autoimmune disease. Multi-drug resistant rheumatoid arthritis patients represent a considerable challenge and require new approaches to treat their underlying autoimmune pathology.Objectives:Based on the action of BiTEs to kill B cells by engaging T cells as effectors we hypothesized that this principle may work also in the treatment of B cell mediated autoimmune diseases including RA.Methods:We treated six patients with multi-drug resistant rheumatoid arthritis (RA) with two cycles of the CD19xCD3 BiTE blinatumomab via a compassionate use program. Eligibility criteria were based on (i) a diagnosis of RA according to the ACR/EULAR 2010 criteria, (ii) evidence for B cell involvement based on positivity of rheumatoid factor (RF) or anti-citrullinated protein antibodies (ACPA) or presence of B cells in the synovial membrane, (iii) active disease with a disease activity score (DAS) 28 over 3.2 units and (iv) treatment resistance to methotrexate and at least three different targeted synthetic (ts) or biologic (b) disease modifying anti-rheumatic drugs (DMARDs). Changes in circulating T and B cells were measured by high dimensional spectral flow cytometry and bioinformatic analysis. Serum cytokines were measured by solid-phase chemiluminescence assay. Ultrasound was performed in all patients at baseline and at 12 week follow-up and scored according to EULAR-OMERACT. Fibroblast activation protein inhibitor (FAPI)-based positron emission tomography/computed tomography (PET-CT) was performed in one patient at baseline and follow up. Ultrasound guided synovial biopsy and immunohistochemistry for quantification of B cells and plasma cells was performed in one patient at baseline and follow up.Results:In all six patients, blinatumomab therapy was safe and well tolerated, with brief increase in body temperature and acute phase proteins during first infusion but no signs of clinically relevant cytokine-release syndrome.Documenting the T cell engagement function, blinatumomab induced a transient reduction of CD4+ and CD8+ T cells in the peripheral blood. Blinatumomab substantially depleted B cells in the peripheral blood. High dimensional analysis documented an immune reset with depletion of activated memory B cells, which were replaced by non-class switched, IgD positive naïve B cells. Blinatumomab lead to a rapid decline in RA clinical disease activity (mean DAS28-CRP 4.72 to 2.28 units), improved synovitis in ultrasound and FAPI-PET-CT and reduced autoantibodies. Synovial biopsy before and after two cycles of blinatumomab therapy documented a successful clearance of CD19+ B cells, CD20+ B cells and even CD138+ plasma cells in the synovial tissue, suggesting that pathologic B cell activation is interrupted.Conclusion:These data demonstrate the principle feasibility to effectively treat B cell mediated autoimmune disease with BiTEs.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of Interests:None declared.

Background:Systemic autoimmune diseases are based on an aberrant activation of B cells. Autologous CD19 chimeric antigen receptor (CAR) T cells allow deep depletion of B cells in humans and represent a new possibility to treat autoimmune disease. Previous observations have suggested that a single infusion of CD19-CAR-T cells is not only well tolerated in patients with SLE and other autoimmune diseases but also induces sustained drug-free remission [1-3]. However, safety and efficacy of CD19-CAR-T cell therapy in autoimmune disease has to be demonstrated in controlled clinical studies.Objectives:To assess the safety and preliminary efficacy of CD19-CAR-T therapy in autoimmune diseases in a controlled clinical study.Methods:CASTLE (CAR-T cells in systemic B cell-mediated autoimmune disease) is a phase I/II basket study that assesses the safety (primary endpoint) and preliminary efficacy (secondary endpoint) of CD19-CAR-T therapy in systemic lupus erythematosus (SLE), idiopathic inflammatory myositis (IIM) and systemic sclerosis (SSc). It consists of a first part with 8 patients followed by a second part with 16 patients. All patients receive standard cyclophosphamide/fludarabine conditioning therapy followed a single infusion of an advanced therapy medicinal product (MB-CART19.1) containing 1x106 CD19-CAR-T cells/kg body weight that were transfected with a lentiviral vector encoding for a 4-1BB based second generation CAR. To be included, patients had to have a diagnosis of SLE, IIM or SSc, (ii) active disease with organ involvement and (iii) failed treatment with a least two immunosuppressive drugs. Safety was assessed by recording cytokine-release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), myelotoxicity and infections during the first 28 days. Preliminary efficacy was assessed by assessing B cell depletion, CAR-T cell expansion and clinical responses.Results:This analysis is on the first part of the CASTLE study comprising 8 patients (5 SLE, 2 SSc, 1 IIM). 6 patients were females, 2 were males. Median age was 33.5 years (range 20-81 years), median disease duration 3 years [range: 1-9 years) and median follow up time 2.4 months (range: 1-5 months). Patients failed on a median number of 4 (range N=3-6) immunosuppressive treatments. From all 8 patients, safety data and B cell/CAR-T cell efficacy data were available, while clinical efficacy data were available from 5/8 patients with sufficiently long follow-up (6 weeks). No higher grade CRS (grade 3 or 4) was observed (grade 0: N=3; grade 1: N=4; grade 2: N=1). No ICANS and no myelotoxicity (grade III/IV neutropenia/leucocytopenia >28 days) were observed. AESI were two late-stage neutropenias that resolved with G-CSF treatment, one flare of SLE before CAR-T cell therapy that required glucocorticoids and two cases of pneumonia (SARS-CoV-2 and CMV) that resolved upon treatment. B cells were completely depleted in all patients within 10 days (Figure 1). CAR-T cells expanded in all patients. Among the 5 patients (3 SLE, 1 SSc, 1 IIM) that had sufficiently long follow up (≥ 6 weeks), three achieved DORIS remission (SLE), one achieved ACR Moderate/Major response (IIM) and one achieved no worsening of lung function (SSc). Furthermore, all patients could successfully stop glucocorticoids and immunosuppressive drugs after CAR-T cell infusion.Conclusion:These data underline the safety of CD19-CAR-T therapy in autoimmune disease. No higher grade CRS or ICANS or no myelotoxicity is observed. Attention has to be given to late-stage neutropenia, exacerbation of the underlying diseases and infections.REFERENCES:[1] Mougiakakos D et al., CD19-Targeted CAR T Cells in Refractory Systemic Lupus Erythematosus. N Engl J Med 2021;385:567-569.[2] Mackensen A. et al., Anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus; Nat Med 2022 Oct; 28(10):2124-2132.[3] Mueller F. et al., A Case Series with Extended Follow-up of CD19 CAR-T cell therapy in Autoimmune Disease; N Engl J Med 2024; in press.Figure 1.Acknowledgements:NIL.Disclosure of Interests:None declared.

