Multi-organ proteomic landscape of COVID-19 autopsies.

The molecular pathology of multi-organ injuries in COVID-19 patients remains unclear, preventing effective therapeutics development. Here, we report a proteomic analysis of 144 autopsy samples from seven organs in 19 COVID-19 patients. We quantified 11,394 proteins in these samples, in which 5,336 were perturbed in the COVID-19 patients compared to controls. Our data showed that cathepsin L1, rather than ACE2, was significantly upregulated in the lung from the COVID-19 patients. Systemic hyperinflammation and dysregulation of glucose and fatty acid metabolism were detected in multiple organs. We also observed dysregulation of key factors involved in hypoxia, angiogenesis, blood coagulation, and fibrosis in multiple organs from the COVID-19 patients. Evidence for testicular injuries includes reduced Leydig cells, suppressed cholesterol biosynthesis, and sperm mobility. In summary, this study depicts a multi-organ proteomic landscape of COVID-19 autopsies that furthers our understanding of the biological basis of COVID-19 pathology.

Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2.

The recent emergence of a novel coronavirus (SARS-CoV-2) in China has caused significant public health concerns. Recently, ACE2 was reported as an entry receptor for SARS-CoV-2. In this study, we present the crystal structure of the C-terminal domain of SARS-CoV-2 (SARS-CoV-2-CTD) spike (S) protein in complex with human ACE2 (hACE2), which reveals a hACE2-binding mode similar overall to that observed for SARS-CoV. However, atomic details at the binding interface demonstrate that key residue substitutions in SARS-CoV-2-CTD slightly strengthen the interaction and lead to higher affinity for receptor binding than SARS-RBD. Additionally, a panel of murine monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) against SARS-CoV-S1/receptor-binding domain (RBD) were unable to interact with the SARS-CoV-2 S protein, indicating notable differences in antigenicity between SARS-CoV and SARS-CoV-2. These findings shed light on the viral pathogenesis and provide important structural information regarding development of therapeutic countermeasures against the emerging virus.

Single-cell profiling identifies mechanisms of inflammatory heterogeneity in chronic rhinosinusitis.

The heterogeneous cellular microenvironment of human airway chronic inflammatory diseases, including chronic rhinosinusitis (CRS) and asthma, is still poorly understood. Here, we performed single-cell RNA sequencing (scRNA-seq) on the nasal mucosa of healthy individuals and patients with three subtypes of CRS and identified disease-specific cell subsets and molecules that specifically contribute to the pathogenesis of CRS subtypes. As such, ALOX15+ macrophages contributed to the type 2 immunity-driven pathogenesis of one subtype of CRS, eosinophilic CRS with nasal polyps (eCRSwNP), by secreting chemokines that recruited eosinophils, monocytes and T helper 2 (TH2) cells. An inhibitor of ALOX15 reduced the release of proinflammatory chemokines in human macrophages and inhibited the overactivation of type 2 immunity in a mouse model of eosinophilic rhinosinusitis. Our findings advance the understanding of the heterogeneous immune microenvironment and the pathogenesis of CRS subtypes and identify potential therapeutic approaches for the treatment of CRS and potentially other type 2 immunity-mediated diseases.

Atlas of currently available human neutralizing antibodies against SARS-CoV-2 and escape by Omicron sub-variants BA.1/BA.1.1/BA.2/BA.3.

SARS-CoV-2 Omicron variant has presented significant challenges to current antibodies and vaccines. Herein, we systematically compared the efficacy of 50 human monoclonal antibodies (mAbs), covering the seven identified epitope classes of the SARS-CoV-2 RBD, against Omicron sub-variants BA.1, BA.1.1, BA.2, and BA.3. Binding and pseudovirus-based neutralizing assays revealed that 37 of the 50 mAbs lost neutralizing activities, whereas the others displayed variably decreased activities against the four Omicron sub-variants. BA.2 was found to be more sensitive to RBD-5 antibodies than the other sub-variants. Furthermore, a quaternary complex structure of BA.1 RBD with three mAbs showing different neutralizing potencies against Omicron provided a basis for understanding the immune evasion of Omicron sub-variants and revealed the lack of G446S mutation accounting for the sensitivity of BA.2 to RBD-5 mAbs. Our results may guide the application of the available mAbs and facilitate the development of universal therapeutic antibodies and vaccines against COVID-19.

The histone demethylase UTX regulates the lineage-specific epigenetic program of invariant natural killer T cells.

