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 antigens (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 GP Ⅱb/Ⅲa were screened and obtained. The expression of GP Ⅱb/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 GP Ⅱb/Ⅲa glycoproteins were detected in the serum of the neonate's mother, and subsequent HLA antibody-specific testing confirmed the presence of antibodies against 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 GP Ⅱb/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 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 antigens (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 GP Ⅱb/Ⅲa were screened and obtained. The expression of GP Ⅱb/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 GP Ⅱb/Ⅲa glycoproteins were detected in the serum of the neonate's mother, and subsequent HLA antibody-specific testing confirmed the presence of antibodies against 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 GP Ⅱb/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.

ObjectiveTransfusion-Related Acute Lung Injury （TRALI） is a common fatal transfusion adverse reaction. Major Histocompatibility Complex （MHC） class I is an important factor involved in the pathogenesis of TRALI； however， the role of complement in itspathogenesis has not been fully elucidated. This study aims to explore the role of complement in MHC class I antibody-mediated TRALI， so as to provide a theoretical basis for clinical prevention and treatment. MethodsThis study established a murine model of transfusion-related acute lung injury （TRALI） based on the "two-hit" theory， with lipopolysaccharide （LPS） as the first hit and MHC class I antibody as the second hit. Male Balb/c mice were randomly divided into seven groups （n=5 per group per experiment）： Naive （blank control）， LPS （first hit only）， Isotype （isotype antibody control）， TRALI （model group）， C5aR1 inhi （C5aR1 antagonist intervention）， C5aR2 inhi （C5aR2 antagonist intervention）， and Anti-C5 （anti-complement C5 antibody intervention）. Rectal temperature was monitored after MHC class I antibody injection. After sample collection， the severity of pulmonary edema was assessed by measuring the lung wet-to-dry weight ratio， histological analysis， and immunohistochemistry. Serum and bronchoalveolar lavage fluid were collected to measure cytokine and complement levels. ResultsMice in the TRALI group exhibited a significant decrease in rectal temperature， an increased lung wet-to-dry weight ratio， elevated serum cytokine levels， and markedly heightened complement C5a levels in bronchoalveolar lavage fluid （P＜0.000 1）. Histopathological examination revealed substantial infiltration of inflammatory cells， predominantly neutrophils accompanied by fewer lymphocytes， plasma cells， and monocytes， along with increased deposition of the membrane attack complex C5b-9 in lung tissues. In contrast， mice treated with anti-C5 antibody demonstrated no significant decrease in rectal temperature. The lung wet-to-dry weight ratio in this group showed no statistical difference compared to either the Naive or Isotype control groups （P＞0.05）. Furthermore， these mice displayed reduced serum cytokine levels， a significant attenuation of inflammatory cell infiltration in the lungs， and a 100% survival rate at the 2-hour time point. However， mice administered either the C5aR1 antagonist or the C5aR2 antagonist failed to be protected and subsequently developed TRALI. ConclusionComplement activation， which forms the membrane attack complex C5b-9， plays a critical role in MHC class I antibody-mediated TRALI. Blocking complement C5 activation can effectively prevent the occurrence of TRALI.

Objective To investigate the impact of statistical uncertainty per control point(SUpCP)on dose calculation on volumetric modulated arc therapy(VMAT)for rectum cancer,and to analyze the accuracy and efficiency of calculation.Methods 19 patients with rectum cancer undergoing radiotherapy were selected.The initial VMAT plans were generated on Monaco TPS using SUpCP=3,then changed SUpCP in the dose calculation process as follow:10 SUpCPs(1～10)for each patient,and totally190 VMAT dose distributions were obtained.For plan evaluation,Dmax,Dmean,D95%,V50,homogeneity index(HI),conformity index(CI)of the planning target volume(PTV),dissymmetric variations of bladder,small intestine and femoral head,and time calculation(Time)were analyzed.Patient specific quality assurance(PSQA),dose deviation of isocenter(ΔDISO)and passing rate of three-dimensional dose distribution(γ33,γ32,γ22)between calculated and delivered radiation doses were measured.Results AsSUpC increased,Dmax and HI of PTV,Dmax of bladder were increased,but D95%and V50 of PTV,Time,γ32 and γ22 were decreased(P<0.05).Dmax and CI of PTV,Dmean of bladder,Dmax and Dmean of small intestine and femoral head,ΔDISO and γ33 showed no statistical significance(P>0.05).When ΔDISO<1%,gamma passing rate>90%for all VMAT plan.When SUpCP<6,Dmax of PTV<110%of the prescribed dose was obtained;while SUpCP>2,time for dose calculation was less than 5 min.Conclusion For VMAT plan of rectum cancer on Monaco TPS using XVMC algorithm,3%～5%of statistical uncertainty per control point for dose calculation,and 3%2 mm or 2%2 mm gamma criteria for three-dimensional dose verification is recommended.This study provides clinical application basis for precise dose calculation of VMAT plan of rectum cancer.

