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The immunological basis of the clinical heterogeneity in autoimmune vasculitis remains poorly understood. In this study, we conduct single-cell transcriptome analyses on peripheral blood mononuclear cells (PBMCs) from newly-onset patients with microscopic polyangiitis (MPA). Increased proportions of activated CD14 + monocytes and CD14 + monocytes expressing interferon signature genes (ISGs) are distinctive features of MPA. Patient-specific analysis further classifies MPA into two groups. The MPA-MONO group is characterized by a high proportion of activated CD14 + monocytes, which persist before and after immunosuppressive therapy. These patients are clinically defined by increased monocyte ratio in the total PBMC count and have a high relapse rate. The MPA-IFN group is characterized by a high proportion of ISG + CD14 + monocytes. These patients are clinically defined by high serum interferon-alpha concentrations and show good response to immunosuppressive therapy. Our findings identify the immunological phenotypes of MPA and provide clinical insights for personalized treatment and accurate prognostic prediction. Autoimmune vasculitis can be heterogeneous in terms of immune cell involvement. Here the authors use a single cell transcriptomics approach to characterise a group of microscopic polyangiitis patients that could be split into two groups typified by monocyte or Interferon associated gene expression.

To identify the dynamics of neutrophil autoimmunity, here we focus on anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis and perform single-cell transcriptome and surface proteome analyses on peripheral white blood cells from patients with new-onset microscopic polyangiitis (MPA). Compared with controls, two neutrophil populations, immature neutrophils and neutrophils with type II interferon signature genes (Neu_T2ISG), are increased in patients with MPA. Trajectory and cell–cell interaction analyses identify Neu_T2ISG as a subset that differentiates from mature neutrophils upon stimulation with IFN-γ and TNF, which synergize to induce myeloperoxidase and Fcγ receptors expression on the neutrophil cell surface and promote ANCA–induced neutrophil extracellular trap formation. Case-by-case analysis indicates that patients with a high proportion of the Neu_T2ISG subset are associated with persistent vasculitis symptoms. A larger cohort analysis shows that serum IFN-γ levels at disease onset correlate with susceptibility to disease relapse. Our findings thus identify neutrophil diversity at the single cell level and implicate a biomarker for predicting relapse in small vessel vasculitis. Neutrophils are early mediators for inflammation, but their functions in autoimmunity is still unclear. By using single cell analyses to compare patients with microscopic polyangiitis (MPA) and controls, here the authors find increased IFN-II-related neutrophils induced by IFN-γ and TNF signaling as a relapse risk marker for small vessel vasculitis.

The mechanisms by which the innate immune system senses damage have been extensively explored in multicellular organisms. In Drosophila , various types of tissue damage, including epidermal injury, tumor formation, cell competition, and apoptosis deficiency, induce sterile activation of the Toll pathway, a process that requires the use of extracellular serine protease (SP) cascades. Upon infection, the SP Spätzle (Spz)-processing enzyme (SPE) cleaves and activates the Toll ligand Spz downstream of two paralogous SPs, Hayan and Persephone (Psh). However, upon tissue damage, it is not fully understood which SPs establish Spz activation cascades nor what damage-associated molecules can activate SPs. In this study, using newly generated uncleavable spz mutant flies, we revealed that Spz cleavage is required for the sterile activation of the Toll pathway, which is induced by apoptosis-deficient damage of wing epidermal cells in adult Drosophila . Proteomic analysis of hemolymph, followed by experiments with Drosophila Schneider 2 (S2) cells, revealed that among hemolymph SPs, both SPE and Melanization Protease 1 (MP1) have high capacities to cleave Spz. Additionally, in S2 cells, MP1 acts downstream of Hayan and Psh in a similar manner to SPE. Using genetic analysis, we found that the upstream SPs Hayan and Psh contributes to the sterile activation of the Toll pathway. While SPE / MP1 double mutants show more impairment of Toll activation upon infection than SPE single mutants, Toll activation is not eliminated in these apoptosis-deficient flies. This suggests that Hayan and Psh sense necrotic damage, inducing Spz cleavage by SPs other than SPE and MP1. Furthermore, hydrogen peroxide, a representative damage-associated molecule, activates the Psh-Spz cascade in S2 cells overexpressing Psh. Considering that reactive oxygen species (ROS) were detected in apoptosis-deficient wings, our findings highlight the importance of ROS as signaling molecules that induce the activation of SPs such as Psh in response to damage.