Invariant natural killer T cells (iNKT cells) are innate-like lymphocytes that protect against infection, autoimmune disease and cancer. However, little is known about the epigenetic regulation of iNKT cell development. Here we found that the H3K27me3 histone demethylase UTX was an essential cell-intrinsic factor that controlled an iNKT-cell lineage-specific gene-expression program and epigenetic landscape in a demethylase-activity-dependent manner. UTX-deficient iNKT cells exhibited impaired expression of iNKT cell signature genes due to a decrease in activation-associated H3K4me3 marks and an increase in repressive H3K27me3 marks within the promoters occupied by UTX. We found that JunB regulated iNKT cell development and that the expression of genes that were targets of both JunB and the iNKT cell master transcription factor PLZF was UTX dependent. We identified iNKT cell super-enhancers and demonstrated that UTX-mediated regulation of super-enhancer accessibility was a key mechanism for commitment to the iNKT cell lineage. Our findings reveal how UTX regulates the development of iNKT cells through multiple epigenetic mechanisms.

CD1a on Langerhans cells controls inflammatory skin disease.

CD1a is a lipid-presenting molecule that is abundantly expressed on Langerhans cells. However, the in vivo role of CD1a has remained unclear, principally because CD1a is lacking in mice. Through the use of mice with transgenic expression of CD1a, we found that the plant-derived lipid urushiol triggered CD1a-dependent skin inflammation driven by CD4(+) helper T cells that produced the cytokines IL-17 and IL-22 (TH17 cells). Human subjects with poison-ivy dermatitis had a similar cytokine signature following CD1a-mediated recognition of urushiol. Among various urushiol congeners, we identified diunsaturated pentadecylcatechol (C15:2) as the dominant antigen for CD1a-restricted T cells. We determined the crystal structure of the CD1a-urushiol (C15:2) complex, demonstrating the molecular basis of urushiol interaction with the antigen-binding cleft of CD1a. In a mouse model and in patients with psoriasis, CD1a amplified inflammatory responses that were mediated by TH17 cells that reacted to self lipid antigens. Treatment with blocking antibodies to CD1a alleviated skin inflammation. Thus, we propose CD1a as a potential therapeutic target in inflammatory skin diseases.

Host range and structural analysis of bat-origin RshSTT182/200 coronavirus binding to human ACE2 and its animal orthologs.

Bat-origin RshSTT182 and RshSTT200 coronaviruses (CoV) from Rhinolophus shameli in Southeast Asia (Cambodia) share 92.6% whole-genome identity with SARS-CoV-2 and show identical receptor-binding domains (RBDs). In this study, we determined the structure of the RshSTT182/200 receptor binding domain (RBD) in complex with human angiotensin-converting enzyme 2 (hACE2) and identified the key residues that influence receptor binding. The binding of the RshSTT182/200 RBD to ACE2 orthologs from 39 animal species, including 18 bat species, was used to evaluate its host range. The RshSTT182/200 RBD broadly recognized 21 of 39 ACE2 orthologs, although its binding affinities for the orthologs were weaker than those of the RBD of SARS-CoV-2. Furthermore, RshSTT182 pseudovirus could utilize human, fox, and Rhinolophus affinis ACE2 receptors for cell entry. Moreover, we found that SARS-CoV-2 induces cross-neutralizing antibodies against RshSTT182 pseudovirus. Taken together, these findings indicate that RshSTT182/200 can potentially infect susceptible animals, but requires further evolution to obtain strong interspecies transmission abilities like SARS-CoV-2.

Epitranscriptomic cytidine methylation of the hepatitis B viral RNA is essential for viral reverse transcription and particle production.

Epitranscriptomic RNA modifications have emerged as important regulators of the fate and function of viral RNAs. One prominent modification, the cytidine methylation 5-methylcytidine (m5C), is found on the RNA of HIV-1, where m5C enhances the translation of HIV-1 RNA. However, whether m5C functionally enhances the RNA of other pathogenic viruses remains elusive. Here, we surveyed a panel of commonly found RNA modifications on the RNA of hepatitis B virus (HBV) and found that HBV RNA is enriched with m5C as well as ten other modifications, at stoichiometries much higher than host messenger RNA (mRNA). Intriguingly, m5C is mostly found on the epsilon hairpin, an RNA element required for viral RNA encapsidation and reverse transcription, with these m5C mainly deposited by the cellular methyltransferase NSUN2. Loss of m5C from HBV RNA due to NSUN2 depletion resulted in a partial decrease in viral core protein (HBc) production, accompanied by a near-complete loss of the reverse transcribed viral DNA. Similarly, mutations introduced to remove the methylated cytidines resulted in a loss of HBc production and reverse transcription. Furthermore, pharmacological disruption of m5C deposition led to a significant decrease in HBV replication. Thus, our data indicate m5C methylations as a critical mediator of the epsilon elements' function in HBV virion production and reverse transcription, suggesting the therapeutic potential of targeting the m5C methyltransfer process on HBV epsilon as an antiviral strategy.

