[Objective] To investigate the molecular mechanism of Syk in phagocytosis induced by CD36 antibodies. [Methods] In vitro, two CD36 monoclonal antibodies, GZ1 (IgG2a) and GZ4 (IgG1), at different concentrations were co-incubated with platelets from CD36-positive blood donors, and the affinity of the antibodies for platelets was assessed using flow cytometry. CD36 positive platelets from donors were labeled with Red-SE. The GZ1 and GZ4 antibodies were added together with dye-labeled platelets to peripheral blood from blood donors for phagocytosis experiments. The expression of the Spleen Tyrosine Kinase (Syk), P-Syk, P38 MAPK and P-P38 were detected using Western blot(WB). In the in vitro phagocytosis inhibition assay, Fcγ receptor (FcγR) antibodies or Syk inhibitor (R406) were pre-incubated with monocytes prior to performing the phagocytosis experiment. In vivo experiments were performed using female C57BL/6J mice. R406 was administered via intraperitoneal injection, followed by tail vein infusion of GZ1 or GZ4 antibodies. Additionally, a treatment group was set up in which the antibodies were infused first, followed by administration of R406. Changes in platelet counts were analyzed using a blood routine analyzer, and antibody binding to platelets was detected by flow cytometry. [Results] GZ1 had higher affinity for CD36positive platelets than GZ4. The phagocytosis rate induced by GZ1 was significantly higher than that of GZ4 [(42.12±2.25)% vs (16.25±6.45)%, P<0.001]. Moreover, GZ1-mediated platelet phagocytosis mainly depends on FcγRⅠ of monocytes, while GZ4-mediated platelet phagocytosis mainly relies on FcγRⅡ of monocytes. WB results showed that the GZ1 group significantly upregulated the expression of Syk and phosphorylated Syk (P-Syk), and induced phosphorylation of the downstream P38 MAPK signaling pathway. Pretreatment of monocytes with R406 inhibited the phagocytosis of antibodyopsonized platelets by monocytes. After injecting GZ1 or GZ4 antibodies into C57BL/6 female mice through the tail vein, GZ1 mAbs had a stronger binding rate to the platelets compared with GZ4 mAbs, leading to a significant decrease in platelet count. After pre-injecting R406 into mice, the platelet count reduction in both the GZ1 and GZ4 groups was alleviated, and the binding rate of antibodies to platelets was also significantly reduced. However, administration of R406 after antibody injection failed to prevent the decrease in platelet counts in mice. [Conclusion] This study indicates that Syk plays an important role in the phagocytosis of anti-CD36 opsonized platelets by monocytes/macrophages and is closely associated with the activation of the P38 MAPK signaling pathway.

Objective: This study investigates the prognostic value and clinical utility of the neutrophil percentage-to-albumin ratio (NPAR) in patients with resected non-small-cell lung cancer (NSCLC). Methods: We retrospectively included 335 patients with NSCLC who underwent lung resection at our institution between January 2017 and October 2018. Optimal cutoffs for preoperative and postoperative day 1 (D1) NPAR were determined using X-tile (version 3.6.1; Yale University, New Haven, CT, USA) to define high and low groups. Overall survival (OS) was evaluated using Kaplan-Meier analysis and Cox proportional hazards models. A perioperative NPAR trajectory (low-low, low-high, high-low, high-high) was constructed to characterize dynamic risk patterns. To mitigate potential bias associated with postoperative measurements, a D1 landmark analysis was performed. A nomogram was developed based on the multivariable model and assessed by calibration at 1, 3, and 5 years. Incremental clinical value beyond TNM stage and surgical approach was evaluated using decision curve analysis (DCA), as well as by 5-year continuous net reclassification improvement (NRI) and integrated discrimination improvement (IDI). Results: The optimal cutoffs for preoperative and postoperative D1 NPAR were 14.5 and 23.1, respectively. In univariate analyses, sex, smoking history, preoperative NPAR, postoperative D1 NPAR, pathologic type, TNM stage, surgical approach, and adjuvant therapy were associated with OS (all p < 0.01). In multivariable Cox regression, high preoperative NPAR (HR 1.896, 95% CI 1.135-3.168; p = 0.014) and high postoperative D1 NPAR (HR 1.905, 95% CI 1.097-3.305; p = 0.014) were independent risk factors, along with TNM stage (Stage II: HR 2.824, 95% CI 1.209-6.595; p = 0.016; Stage III: HR 9.470, 95% CI 4.935-18.171; p < 0.001) and open surgery (HR 2.350, 95% CI 1.341-4.117; p = 0.003). Trajectory analysis further stratified risk, with the high-high group showing the poorest survival (adjusted HR 3.48, 95% CI 1.43-8.47; p = 0.006). The association of postoperative NPAR persisted in the D1 landmark analysis (HR 1.836, 95% CI 1.071-3.148; p = 0.027). Adding NPAR to TNM stage and surgical approach improved 5-year risk reclassification (continuous NRI 0.377, 95% CI 0.094-0.659; IDI 0.028, 95% CI -0.002-0.054) and increased net benefit on DCA. The nomogram demonstrated acceptable calibration at 1, 3, and 5 years. Conclusions: This study demonstrates that NPAR serves as an independent prognostic marker for long-term outcomes in patients with NSCLC. The use of NPAR offers clinicians a comprehensive and precise tool for assessing patient prognosis.

