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Objectives Clinical observations in patients with dermatomyositis (DM) and autoantibodies against the melanoma differentiation–associated protein 5 (MDA5) suggest that the autoantibodies contribute to the pathogenesis of MDA5(+) DM. To gain insight into the role of the anti-MDA5 autoantibodies, we aimed to identify their binding sites on the different domains of the MDA5 protein. Methods We developed an in-house ELISA to assess the reactivity against the MDA5 domains (conformational epitopes) in plasma (n  = 8) and serum (n  = 24) samples from MDA5(+) patients with varying clinical manifestations and disease outcomes. The reactivities were also assessed using western blot (linearized epitopes). An ELISA-based depletion assay was developed to assess cross-reactivity among the different MDA5 domains. Results All eight plasma samples consistently showed reactivity towards conformational and linearized epitopes on the helicase domains of the MDA5 protein. The ELISA-based depletion assay suggests that anti-MDA5 autoantibodies specifically target each of the three helicase domains. Twenty-two of the 24 serum samples showed reactivity in the in-house ELISA and all 22 displayed reactivity towards the helicase domains of the MDA5 protein. Conclusions Our data revealed that the main immunogenic targets of anti-MDA5 autoantibodies from MDA5(+) patients are the helicase domains. Considering that the helicase domains are responsible for the enzymatic activity and subsequent triggering of an inflammatory response, our findings suggest that binding of anti-MDA5 autoantibodies could alter the canonical activity of the MDA5 protein and potentially affect the downstream induction of a pro-inflammatory cascade. [ABSTRACT FROM AUTHOR]

Unlike current therapy for refractory ILD in antisynthetase syndrome, such as rituximab or cyclophosphamide, CD19-targeting CAR T-cell therapy may lead to B-cell depletion, potentially breaking the crosstalk between B- and T-cell responses. Although CD19-targeting CAR T-cell therapy seemed highly efficient in a patient with antisynthetase syndrome that was refractory to conventional treatment, this approach has some limitations. Thus, B-cell suppression with CD19-targeting CAR T-cell therapy combined with T-cell inhibition could be an option to achieve disease remission in refractory antisynthetase syndrome. [Extracted from the article]

Delivering labile CpG ODNs within liposomes are advantageous. Liposomes protects the cargo from premature digestion, furthermore, it retains ODN activity/stability until it reaches relevant target cells. Although both are strictly dependent on TLR9, two structurally distinct classes of CpG ODNs are capable of activating human pDCs differentially. While K-type ODN trigger pDCs to differentiate, mature and secrete TNF-α, D-type ODN initiate IRF-dependent IFN-α production. Strikingly, when K- and D-type ODNs are co-incubated in their free forms, K-ODN masks the activity of D-ODN. However, to induce CpG ODN dependent-immunotherapy both K- and D-specific immune activations are indispensable. In this study, we hypothesized that encapsulating K-type and D-type CpG ODN in different liposomes could modify their subcellular distribution and lead to a synergistic immune activation rather than antagonistic obstruction. Data revealed that simultaneous stimulation with neutral D-ODN loaded liposomes along with K-ODN loaded within cationic liposomes improved rather than masking D-specific IFNα production from PBMCs. Of ~90% of all PBMCs reproduced this activation (n=10 donors). Intracellular cytokine staining and ELISPOT studies supported this heightened immune activation (TNF-α and IFN-α) of liposomal combinations of K- and D-type CpG ODNs. Similar findings were detected in mice experiments. Furthermore, the liposomal K- and D-ODN combinations were used in immunization studies against ovalbumin (OVA). Results implicated that when either neutral (ND) or anionic liposomal (AD) D-ODN is combined either with sterically stabilized cationic liposomal K (SSCL-K) or cationic- K-ODN (CK), anti OVA-specific IgG2a/IgG1 responses were over 1.0, thereby a pronounced Th1-biased anti-OVA immunity was established. To check whether this improved Th1-immunity is specific enough to control OVA-dependent B16 melanoma development in mice we next challenged immunized animals with OVA expressing B16 tumor cells. Only mice that were immunized with ND+SSCL-K vaccine combination, had the lowest tumor size (145.7±87.4) compared to naïve (1372.8±840.6) or unencapsulated D+K (410.9±287.1) immunized groups @ D=15 post-tumor challenge. This study demonstrated that superior features of D and K type CpG ODN could be combined if suitable liposomes were employed for loading, and extends the breadth of CpG ODN-dependent immunotherapy which otherwise is not possible. Overall, our data implicate that an effective preventive liposomal cancer vaccine against melanoma could be developed. [ABSTRACT FROM AUTHOR]