Genome-wide CRISPR screenings identified SMCHD1 as a host-restricting factor for AAV transduction.

AAV-mediated gene therapy typically requires a high dose of viral transduction, risking acute immune responses and patient safety, part of which is due to limited understanding of the host-viral interactions, especially post-transduction viral genome processing. Here, through a genome-wide CRISPR screen, we identified SMCHD1 (Structural Maintenance of Chromosomes Hinge Domain 1), an epigenetic modifier, as a critical broad-spectrum restricting host factor for post-entry AAV transgene expression. SMCHD1 knock-down by RNAi and CRISPRi or knock-out by CRISPR all resulted in significantly enhanced transgene expression across multiple viral serotypes, as well as for both single-strand and self-complementary AAV genome types. Mechanistically, upon viral transduction, SMCHD1 effectively repressed AAV transcription by the formation of an LRIF1-HP1-containing protein complex and directly binding with the AAV genome to maintain a heterochromatin-like state. SMCHD1-KO or LRIF1-KD could disrupt such a complex and thus result in AAV transcriptional activation. Together, our results highlight the host factor-induced chromatin remodeling as a critical inhibitory mechanism for AAV transduction and may shed light on further improvement in AAV-based gene therapy.

OARD: Open annotations for rare diseases and their phenotypes based on real-world data.

Diagnosis for rare genetic diseases often relies on phenotype-driven methods, which hinge on the accuracy and completeness of the rare disease phenotypes in the underlying annotation knowledgebase. Existing knowledgebases are often manually curated with additional annotations found in published case reports. Despite their potential, real-world data such as electronic health records (EHRs) have not been fully exploited to derive rare disease annotations. Here, we present open annotation for rare diseases (OARD), a real-world-data-derived resource with annotation for rare-disease-related phenotypes. This resource is derived from the EHRs of two academic health institutions containing more than 10 million individuals spanning wide age ranges and different disease subgroups. By leveraging ontology mapping and advanced natural-language-processing (NLP) methods, OARD automatically and efficiently extracts concepts for both rare diseases and their phenotypic traits from billing codes and lab tests as well as over 100 million clinical narratives. The rare disease prevalence derived by OARD is highly correlated with those annotated in the original rare disease knowledgebase. By performing association analysis, we identified more than 1 million novel disease-phenotype association pairs that were previously missed by human annotation, and >60% were confirmed true associations via manual review of a list of sampled pairs. Compared to the manual curated annotation, OARD is 100% data driven and its pipeline can be shared across different institutions. By supporting privacy-preserving sharing of aggregated summary statistics, such as term frequencies and disease-phenotype associations, it fills an important gap to facilitate data-driven research in the rare disease community.

Autism patient-derived SHANK2BY29X mutation affects the development of ALDH1A1 negative dopamine neuron.

Autism spectrum disorder (ASD) encompasses a range of neurodevelopmental conditions. Different mutations on a single ASD gene contribute to heterogeneity of disease phenotypes, possibly due to functional diversity of generated isoforms. SHANK2, a causative gene in ASD, demonstrates this phenomenon, but there is a scarcity of tools for studying endogenous SHANK2 proteins in an isoform-specific manner. Here, we report a point mutation on SHANK2, which is found in a patient with autism, located on exon of the SHANK2B transcript variant (NM_133266.5), hereby SHANK2BY29X. This mutation results in an early stop codon and an aberrant splicing event that impacts SHANK2 transcript variants distinctly. Induced pluripotent stem cells (iPSCs) carrying this mutation, from the patient or isogenic editing, fail to differentiate into functional dopamine (DA) neurons, which can be rescued by genetic correction. Available SMART-Seq single-cell data from human midbrain reveals the abundance of SHANK2B transcript in the ALDH1A1 negative DA neurons. We then show that SHANK2BY29X mutation primarily affects SHANK2B expression and ALDH1A1 negative DA neurons in vitro during early neuronal developmental stage. Mice knocked in with the identical mutation exhibit autistic-like behavior, decreased occupancy of ALDH1A1 negative DA neurons and decreased dopamine release in ventral tegmental area (VTA). Our study provides novel insights on a SHANK2 mutation derived from autism patient and highlights SHANK2B significance in ALDH1A1 negative DA neuron.