Confirmation of HLA-C*07:262 by third-generation sequencing, differing from C*07:02:01:01 by a single nucleotide substitution in exon 2.

Anti-CD36 isoantibodies can induce platelet transfusion refractoriness and fetal neonatal immune thrombocytopenia. However, the mechanism of platelet clearance mediated by these antibodies (Abs) in such disorders remains unknown.We analyzed platelet clearance caused by mouse and human CD36 monoclonal Abs GZ1 IgG1 and IgG2 subclasses in vitro and in vivo.Platelet clearance was evaluated in vitro by platelet phagocytosis assays and in vivo by monoclonal Ab GZ1 administration to C57BL/6J mice. Platelet activation, apoptosis, and desialylation were analyzed by flow cytometry.Both anti-CD36 Abs subclasses caused lower platelet clearance than anti-αIIbβ3 owing to the FcγR occupancy of monocytes by anti-CD36 Abs. This reaction could lead to mild thrombocytopenia, compared with the severe thrombocytopenia induced by anti-αIIbβ3 platelet-specific Abs. IgG subclass-mediated platelet clearance was inhibited by anti-FcγR Abs and intravenous immunoglobulin (IVIG). The human IgG2 Ab subclass caused lower platelet clearance than IgG1. IgG1- and IgG2-mediated platelet phagocytosis was inhibited by anti-FcγRI and anti-FcγRII, respectively. Unlike IgG1, the IgG2 Ab subclass induced platelet activation, apoptosis, and desialylation and platelet clearance by endothelial cells via Fc-independent pathway.IgG1 and IgG2 subclasses of anti-CD36 Abs triggered platelet clearance primarily via the Fc-dependent pathway. The IgG2 Ab subclass, however, additionally induced platelet clearance via the Fc-independent pathway. These results indicate that the anti-CD36 Ab IgG subclass influences platelet clearance efficiency and may therefore determine the severity of immune thrombocytopenia caused by anti-CD36 antibodies.

Acute myeloid leukemia (AML) is a clinically aggressive and genetically heterogeneous hematologic malignancy with persistently poor clinical outcomes, largely due to the resistance to conventional chemotherapeutic agents including cytarabine (Ara-C). Emerging evidence indicates that the upregulation of the scavenger receptor CD36 is associated with AML relapse and drug resistance. In this study, we showed that high expression of CD36 is associated with poor prognosis for patients with AML. We then evaluated the therapeutic potential of a CD36-targeted monoclonal antibody (mAb) in combination with Ara-C, while investigating the molecular mechanisms underlying their synergistic interaction. Our results demonstrated that the combination of anti-CD36 mAb (GZ1) and Ara-C significantly enhanced the sensitivity of AML cells to Ara-C, reduced tumor burden and prolonged survival in both in vitro and in vivo models. Further analysis revealed that the efficacy of mAb GZ1 in combination with Ara-C was dependent on the IgG subtype, with IgG2a and IgG1 exhibiting stronger synergistic effects compared to the IgG3 subclass. Additionally, the study on an Fc-silenced mutant (GZ1 LALAPG) indicated that Fc-mediated functions played a critical role in the enhanced therapeutic effects. These findings highlighted the potential of anti-CD36 mAb in combination with Ara-C as a novel treatment strategy for overcoming drug resistance in AML.