Exosomes are a class of naturally occurring nanovesicles that are secreted virtually by all cell types. Recent studies revealed that due to their low toxicity/immunogenicity as well as their nanosize, exosomes are excellent vehicles for protein/peptide drug, gene or even oligonucleotide delivery. Targeted drug delivery proved to be a critical topic in achieving efficient cancer therapy. In recent decades, endogenous vehicles, such as protein and polysaccharide nanoparticles were explored extensively for drug delivery to obtain better therapeutic effects because of their low toxicity and biocompatibility. Here, we describe a mild method to externally load magnetic iron oxide nanoparticles (FeO NPs) within exosomes. The modified exosomes could be driven more effectively to target cells under magnetic field, thereby expand exosome directed therapy opportunities. Exosomes were loaded with different doses of FeO NPs via rehydration-dehydration method. Internalization of FeO NP loaded and unloaded exosomes were conducted both under magnetic and no-magnetic field at various time intervals. In some experiments in addition to FeO NP loading D-type CpG ODN was also co-encapsulated within exosomes and internalization as well as stimulatory activities were analyzed either on RAW264.7 macrophages or on spleen cells. Data revealed that exosome uptake by macrophages improved upto 35 fold in a dose dependent manner in magnetic field compared to exosomes devoid of FeO NP within 30 minutes of incubation. Moreover, co-encapsulation of D-type CpG ODN with FeO NP within exosomes significantly increased ODN internalization by 30 minutes of incubation especially at lower ODN dose in comparison to FeO NP free form of exosomes. Immunostimulatory activities of CpG ODN under magnetic field following co-encapsulation within exosomes along with FeO NP found to be significantly magnified in addition to rapid cytokine induction rate. In conclusion, this study showed that FeO NPs increased internalization of exosomes, thereby facilitated rapid and intense delivery of its cargo to target cells under magnetic field. Loading FeO NPs within exosomes offers an effective theranostic approach for developing targeted exosomes that could sustain their translational impact. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs), secreted by almost all living cells, have gained significant attention for their role in intercellular communication and their potential as versatile carriers for biotherapeutics. However, the clinical translation of EV-based therapies faces significant challenges, primarily due to the lack of efficient methods for loading biotherapeutic agents into EVs. This study introduces a simple, reproducible strategy for the simultaneous incorporation of various biotherapeutics within EVs. The process is gentle and preserves the essential physicochemical and biological characteristics of EVs, thereby protecting labile ligands from premature degradation and elimination. The binding and uptake efficiency of EVs by target cells reached approximately 97 % within 24 h of incubation. Administration of EVs loaded with oligodeoxynucleotides (ODN) resulted in a 4-fold increase in IFNγ + CD4 + T cells and a 5-fold increase in IFNγ + CD8 + T cells in the spleens and lymph nodes. Additionally, the co-administration of EVs with ODN and ovalbumin (OVA) induced elevated Th1-biased antibody responses and antigen-specific cytotoxic T-cell responses, providing long-lasting complete protection in 60 % of mice against T-cell thymoma challenge. Furthermore, EVs associated with three different ligands (OVA, CpG-ODN, and α-GalCer) effectively regressed established murine melanoma and significantly improved survival rates in mice. This study presents a powerful and promising approach to overcoming the limitations of EV-based cancer vaccines, advancing the development of effective cancer immunotherapies. SUMMARY: Immunization with EVs that are co-associated with antigen and biotherapeutic cargo through a lyophilization-based technique elicits potent anti-cancer immunity., Competing Interests: Declaration of competing interest The funding bodies played no role in study design, data collection, decision to publish, or preparation of the manuscript. The authors (I.G. & M.G.) declare that they have the inventor rights on CpG ODN related patents., (Copyright © 2024. Published by Elsevier B.V.)