Liproxstatin-1 alleviates cartilage degradation by inhibiting chondrocyte ferroptosis in the temporomandibular joint.

BGROUND INFORMATION: Ferroptosis contributes to temporomandibular joint osteoarthritis (TMJOA) lesion development and is still poorly understood. RESULTS: In this study, we used different TMJOA animal models to examine whether ferroptosis was related to disease onset in TMJOA induced by monosodium iodoacetate (MIA), IL-1ß, occlusion disorder (OD), and unilateral anterior crossbite (UAC). Immunohistochemical staining and Western blot analysis were used to detect ferroptosis- and cartilage degradation-related protein expression. Our results revealed reduced levels of the ferroptosis-related protein GPX4 in the cartilage layer, but the levels of ACSL4 and P53 were increased in the condyle. Injection of the ferroptosis inhibitor liproxstatin-1 (Lip-1) effectively decreased ACSL4, P53 and TRF expression. In vitro, IL-1ß reduced cartilage extracellular matrix expression in mandibular condylar chondrocytes (MCCs). Lip-1 maintained the morphology and function of mitochondria and ameliorated the exacerbation of lipid peroxidation and reactive oxygen species (ROS) production induced by IL-1ß. CONCLUSION: These results suggest that chondrocyte ferroptosis plays an important role in the development and progression of TMJOA. SIGNIFICANCE: Inhibiting condylar chondrocyte ferroptosis could be a promising therapeutic strategy for TMJOA.

Synergistic integration of mRNA-LNP with CAR-engineered immune cells: Pioneering progress in immunotherapy.

Chimeric antigen receptor T cell (CAR-T) therapy has emerged as a revolutionary approach in the treatment of malignancies. Despite its remarkable successes, this field continues to grapple with challenges such as scalability, safety concerns, limited therapeutic effect, in vivo persistence, and the need for precise control over CAR expression. In the post-pandemic era of COVID-19 vaccine immunization, the application of messenger RNA (mRNA) encapsulated within lipid nanoparticles (LNPs) has recently garnered significant attention as a potential solution to address these challenges. This review delves into the dynamic landscape of mRNA-LNP technology and its potential implications for CAR-engineered immune cell-based immunotherapy.

Transcription factor EB coordinates environmental cues to regulate T regulatory cells' mitochondrial fitness and function.

T regulatory (Treg) cells are essential for self-tolerance whereas they are detrimental for dampening the host anti-tumor immunity. How Treg cells adapt to environmental signals to orchestrate their homeostasis and functions remains poorly understood. Here, we identified that transcription factor EB (TFEB) is induced by host nutrition deprivation or interleukin (IL)-2 in CD4+ T cells. The loss of TFEB in Treg cells leads to reduced Treg accumulation and impaired Treg function in mouse models of cancer and autoimmune disease. TFEB intrinsically regulates genes involved in Treg cell differentiation and mitochondria function while it suppresses expression of proinflammatory cytokines independently of its established roles in autophagy. This coordinated action is required for mitochondria integrity and appropriate lipid metabolism in Treg cells. These findings identify TFEB as a critical regulator for orchestrating Treg generation and function, which may contribute to the adaptive responses of T cells to local environmental cues.

PCSK9 inhibition interrupts the cross-talk between keratinocytes and macrophages and prevents UVB-induced skin damage.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an enzyme that promotes the degradation of low-density lipoprotein receptors. It is involved in hyperlipidemia as well as other diseases, such as cancer and skin inflammation. However, the detailed mechanism for PCSK9 on ultraviolet B (UVB)-induced skin lesions was not clear. Thus, the role and possible action mechanism of PCSK9 in UVB-induced skin damage in mice were studied here using siRNA and a small molecule inhibitor (SBC110736) against PCSK9. Immunohistochemical staining revealed a significant increase in PCSK9 expression after UVB exposure, indicating the possible role of PCSK9 in UVB damage. Skin damage, increase in epidermal thickness, and keratinocyte hyperproliferation were significantly alleviated after treatment with SBC110736 or siRNA duplexes, compared with that in the UVB model group. Notably, UVB exposure triggered DNA damage in keratinocytes, whereas substantial interferon regulatory factor 3 (IRF3) activation was observed in macrophages. Pharmacologic inhibition of STING or cGAS knockout significantly reduced UVB-induced damage. In the co-culture system, supernatant from UVB-treated keratinocyte induced IRF3 activation in macrophages. This activation was inhibited with SBC110736 and by PCSK9 knockdown. Collectively, our findings reveal that PCSK9 plays a critical role in the crosstalk between damaged keratinocytes and STING activation in macrophages. The interruption of this crosstalk by PCSK9 inhibition may be a potential therapeutic strategy for UVB-induced skin damage.