Background:In fetal and neonatal alloimmune thrombocytopenia (FNAIT), maternal antibodies react with alloantigen expressed on fetal platelets, leading to their clearance via antibody-dependent phagocytosis. In Caucasians, most FNAIT cases are caused by anti-HPA-1a antibodies. In contrast, anti-HLA class I antibodies are rarely found in FNAIT, but are frequently implicated in cases of platelet transfusion refractoriness (PTR). This phenomenon leads to ongoing debate regarding the role of anti-HLA class I antibodies in FNAIT. In this study, we investigated the platelet clearance mediated by anti-HLA class I antibodies in whole blood both in vitro and in vivo. Methods:Clearance of opsonized platelet was analyzed by platelet phagocytosis assay and by antibody administration to Balb/c female mice. Results:To mimic FNAIT conditions, whole blood was pretreated with anti-HLA antibodies before the phagocytosis of anti-HPA-1a antibody-opsonized platelets. Compared to untreated whole blood, anti-HLA-ABC and anti-HLA-DR IgG antibodies inhibited the phagocytosis of anti-HPA-1a-antibody-opsonized platelets. Similar results were obtained with purified monocytes, indicating that anti-HLA-ABC antibodies bound to monocytes can interfere with antibody-mediated platelet phagocytosis. Furthermore, the administration of anti-MHC-I antibodies to mice led to a significant decrease in the platelet count within 24 h. However, anti-αIIbβ3 antibody administration resulted in significantly higher platelet clearance over different time points. Analysis of antibody-bound platelets showed the presence of anti-αIIbβ3 antibodies on the platelet surface, but not on monocytes. In contrast, anti-MHC-I antibodies were found on both platelets and monocytes. Interestingly, monocytes exhibited higher levels of anti-MHC-I binding than platelets (87.0% vs. 25.5%), most likely because platelets express significantly fewer HLA class I antigens than monocytes, as indicated by our flow cytometric analysis of whole blood. Conclusions:These results indicated that anti-MHC-I antibodies preferentially bind to monocytes rather than platelets in whole blood and can be cleared by monocytes via endocytosis. Furthermore, we found that the presence of anti-HLA class I antibodies did not significantly influence platelet clearance induced by anti-αIIbβ3 antibodies. The question of whether these observations can explain the controversial opinions regarding the relative roles of anti-HLA class I and anti-αIIbβ3 antibodies in FNAIT requires further assessment in a murine model of FNAIT.

Genetic testing is widely recommended in the diagnosis and management of breast cancer. This study aimed to investigate the knowledge, attitude, and practice (KAP) toward genetic testing in Chinese patients with breast cancer. This multicenter cross-sectional study enrolled breast cancer patients in seven public hospitals in Shanghai, China, between November 2022 and January 2023. A self-administered web-based questionnaire was used to collect the participants’ demographic information and their KAP regarding genetic testing. A total of 592 valid questionnaires were collected in this study; 145 (24.49%) patients underwent genetic testing, and 20.61% of the patients never learned about genetic testing. The knowledge, attitude, and practice scores were 4.59 ± 4.49 (22.95%, possible range: 0–20), 16.72 ± 2.92 (83.60%, possible range: 0–20), and 23.67 ± 5.18 (73.97%, possible range: 0–32), respectively. Multivariable logistic regression showed that knowledge (OR = 1.21, 95%CI: 1.51–1.28, P < 0.001), attitude (OR = 1.10, 95%CI: 1.01–1.19, P = 0.027), Jiangsu Province (OR = 0.40, 95%CI: 0.19–0.84, P = 0.016), monthly income of 5000–10,000 CNY (OR = 0.46, 95%CI: 0.25–0.86, P = 0.015), disease duration of 5–10 years (OR = 0.50, 95%CI: 0.27–0.94, P = 0.030) and disease duration of ≥ 10 years (OR = 0.26, 95%CI: 0.11–0.60, P = 0.002), triple-negative subtype (OR = 3.45, 95%CI: 1.51–7.85, P = 0.003) were independently associated with patients’ behavior of undergoing genetic testing. The structural equation modeling showed that knowledge directly positively influenced attitude (β = 0.343, P < 0.001), while attitude directly positively influenced practice (β = 0.942, P < 0.001). Chinese patients with breast cancer demonstrated poor knowledge, positive attitude, and suboptimal practice toward genetic testing. More education and counseling on genetic testing for patients are necessary.