The autoimmune disease systemic sclerosis (SSc) presents with multiple organ manifestations that often complicate management strategies. To explore variations in immune cell subsets and their link to clinical heterogeneity, here we perform single-cell profiling of peripheral blood mononuclear cells (PBMC) from 21 SSc patients who never received immunosuppressive therapy. We identify a subset of EGR1 + CD14 + monocytes in patients with scleroderma renal crisis (SRC). This subset activates NF-kB signaling and differentiates into tissue-damaging macrophages, which accumulate at sites of tissue injury. Furthermore, we identify a CD8 + T cell subset with type II interferon signature in the peripheral blood and the lung tissue of patients with progressive interstitial lung disease (ILD), suggesting that chemokine-driven migration of these cells contributes to ILD progression. Thus, our single-cell analysis reveals distinct immune cell abnormalities associated with clinical organ manifestations, providing insights into tailored treatment strategies. Clinical manifestations in patients with systemic sclerosis (SSc) are highly heterogeneous, and identifying the root causes of clinical symptoms is challenging. Here, by performing single-cell profiling of peripheral blood from SSc patients, the authors identify distinct immune abnormalities associated with acute organ complications, including interstitial lung disease and scleroderma renal crisis.

Alternative splicing is an important mechanism in the process of eukaryotic nuclear mRNA precursors producing multiple protein products from a single gene. Although group I self-splicing introns usually perform regular splicing, limited examples of alternative splicing have also been reported. The exon-skipping type of splicing has been observed in genes containing two group I introns. To characterize splicing patterns (exon-skipping/exon-inclusion) of tandemly aligned group I introns, we constructed a reporter gene containing two Tetrahymena introns flanking a short exon. To control splicing patterns, we engineered the two introns in a pairwise manner to design pairs of introns that selectively perform either exon-skipping or exon-inclusion splicing. Through pairwise engineering and biochemical characterization, the structural elements important for the induction of exon-skipping splicing were elucidated.

Glucagon-like peptide-1 (GLP-1) released from intestinal L cells in response to nutrient ingestion inhibits both gastrointestinal emptying and gastric acid secretion and promotes satiety. The main biological effect of GLP-1 is the stimulation of insulin secretion (thereby fulfilling the criterion for an incretin hormone) in order to reduce blood glucose levels in mammalian species. Chicken GLP-1 receptor (cGLP-1R) has also been identified in various tissues by gene expression analysis. Although certain effects of GLP-1 in mammals and birds are consistent, e.g., inhibition of food intake, whether GLP-1 has the same insulinotropic activity in chickens as in mammals is debated. Moreover, the expression of cGLP-1R in chicken pancreatic B cells has not been reported. The localization of cGLP-1R and its mRNA in pancreatic islets is studied by triple-immunofluorescence microscopy and in situ hybridization. Triple-immunofluorescence microscopy with antisera against cGLP-1R, somatostatin and insulin or glucagon revealed that cGLP-1R protein was exclusively localized in D cells producing somatostatin in chicken pancreatic islets. The D cells were localized in peripheral areas of the pancreatic islets and cGLP-1R mRNA was detected in the same areas, indicating that cGLP-1R mRNA was also expressed in D cells. This is the first report to demonstrate that cGLP-1R is expressed by D cells, not B cells as in mammals. Our study suggests that chicken GLP-1 performs its insulinotropic activity by a different mode of action from that of the mammalian hormone.

Glucagon-like peptide (GLP)-1 and neurotensin (NT) are distributed throughout the chicken ileum. Here, we attempt to determine if GLP-1 and NT co-localize in the chicken ileum by using immunofluorescence, immunocytochemistry and in situ hybridization techniques. Three types of enteroendocrine cells, GLP-1 + /NT + , GLP-1 + /NT − and GLP-1 − /NT + cells, were detected in the mucosal epithelium by the double immunofluorescence method. The ratio of GLP-1 + /NT + cells at the crypts in the distal ileum was significantly higher than that in the proximal ileum. The ratios of the three cell types were similar along the crypt–villous axis in the proximal ileum but the percentage of GLP-1 + /NT + cells significantly decreased at the middle part of villi relative to crypts and the bottom part of villi in the distal ileum. Enteroendocrine cells that were immunoreactive to both GLP-1 and NT peptides and showed both proglucagon and NT precursor mRNA signals were found in the crypts of the distal ileum but not in the villous epithelium. The results from performing an immunocytochemical method with colloidal gold indicated that the GLP-1 content within GLP-1 + /NT + cell secretory granules decreased stepwise from the crypt to the middle part of the villus but the NT content in these granules increased in this direction. These findings reveal that the cells producing both GLP-1 and NT are mainly localized in the crypts of the chicken ileum but these endocrine cells specialize in NT-producing cells at the villous epithelium of the distal ileum.