Trastuzumab-functionalized bionic pyrotinib liposomes for targeted therapy of HER2-positive breast cancer.

In this study, we prepared a bionic nanosystem of trastuzumab-functionalized SK-BR-3 cell membrane hybrid liposome-coated pyrotinib (Ptb-M-Lip-Her) for the treatment of HER2-positive breast cancer. Transmission electron microscopy, dynamic light scattering, polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting were used to verify the successful preparation of Ptb-M-Lip-Her. In vitro drug release experiments proved that Ptb-M-Lip-Her had a sustained release effect. Cell uptake experiments and in vivo imaging experiments proved that Ptb-M-Lip-Her had good targeting ability to homologous tumor cells (SK-BR-3). The results of cell experiments such as MTT, flow cytometry, immunofluorescence staining and in vivo antitumor experiments showed that Ptb-M-Lip-Her could significantly promote apoptosis and inhibit the proliferation of SK-BR-3 cells. These results clearly indicated that Ptb-M-Lip-Her may be a promising biomimetic nanosystem for targeted therapy of HER2-positive breast cancer.

The Oral Administration of Lactobacillus delbrueckii subsp. lactis 557 (LDL557) Ameliorates the Progression of Monosodium Iodoacetate-Induced Osteoarthritis.

Low-grade body inflammation is a major cause of osteoarthritis (OA), a common joint disease. Gut dysbiosis may lead to systemic inflammation which can be prevented by probiotic administration. The Lactobacillus delbrueckii subsp. lactis 557 (LDL557) has been demonstrated to have beneficial effects for anti-inflammation. This study investigated the effects of LDL557 on OA progress using monosodium iodoacetate (MIA)-induced OA of rats. Live or heat-killed (HK)-LDL557 of a low or high dose was administrated for two weeks before MIA-induced OA, and then continuously administrated for another six weeks. After taking supplements for eight weeks, OA progress was analyzed. Results showed that MIA induced knee joint swelling, chondrocyte damage, and cartilage degradation, and supplementation with a high dose of LDL557 reduced MIA-induced knee joint swelling, chondrocyte damage, and cartilage degradation. Additionally, MIA increased serum levels of the matrix-degrading enzyme MMP-13, while a high dose of HK-LDL557 decreased it for the controls. Simultaneously, bone turnover markers and inflammatory cytokines of serum were assayed, but no significant differences were found except for a TNF-α decrease from a low dose of live LDL557. These results demonstrated that supplementation with high doses of live LDL557 or HK-LDL557 can reduce the progression of MIA-induced OA in rats.

A novel scoring model for predicting efficacy and guiding individualised treatment in immune thrombocytopaenia.

Despite diverse therapeutic options for immune thrombocytopaenia (ITP), drug efficacy and selection challenges persist. This study systematically identified potential indicators in ITP patients and followed up on subsequent treatment. We initially analysed 61 variables and identified 12, 14, and 10 candidates for discriminating responders from non-responders in glucocorticoid (N = 215), thrombopoietin receptor agonists (TPO-RAs) (N = 224), and rituximab (N = 67) treatments, respectively. Patients were randomly assigned to training or testing datasets and employing five machine learning (ML) models, with eXtreme Gradient Boosting (XGBoost) area under the curve (AUC = 0.89), Decision Tree (DT) (AUC = 0.80) and Artificial Neural Network (ANN) (AUC = 0.79) selected. Cross-validated with logistic regression and ML finalised five variables (baseline platelet, IP-10, TNF-α, Treg, B cell) for glucocorticoid, eight variables (baseline platelet, TGF-ß1, MCP-1, IL-21, Th1, Treg, MK number, TPO) for TPO-RAs, and three variables (IL-12, Breg, MAIPA-) for rituximab to establish the predictive model. Spearman correlation and receiver operating characteristic curve analysis in validation datasets demonstrated strong correlations between response fractions and scores in all treatments. Scoring thresholds SGlu ≥ 3 (AUC = 0.911, 95% CI, 0.865-0.956), STPO-RAs ≥ 5 (AUC = 0.964, 95% CI 0.934-0.994), and SRitu = 3 (AUC = 0.964, 95% CI 0.915-1.000) indicated ineffectiveness in glucocorticoid, TPO-RAs, and rituximab therapy, respectively. Regression analysis and ML established a tentative and preliminary predictive scoring model for advancing individualised treatment.