INTRODUCTION: Transfusion-related acute lung injury (TRALI) is a life-threatening syndrome that occurs within six hours of a blood transfusion. MATERIALS AND METHODS: The objective of this study was to investigate the characteristics, clinical manifestations, outcomes, and risk factors of TRALI in patients who underwent surgery at a single medical centre of China. The retrospective study included 5,846 patients who received intraoperative and postoperative blood transfusions. All donors implicated in TRALI cases were tested for anti-HLA class I and II antibodies using LABScreen Multi assay and for granulocyte antibodies using granulocyte agglutination test. RESULTS: Four patients (4/5846, 0.068%) developed TRALI, with specific incidence of 0.26/100,000 blood product; 9/10,000 units of packed red blood cells (PRBC) (1/1,065); 16/10,000 units of fresh frozen plasma (FFP) (1/645); and 5/10,000 units of Apheresis platelets (AP)(1/2,120). In one patient, TRALI was found to be secondary to a human leukocyte antigen (HLA) class I antibody (anti-HLA-A*11); in another, it was secondary to an HLA class II antibody (anti-HLA-DRB*04). The one with possibility of reverse TRALI was identified as a result of anti-A*24. One of the four patients was non-immunologically mediated TRALI. CONCLUSIONS: To reduce the incidence and prevent TRALI, it is recommended that male donors and female donors who have never been pregnant or have tested negative for HLA antibodies since their last pregnancy should be selected for blood donation as much as possible. In addition, it is important to initiate clinical intervention and treatment at an early stage in order to improve the patient's prognosis.

Transfusion-related acute lung injury (TRALI) is a leading cause of blood transfusion-triggered mortality. Recently, we demonstrated the critical role of Fc-dependent complement activation in anti-CD36-mediated murine TRALI. In this study, we found that C5-/- mice were protected, and administration of anti-C5 rescued wild-type mice from anti-CD36-mediated TRALI. However, C5aR1-/- mice were not protected against anti-CD36-mediated TRALI, implying a possible role of C5b-9 (membrane attack complex [MAC]). Accordingly, elevated levels of MAC were detected in bronchoalveolar lavage fluid and lung tissue of mice with anti-CD36-induced TRALI. Inhibition of MAC formation by administration of anti-C7-blocking monoclonal antibody alleviated TRALI in mice, suggesting the critical role of the MAC in the pathology of anti-CD36-mediated TRALI. Furthermore, anti-C7 treatment also led to favorable outcome in murine TRALI induced by anti-major histocompatibility complex class 1, indicating the potential broader applicability of MAC inhibitors in the treatment of antibody-mediated TRALI. Therefore, this approach may be promising to further explore the treatment of patients with TRALI.

Objective To analyze CD36 gene by PacBio Sequel Ⅱ the third-generation sequencing technology(TGS),including non-coding sequence,and to investigate the molecular mechanism of CD36 deficiency.Methods Flow cytometry was performed in the southern Chinese population to detect the CD36 phenotype.Among them,15 cases of CD36 type Ⅰ defi-ciency,15 cases of CD36 type Ⅱ deficiency,and 10 positive samples were selected.The TGS of the CD36 gene was per-formed and statistical analysis was conducted.Results 40 samples(including 15 cases of type Ⅰ deficiency,15 cases of type Ⅱ deficiency,and 10 positive samples)were subjected by TGS of CD36 full-length sequences(except part of intron1).A total of 180 polymorphic loci were identified.Among them,13 kinds were in the coding region,the rest were in non-coding region,with most mutations located in regulatory regions such as the 5'-UTR and 3'-UTR.Conclusion The high polymorphism of CD36 non-coding regions,particularly in regulatory sequences,provides mechanistic insights into type Ⅱ CD36 deficiency.