Objectives: To determine prevalence and clinical associations of anti-FHL1 autoantibodies in patients with idiopathic inflammatory myopathies (IIM), and to evaluate autoantibody levels over time., Methods: Sera at the time of diagnosis from patients with IIM (n = 449), autoimmune disease controls (DC, n = 130), neuromuscular diseases (NMD, n = 16) and healthy controls (HC, n = 100) were analyzed for anti-FHL1 autoantibodies by Enzyme-Linked ImmunoSorbent Assay (ELISA). Patients with IIM FHL1+ and FHL1- were included in a longitudinal analysis. Serum levels were correlated to disease activity., Results: Autoantibodies to FHL1 were more frequent in patients with IIM (122/449, 27%) compared with DC (Autoimmune DC and NMD, 13/146, 9%, p< 0.001) and HC (3/100,3%, p< 0.001). Anti-FHL1 levels were higher in IIM [median (IQR)=0.62 (0.15-1.04)] in comparison with DC [0.22 (0.08-0.58)], HC [0.35 (0.23-0.47)] and NMD [0.48 (0.36-0.80)] p< 0.001. Anti-FHL1+ patients with IIM were younger at time of diagnosis compared with the anti-FHL1- group (p= 0.05) and were seronegative for other autoantibodies in 25%.In the first follow-up anti-FHL1+ sample 20/33 (60%) positive at baseline had turned negative for anti-FHL1 autoantibodies. Anti-FHL1 autoantibodies rarely appeared after initiating treatment. Anti-FHL1 autoantibody levels correlated with CK (r = 0.62, p= 0.01), disease activity measure MYOACT (n = 14, p= 0.004) and inversely with manual muscle test-8 (r=-0.59, p= 0.02) at baseline., Conclusions: Anti-FHL1 autoantibodies were present in 27% of patients with IIM, of these 25% were negative for other autoantibodies. Other autoimmune diseases had lower frequencies and levels. Anti-FHL1 levels often decreased with immunosuppressive treatment, correlated with disease activity measures at diagnosis and rarely appeared after start of treatment., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Objectives: Clinical observations in patients with dermatomyositis (DM) and autoantibodies against the melanoma differentiation-associated protein 5 (MDA5) suggest that the autoantibodies contribute to the pathogenesis of MDA5(+) DM. To gain insight into the role of the anti-MDA5 autoantibodies, we aimed to identify their binding sites on the different domains of the MDA5 protein., Methods: We developed an in-house ELISA to assess the reactivity against the MDA5 domains (conformational epitopes) in plasma (n = 8) and serum (n = 24) samples from MDA5(+) patients with varying clinical manifestations and disease outcomes. The reactivities were also assessed using western blot (linearized epitopes). An ELISA-based depletion assay was developed to assess cross-reactivity among the different MDA5 domains., Results: All eight plasma samples consistently showed reactivity towards conformational and linearized epitopes on the helicase domains of the MDA5 protein. The ELISA-based depletion assay suggests that anti-MDA5 autoantibodies specifically target each of the three helicase domains. Twenty-two of the 24 serum samples showed reactivity in the in-house ELISA and all 22 displayed reactivity towards the helicase domains of the MDA5 protein., Conclusions: Our data revealed that the main immunogenic targets of anti-MDA5 autoantibodies from MDA5(+) patients are the helicase domains. Considering that the helicase domains are responsible for the enzymatic activity and subsequent triggering of an inflammatory response, our findings suggest that binding of anti-MDA5 autoantibodies could alter the canonical activity of the MDA5 protein and potentially affect the downstream induction of a pro-inflammatory cascade., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

CpG Oligonucleotides (ODN) are immunomodulatory synthetic oligonucleotides specifically designed to stimulate Toll-like receptor 9. TLR9 is expressed on human plasmacytoid dendritic cells and B cells and triggers an innate immune response characterized by the production of Th1 and pro-inflammatory cytokines. This chapter reviews recent progress in understanding the mechanism of action of CpG ODN and provides an overview of human clinical trial results using CpG ODN to improve vaccines for the prevention/treatment of cancer, allergy, and infectious disease.