Brain structural covariance networks (SCNs) based on pairwise statistical associations of cortical thickness data across brain areas reflect underlying physical and functional connections between them. SCNs capture the complexity of human brain cortex structure and are disrupted in neurodegenerative conditions. However, the longitudinal assessment of SCN dynamics has not yet been explored, despite its potential to unveil mechanisms underlying neurodegeneration. Here, we evaluated the changes of SCNs over 12 months in patients with a first inflammatory-demyelinating attack of the Central Nervous System and assessed their clinical relevance by comparing SCN dynamics of patients with and without conversion to multiple sclerosis (MS) over one year. All subjects underwent clinical and brain MRI assessments over one year. Brain cortical thicknesses for each subject and time point were used to obtain group-level between-area correlation matrices from which nodal connectivity metrics were obtained. Robust bootstrap-based statistical approaches (allowing sampling with replacement) assessed the significance of longitudinal changes. Patients who converted to MS exhibited significantly greater network connectivity at baseline than non-converters (p = 0.02) and a subsequent connectivity loss over time (p = 0.001–0.02), not observed in non-converters’ network. These findings suggest SCN analysis is sensitive to brain tissue changes in early MS, reflecting clinically relevant aspects of the condition. However, this is preliminary work, indicated by the low sample sizes, and its results and conclusions should be treated with caution and confirmed with larger cohorts.

BackgroundSubclinical tenosynovitis arose as a primary sign of inflammation already in the pre-clinical phases of Rheumatoid Arthritis (RA) and tenosynovitis is a common feature of active state and might persist although sustained remission is achieved.ObjectivesTo assess the histological features of (teno)synovium from the extensor digitorum tendons of the hand across RA trajectory and to compare it with paired synovial tissue samples.MethodsThirty-seven patients underwent minimally invasive ultrasound (US)-guided biopsy of the tenosynovium of extensor digitorum tendons [n=16 with clinically suspected arthralgia (CSA), n=19 with RA (n=4 naïve, n=6 resistant to csDMARDS, n=8 to bDMARDs, and n=1 in sustained remission respectively)]. Twenty-three out of 37 patients presenting CSA or clinically evident synovitis in a peripheral joint (whether anatomically adjacent or not to the tendon of interest) underwent US-guided synovial tissue (ST) biopsy also in that location. The Krenn synovitis score (KSS) and the inflammatory infiltrate using CD68/CD21/CD3/CD20 immunohistochemical (IHC) analysis were assessed for each sample.ResultsTenosynovium belonging to the extensor digitorum compartment of the wrist was successfully collected using a minimally invasive US-guided technique that enabled to retrieve representative samples in terms of lining and sublining layers presence in 86.5% and 100% of cases, respectively. Considering the pre-arthritis subgroup, KSS was similar in ACPA/RFpos vs ACPA/RFneg CSA (p=0.5814) and KSS was contingent with RA activity, being significantly higher in naïve or resistant RA (ANOVA p<0.001) compared to CSA. Moreover, the comparison between (teno)synovial and synovial tissue paired samples from two adjacent anatomical locations revealed a similar KSS value (p>0.999) and IHC showed comparable enrichment in terms of lining and sublining CD68pos, sublining CD20pos and CD3pos cells (for all comparisons p>0.05) between the two sites, in naive and c- or b-DMARDs resistant RA patients. The procedure was well tolerated, and no tendon injury occurred within 10 months follow-up. One patient developed local hematoma that recovered after topic treatment.Conclusion(Teno)synovium is a retrievable tissue through a minimally invasive procedure across RA trajectory and its inflammation is directly related to the disease state. Semiquantitative analysis showed comparable inflammatory degree and inflammatory cellular composition in adjacent anatomical sites of the same person.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of InterestsNone Declared.