Background: Natural killer (NK) cells are key effectors of innate immunity with broad-spectrum anti-tumor activity. However, peripheral blood-derived NK (PBNK) cells are typically quiescent, which limits their therapeutic utility. This study aimed to develop an efficient strategy for the in vitro activation and expansion of PBNK cells and then evaluate their potential anti-tumor efficacy in vitro and vivo. Methods: NK cells were isolated from healthy blood donors' peripheral blood and stimulated with anti-CD16 and anti-CD137 antibodies in the presence of interleukin-2 (IL-2) and interleukin-15 (IL-15) under serum-free conditions, generating super NK (SNK) cells. The expression levels of activating and inhibitory receptors on the expanded SNK cells were assessed by flow cytometry. Cytotoxicity against tumor cells was assessed at various effector-to-target (E:T) ratios in vitro. In vivo, anti-tumor efficacy was evaluated in K562-engrafted NSG mice. RNA sequencing was performed to identify differentially expressed genes (DEGs) between SNK and PBNK cells. Results: Stimulation with anti-CD16 and anti-CD137 antibodies resulted in significant expansion of donor-derived NK cells, with over 861.9 ± 48.84-fold expansion (n = 5) within 15 days of culture. SNK cells exhibited significantly elevated expression of activating receptors, including NKG2D. Functionally, SNK cells demonstrated superior cytotoxicity compared with PBNK cells across all tested E:T ratios in vitro and higher expressions of the effector molecules interferon-gamma (IFN-γ) and granzyme B (Gzm B). In vivo, adoptive SNK cell transfer resulted in significant tumor suppression and prolonged survival in a dose-dependent manner. Transcriptomic analysis revealed significant enrichment of DEGs associated with cytokine and chemokine signaling, immune activation, and cytotoxic effector function compared with the PBNK cells. Conclusions: Anti-CD16/CD137 antibody stimulation, in combination with IL-2 and IL-15, facilitates robust activation and rapid expansion of functionally enhanced NK cells from peripheral blood. The resulting SNK cells demonstrated enhanced anti-tumor efficacy both in vitro and in vivo and may be used as allogeneic NK cell-based immunotherapy in future cancer treatment strategies.

Objective To explore the role of the c.598G>A mutation of the ITGB3 gene in the occurrence of fetal and neonatal alloimmune thrombocytopenia(FNAIT)through its expression in vitro.Methods The platelet antibodies in the sera of the affected neonate and her mother were detected using commercial enzyme-linked immunosorbent assay(ELISA),solid-phase agglutination,flow cytometry and the gold standard monoclonal antibody-specific immobilization of platelet anti-gens(MAIPA).The common human platelet antigen(HPA)genotypes of the neonate and her parents were obtained using the HPA-SSP method.The presence of mutations was analyzed by sequencing the exons of the ITGB3 and ITGA2B genes.The target gene of ITGB3 was obtained by PCR amplification using the existing human platelet cDNA.The wild-type ITGB3 eukaryotic expression vector was constructed by TA cloning technology.The 598G>A mutant ITGB3 eukaryotic expression vector was obtained by point mutation,and the plasmid DNA was co-transfected with that of ITGA2B(αⅡb)into HEK293 cells.The transfected cells stably expressing GPⅡb/Ⅲa were screened and obtained.The expression of GPⅡb/Ⅲa in 598G>A mutant transfected cells and the presence of antibodies against this mutation in the serum of mother were detected by flow cytometry and MAIPA.Results Antibodies against HLA-class Ⅰ and GPⅡb/Ⅲa glycoproteins were detected in the serum of the neonate's mother,and subsequent HLA antibody-specific testing confirmed the presence of antibodies a-gainst HLA-B∗57∶01 and A∗02∶05.ITGB3 sequencing showed that the neonate and her father carried the c.598G>A point mutation,which results in the change of glutamate to lysine at position 200.Antibodies against GPⅡb/Ⅲa glycoproteins were not detected using constructed c.598G>A mutant transfected cells reacted with the maternal serum.Conclusion The in vitro expression and analysis of the ITGB3 c.598G>A mutation did not support a role for this mutation in the pathogenesis of FNAIT.The establishment of this method facilitates the discovery of new platelet low-frequency antigens,and provides a theoretical foundation for the detection of antibodies against platelet antigens associated with patients with adverse pregnancy and childbirth histories.