The early and rapid response to daratumumab in children with chronic refractory immune thrombocytopenia from a referral single centre of China.

Chronic refractory primary immune thrombocytopenia (CRITP) is currently defined as refractory to multiple therapeutic of second-line agents with or without splenectomy, faced with the threat of severe bleeding and challenging to obtain effective treatment. Although stable and effective drug therapy is needed, it is tough to find one. Daratumumab (Dara), an anti-CD38 monoclonal antibody presented the target cloned plasma cells in multiple myeloma, has also been reported to be effective in refractory autoimmune cytopenia in some case or series reports and ongoing clinical trials for adult patients with CRITP. Here, we report the early and durable response of Dara combination with avatrombopag in three CRITP patients (2 male and 1 female aged 12, 5 and 7 years, respectively) in our centre, with a follow-up period of more than 25 weeks. Before Dara, the duration of immune thrombocytopenia was 9, 1.4 and 4 years, respectively, a baseline platelet count of 4, 6, 9 × 109/L, the bleeding score was all above level 2 and the number of previous drugs was >3. The time to response (R: Plt ≥30 × 109/L with at least a twofold increase in the baseline count) of Dara was on Day 45, 6 and 4 and achieved complete response (CR: Plt ≥100 × 109/L) on Day 51, 6 and 8, the sustained response (SR: Plt >30 × 109/L following Dara at ≥75% of the platelet count assessment at follow-up end-point since the patient achieved response) was 48, 175 and 204 days with the follow-up time of 39.1, 25.9 and 29.7 weeks. The bleeding score decreased from grade 3 to grade 0 during follow-up. No significant treatment-related adverse events were found during follow-up. Dara combination with avatrombopag may be a safe and efficacious therapy for children with CRITP, but it needs to be further explored.

Efficacy and safety of avatrombopag in Chinese children with persistent and chronic primary immune thrombocytopenia: A multicentre observational retrospective study in China.

Avatrombopag (AVA) is a novel thrombopoietin receptor agonist (TPO-RA) that has been recently approved as a second-line therapy for immune thrombocytopenia (ITP) in adults; however, its safety and efficacy data in children are lacking. Here, we demonstrated the efficacy and safety of AVA as second-line therapy in children with ITP. A multicentre, retrospective, observational study was conducted in children with persistent or chronic ITP who did not respond to or relapsed from previous treatment and were treated with AVA for at least 12 weeks between August 2020 and December 2022. The outcomes were the responses (defined as achieving a platelet count ≥30 × 109/L, twofold increase in platelet count from baseline and absence of bleeding), including rapid response within 4 weeks, sustained response at weeks 12 and 24, bleeding control and adverse events (AEs). Thirty-four (18 males) patients with a mean age of 6.3 (range: 1.9-15.3) years were enrolled. The median number of previous treatment types was four (range: 1-6), and 41.2% patients switched from other TPO-RAs. Within 4 weeks, overall response (OR) was achieved in 79.4% patients and complete response (CR, defined as a platelet count ≥100 × 109/L and the absence of bleeding) in 67.7% patients with a median response time of 7 (range: 1-27) days. At 12 weeks, OR was achieved in 88.2%, CR in 76.5% and sustained response in 44% of patients. At 24 weeks, 22/34 (64.7%) patients who achieved a response and were followed up for 24 weeks were evaluated; 12/22 (54.55%) achieved a sustained response. During AVA therapy, median platelet counts increased by week 1 and were maintained throughout the treatment period. The proportion of patients with grade 1-3 bleeding decreased from 52.95% at baseline to 2.94% at 12 weeks, while concomitant ITP medications decreased from 36.47% at baseline to 8.82% at 12 weeks, with only 9 (26.47%) patients receiving rescue therapy 23 times within 12 weeks. There were 61.8% patients with 59 AEs: 29.8% with Common Terminology Criteria for Adverse Events grade 1 and the rest with grade 2. These findings show that AVA could achieve a rapid and sustained response in children with persistent or chronic ITP as a second-line treatment, with good clinical bleeding control and reduction of concomitant ITP therapy, without significant AEs.