Monitoring Large Language Model (LLM) outputs is crucial for mitigating risks from misuse and misalignment. However, LLMs could evade monitoring through steganography: Encoding hidden information within seemingly benign generations. In this paper, we evaluate the steganography capabilities in frontier LLMs to better understand the risk they pose. We focus on two types of steganography: passing encoded messages and performing encoded reasoning. We find that current models are unable to encode short messages in their outputs without a monitor noticing under standard affordances. They can succeed, however, if given additional affordances like using an unmonitored scratchpad and coordinating on what encoding scheme to use. We additionally find early signs that models can perform basic encoded reasoning in a simple state-tracking problem. This includes some ability to reason with their own and pre-defined schemes, including encoding schemes such as Hexadecimal. Despite this, they can rarely hide reasoning subtly within a cover task to fool a monitor. Overall, our results indicate that current LLMs exhibit nascent steganographic capabilities. While these capabilities are likely insufficient to bypass well-designed monitors at present, this could change in the future.

Emicizumab, a factor (F)VIIIa-function mimetic bispecific antibody (BsAb) to FIXa and FX, has become an indispensable treatment for people with hemophilia A (PwHA). Although emicizumab is very potent, long-term outcomes from the clinical studies suggest that a small proportion of PwHA still experiences bleeds. Additionally, non-clinical studies indicate that the maximum cofactor activity of emicizumab is lower than international standard activity (100 IU/dL of FVIII). An increased cofactor activity BsAb would benefit such patients. Here, we report NXT007, a BsAb binding FIXa and FX developed through further engineering of emicizumab. Emicizumab has a common light chain, but through advances in antibody engineering, we were able to create a more potent BsAb with two new non-common light chains. After extensive optimization of the heavy and light chains, the resulting BsAb, NXT007, exerted in vitro thrombin generation (TG) activity in hemophilia A plasma equivalent to 100 IU/dL of FVIII when triggered by tissue factor. NXT007 demonstrated potent hemostatic activity in an acquired hemophilia A model in non-human primates at a much lower dosage than emicizumab, consistent with an around 30-fold dose shift in the in vitro TG activity between NXT007 and emicizumab. Moreover, together with Fc engineering that enhanced FcRn binding and reduced in vivo clearance, we demonstrate that NXT007 could be effective at a much lower dosage with a longer dosing interval compared to emicizumab. These non-clinical results suggest that NXT007 could maintain a non-hemophilic range of coagulation potential in PwHA and provides a rationale for its clinical testing.

Emicizumab, a factor (F)VIIIa-function mimetic therapeutic bispecific antibody (BsAb) to FIXa and FX, has become an indispensable treatment for people with hemophilia A (PwHA). However, non-clinical studies suggest that the maximum cofactor activity of emicizumab is lower than international standard activity (100 IU/dL of FVIII), leaving room for further improvement. Since not all PwHA experienced zero treated bleeds, increased cofactor activity would be beneficial for such patients. Here, we report NXT007, a BsAb against FIXa and FX developed through further engineering of emicizumab. While emicizumab has a common light chain, advances in antibody engineering enabled us to identify a more potent BsAb with two distinct new light chains, and following extensive mutational optimization of the two emicizumab-derived heavy chains and two light chains, the resulting NXT007 exerted in vitro thrombin generation (TG) activity in hemophilia A plasma corresponding to that at 100 IU/dL of FVIII when coagulation is triggered by tissue factor. NXT007 demonstrated potent hemostatic activity in an acquired hemophilia A model in non-human primates at much lower dosage than emicizumab, consistent with an around 30-fold dose shift in the in vitro TG activity between NXT007 and emicizumab. Moreover, together with Fc engineering that enhanced FcRn binding and reduced in vivo clearance, we demonstrate that NXT007 could be effective at much lower dosage with a longer dosing interval compared to emicizumab. These non-clinical results suggest that NXT007 is expected to maintain a non-hemophilic range of coagulation potential in PwHA and provides a rationale for its clinical testing.Competing Interest StatementThe authors have declared no competing interest.