Objective To analyze the structure of CD36,search the possible B cell epitopes and prepare multi-antigen peptides(MAP)with B cell epitopes,so as to provide a preliminary experimental basis for the preparation of CD36 antibod-ies using MAP with B cell epitopes.Methods The potential B cell epitopes of CD36 were analyzed by bioinformatics meth-ods,including physical and chemical properties,secondary structure,potential phosphorylation and glycosylation sites.Eight-branch MAP with CD36 B cell epitopes were synthesized by FMOC using polylysine as the core matrix.The purity of MAPs was analyzed by reverse high-performance liquid chromatography chromatography(RP-HPLC),and the molecular weight of MAPs was determined by mass spectrometry.Results CD36 is a stable and hydrophilic alkaline protein,with multiple phosphorylation and glycosylation sites and strong antigenicity,and its secondary structure is mainly characterized by irregular curls.Four potential B cell epitopes were obtained and 4 MAPs containing potential B cell epitopes were pre-pared.RP-HPLC analysis showed that the purity of the MAPs were above 85%,and the molecular weight of 3 MAPs was consistent with the expected theoretical molecular weight.Conclusion CD36 on platelet has strong antigenicity.MAPs con-taining CD36 B cell epitopes can provide the experimental basis for the preparation and related research of CD36 antibodies.

Objective To prepare a Fc-silent chimeric antibody against human CD36 and analyse its bioactivity and phagocytic effect on CD36(+) platelets. Methods The genes encoding VH and VL fragments of the monoclonal antibody (mAb) 32-106 were obtained through RNA extraction from hybridoma cell lines, PCR amplification and sequence analysis. By using gene synthesis and recombination techniques, the vectors expressing light and heavy chains of the chimeric antibody against human CD36 were constructed. Stable cell lines secreting the chimeric antibody were established by selective culture using Zeocin and Blasticidin. Antibodies were purified by an affinity chromatography column. The purity and molecular weight of the chimeric antibody were detected by SDS-PAGE. The activity of the antibody binding to CD36 antigen was determined by ELISA and flow cytometry. The ability of the chimeric antibody to participate in CD36(+) platelet phagocytosis was analyzed by a platelet phagocytosis assay. Results The vectors expressing light and heavy chains of chimeric antibody were successfully constructed. After co-transfection into HEK293 cells, stable cell lines secreting chimeric antibody were obtained by screening and cloning. The high purity and correct molecular weight of the chimeric antibody were confirmed by protein silver staining. The results of Flow cytometry and ELISA showed that the chimeric antibody had the activity of binding to human CD36 antigen. Platelet phagocytosis assay showed that the Fc-silent chimeric antibody against human CD36 basically lost the ability of mediating monocyte phagocytosis of CD36(+) platelets. Conclusion In this study, the Fc-silent chimeric antibody against human CD36 was successfully prepared, and its loss of ability to mediate CD36(+) platelet clearance was confirmed in vitro, which provides a preliminary basis for the study of modified antibodies for the therapy of CD36-antibody-mediated fetal and neonatal alloimmune thrombocytopenia(FNAIT).

Abstract OBJECTIVE: Individuals with a cluster of differentiation 36 (CD36) deficiency are at risk of developing anti-CD36 antibodies after platelet transfusion or pregnancy. Therefore, we investigated CD36 expression on platelets of blood donors in Zhongshan city, and analyzed the frequency of CD36 deficiency and gene polymorphism. METHODS: A total of 846 healthy donors were randomly selected. CD36 expression on platelets was tested and monocytes from CD36-negative samples were further analyzed to determine the type of CD36 deficiency through flow cytometry. Genetic variation was analyzed through genomic DNA sequencing of exons 3–14 in the protein-coding region of CD36. Anti-CD36 antibody detection for the donors with CD36 deficiency was performed. RESULTS: The frequency of CD36-negative platelets in blood donors was 1.77% (15/846), of which Type I deficiency accounted for 0.12% (1/846) and Type II accounted for 1.65% (14/846). Seven types of gene mutations in eight cases of CD36-negative samples were discovered: 329_330 del AC ( n = 2), 287 G > C ( n = 1), 220 C > T and 1228_1239 ATTGTGCCTATT ( n = 1), 1228_1239 ATTGTGCCTATT ( n = 1), 538 T > C and 879 T > C ( n = 1), 287 G > C and 1163 A > T ( n = 1), and 429 + 3 dup G ( n = 1). Moreover, the anti-CD36 antibody was detected in the plasma of donors with Type I CD36 deficiency. CONCLUSION: A new mutation site in exon 5 (429 + 3 dup G) was identified. The establishment of a CD36-negative database can provide compatible platelets to improve the efficacy of transfusion.

CD36, also known as glycoprotein IV, is classified into two distinct subgroups based on the presence or absence of its expression on monocytes. The CD36 gene spans approximately 50,000 base pairs. Historically, research has focused on identifying CD36 mutations through Sanger sequencing and next-generation sequencing (NGS), with limited exploration of haplotypes. In this study, we collected blood samples from donors with type I and type II CD36 deficiencies as well as from healthy controls, and employed single-molecule long-read sequencing (also known as Third-Generation Sequencing) of genomic DNA to analyze the genetic basis of CD36. The study identified 180 genetic variants, 12 of which were found to alter the amino acid sequence. Notably, four of these mutations (c.220 C > T; c.329_330delAC; c.430-1 G > C; c.1006 + 2 T > G) are premature termination mutations that lead to protein truncation. Using Fisher’s exact test, we statistically analyzed a specific haplotype, c.-132A > C and c.329_330delAC, along with their clinical phenotypes, revealing a strong association between these variants in the 5’ block and type I CD36 deficiency. We analyzed the CD36 gene sequences in platelet donors and patients with PTR (platelet transfusion refractoriness) and FNAIT (fetal and neonatal alloimmune thrombocytopenia), conducting a detailed haplotype analysis associated with type I CD36 deficiency and FNAIT.

Antibodies against human CD36 are responsible for several immune-mediated disorders. The detection of anti-CD36 antibodies using the standard monoclonal antibody (mAb) immobilization of platelet antigens (MAIPA) assay is hampered by a high frequency of false-negative results, most likely due to competitive inhibition of the mAb used as the capture antibody. We generated a panel of mouse mAbs against CD36 and seven hybridomas (GZ-3, GZ-13, GZ-70, GZ-143, GZ-413, GZ-507, and GZ-608), which were selected for MAIPA assays, as they reacted with mouse and human CD36. Fourteen anti-CD36 sera were assayed; all of which showed a positive reaction in a PakPlus (Immucor GTI Diagnostics, Inc., Waukesha, WI, USA) ELISA-based screening (optical density: 0.257-2.292). When the reference anti-CD36 mAb FA6-152 was used in the MAIPA assay, only 6/14 (42.9%) sera displayed a positive reaction. In contrast, anti-CD36 antibodies were detected in 13/14 (92.9%) sera when GZ-70 and GZ-608 mAbs were used. This significant improvement resulted in the identification of anti-CD36 antibodies by an antigen capture assay. Since patient's platelets possibly carrying rare native antigens are used, this method will facilitate the identification of new platelet antibodies against CD36 that are involved in immune-mediated thrombocytopenia and other diseases, such as transfusion-related acute lung injury.

Anti-CD36 Abs have been suggested to induce transfusion-related acute lung injury (TRALI) upon blood transfusion, particularly in Asian populations. However, little is known about the pathological mechanism of anti-CD36 Ab–mediated TRALI, and potential therapies have not yet been identified. Here, we developed a murine model of anti-CD36 Ab–mediated TRALI to address these questions. Administration of mouse mAb against CD36 (mAb GZ1) or human anti-CD36 IgG, but not GZ1 F(ab′)2 fragments, induced severe TRALI in Cd36+/+ male mice. Predepletion of recipient monocytes or complement, but not neutrophils or platelets, prevented the development of murine TRALI. Moreover, plasma C5a levels after TRALI induction by anti-CD36 Abs increased more than 3-fold, implying a critical role of complement C5 activation in the mechanism of Fc-dependent anti-CD36–mediated TRALI. Administration of GZ1 F(ab′)2, antioxidant (N-acetyl cysteine, NAC), or C5 blocker (mAb BB5.1) before TRALI induction completely protected mice from anti-CD36–mediated TRALI. Although no significant amelioration in TRALI was observed when mice were injected with GZ1 F(ab′)2 after TRALI induction, significant improvement was achieved when mice were treated postinduction with NAC or anti-C5. Importantly, anti-C5 treatment completely rescued mice from TRALI, suggesting the potential role of existing anti-C5 drugs in the treatment of patients with TRALI caused by anti-CD36.

Objective To explore the pathogenesis of fetal edema caused by CD36 antibody in fetal/neonatal alloimmune thrombocytopenia (FNAIT), and to provide reference for clinical prevention and treatment. Methods The established CD36 monoclonal antibody was incubated with human peripheral blood mononuclear cells (PBMC), and the concentrations of cytokines (TNF-α and IL-1β) in the supernatant of cell culture were detected by ELISA. The permeability of endothelial cells were investigated by detecting the fluorescence intensity of FITC-albumin by incubating cytokine-rich cell supernatant with human umbilical vein endothelial cells (HUVEC). Results Flow cytometry showed that CD36 monoclonal antibody could bind to human monocytes. Compared with isotype IgG control, increased cytokine TNF-α (pg/mL) (407.73±20.40 vs 29.38 ±4.72, P<0.05) and IL-1β (pg/mL) (247.14±83.59 vs 53.68±26.96, P<0.05) were detected in the supernatant of cell culture after incubation of CD36 monoclonal antibody with human PBMC. Detection of fluorescence intensity of FITC-albumin in transwell cultured HUVEC showed that cytokine-rich cell supernatant derived from CD36 monoclonal antibody incubated with human PBMC can increase the permeability of endothelial cells significantly (CD36 antibody vs isotype IgG, MFI value: 492±16 vs 320±11, P<0.05). Conclusion The effect of CD36 monoclonal antibody on PBMC can increase HUVEC permeability, which may be one of the pathogenesis of fetal edema with FNAIT.

目的 了解广州地区人类白细胞抗原(HLA)-Ⅰ类抗体所致血小板输注无效(PTR)患者的HLA抗体特异性及高频抗原决定簇.方法 采用ELISA方法对209例PTR患者的HLA-Ⅰ类抗体进行检测.运用Luminex检测平台,通过HLA特异性抗体检测试剂盒(LIFECODES LSA? CLASS Ⅰ)检测患者血清中针对HLA-A、B及Cw的特异性抗体;经MATCH IT!和HLA Matchmaker软件分析,获得患者HLA-Ⅰ类抗体特异性及相应epitopes.统计分析PTR患者血清中针对HLA-A、B、Cw位点的特异性抗体及epitopes的阳性频率.结果 209份HLA-Ⅰ类抗体阳性PTR患者标本中共检出95种特异性抗体,其中针对HLA-A位点的抗体29种,以A*24:02(42.58％)最常见;检出针对HLA-B位点的抗体 48 种,阳性频率较高的有 B*27:08(50.72％)、B*82:02(45.45％)、B*15:12(44.98％)、B*07:02(44.02％)、B*35:01(42.58％)、B*35:08(42.58％)和B*27:05(42.11％);针对血小板低表达Cw位点的抗体仅检出18种,常见C*07:01(19.62％)和C*07:02(18.66％).运用 HLA Matchmaker 软件分析,共检出 182 种 HLA-Ⅰ 类 epitopes,以 163 LG(19.28％)、71 KA(16.27％)、30 G(15.06％)、163 EW(11.45％)和 127 K(11.45％)相对高频.结论 获得广州地区PTR患者体内HLA-Ⅰ类抗体的分布特点,对血小板供者库的建立以及Eplet配型方法的开展提供理论依据.