Objective To investigate the expression of targeted regulation of microRNA-146a (miR-146a) on E-box zinc finger protein 1 gene (ZEB1) and its related molecular mechanisms involved in neuroprotection and autophagy inhibition in a rat model of cerebral hemorrhage. Methods Forty 8-week-old male SD rats were randomly divided into four groups: sham operation group, model group, miR-146a overexpression group and miR-146a underexpression group, with 10 rats in each group. Except for the sham operation group, the other three groups were induced to establish an aneurysmal spontaneous intracerebral hemorrhage rat model using type VII collagenase. Rats of four groups were fed a high-salt diet for 6 weeks and euthanized after an additional feeding for 20 weeks. Neurological function [modified neurologic severity score (mNSS)] and brain water content were compared among the four groups. FJC staining was used to detect the number of degenerated neurons; TUNEL staining was used to detect apoptosis; hematoxylin-eosin (HE) staining was used to observe pathological changes in brain tissue; quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-146a and ZEB1 mRNA; Western blot was used to detect the expression of ZEB1, autophagy-related proteins human recombinant autophagy effector protein (Beclin 1) and microtubule-associated protein 1 light chain 3 (LC3). Results Compared to the sham surgery group, the model group showed a significant increase in mNSS score, brain water content, the number of degenerated neurons and cell apoptosis rate, and the expression level of miR-146a was significantly decreased, while the expressions of ZEB1 mRNA, as well as ZEB1, Beclin 1and LC3 proteins, were significantly increased in the model group (P < 0.05). Compared to the model group, the miR-146a overexpression group exhibited a significant decrease in mNSS score, brain water content, the number of degenerated neurons and cell apoptosis rate, and the expression level of miR-146a was significantly increased, while the expressions of ZEB1 mRNA, as well as ZEB1, Beclin 1 and LC3 proteins were significantly reduced in the miR-146a overexpression group (P < 0.05). Compared to the model group, the miR-146a low-expression group showed a further increase in mNSS score, brain water content, the number of degenerated neurons and cell apoptosis rate, and the expression level of miR-146a was further decreased, while the expressions of ZEB1 mRNA, as well as ZEB1, Beclin 1 and LC3 proteins were further elevated in the miR-146a low-expression group (P < 0.05). Conclusion Upregulating miR-146a can exert neuroprotective effects by targeting and inhibiting ZEB1 gene and protein expression, as well as regulating autophagy activity.

【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.

【Objective】 To search compatible and suitable platelets for platelet transfusion refractoriness (PTR) patient caused by compound antibodies against HLA and CD36. 【Methods】 ELISA method was used to detect the antibody against platelet antigens and the specificity of HLA-I antibody in PTR patients. The specificity of HLA-I antibody and corresponding epitopes of patients were analyzed using MATCH IT! and HLA Matchmaker software. The HLA genotype of both donor and patient was obtained by HLA-SSO method. Compatible or suitable donor platelets for PTR patients were searched through cross-reactive group (CREG) of HLA-I and HLA epitope-matched approach (Eplet). The matching degree was identified using monoclonal antibody-specific immobilization of platelet antigens (MAIPA) and the platelet suspension immunofluores-cence test (PIFT). Finally, the transfusion effect was evaluated according to the corrected count increment (CCI). 【Results】 Compound antibodies against both CD36 and HLA-I antigens were detected in two PTR patients, and their phenotype of CD36 was conformed to be type I deficient. Through LSA testing, high-frequency of HLA -I antibodies was found in these two patients, and the panel reactive antibody in patients 1 and 2 was 56% (54/96) and 53% (51/96), respectively. According to HLA-CREG and Eplet matching strategies, one donor of grade C-matching with patient 1 and one donor of grade D-matching with patient 2 were screened from the CD36 deficiency donor bank, respectively. And the selected donors avoided the antigen of HLA-I antibody epitope. These results of MAIPA and PIFT also confirmed that no immune response was detected between the patient and the donor. And a CCI of >4.5 within 24 hour of transfusion of compatible platelets was obtained in patient 2. 【Conclusion】 For PTR patients caused by HLA and CD36 compound antibodies, a combination strategy including serological cross-matching, HLA-CREG and Eplet approach should be used to select the CD36 deficient donor platelets which evaded the antigen corresponding to HLA-I antibodies and had the compatible HLA epitopes.

Objective: To understand the prevalence rate of pre myopia among preschoolers and its associated factors, so as to provide a basis for the formulation of myopia prevention and control policies. Methods: In May 2022, a tatol of 1 070 preschoolers in the Science City Area of Mianyang City were selected by using a cluster sampling method. The visual acuity of preschool children was obtained through refractive examination, and a questionnaire survey was conducted to investigate associated factors of myopia in the preclinical stage by Chi square test and multivariate Logistic regression analysis. Results: The prevalence rate of pre myopia in preschoolers was 67.01%, including 66.30% for boys and 67.76% for girls. There was no significant difference by sex( χ 2=0.26, P > 0.05). Multivariate Logistic regression analysis showed that at least one parent with myopia, too much near work activities, time spent on outdoor activity <2 h per day, lack of sleep, and screen time ≥1 h per day were positively correlated with the occurrence of pre myopia( OR =1.62, 1.46, 1.46, 1.38, 1.53, P <0.01). Conclusion The prevalence of pre myopia among preschool children in Science City Area is high. It is suggested that parents, kindergartens and relevant departments should pay attention to the visual status of preschoolers, increase outdoor activities, ensure adequate sleep and train preschoolers to develop scientific eye hygiene habits.

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.

OBJECTIVE: To compare the performance of Clear Cell Likelihood Score (ccLS) v1.0 and v2.0 in diagnosing clear cell renal cell carcinoma (ccRCC) from small renal masses (SRM). METHODS: We retrospectively analyzed the clinical data and MR images of patients with pathologically confirmed solid SRM from the First Medical Center of the Chinese PLA General Hospital between January 1, 2018, and December 31, 2021, and from Beijing Friendship Hospital of Capital Medical University and Peking University First Hospital between January 1, 2019 and May 17, 2021. Six abdominal radiologists were trained for use of the ccLS algorithm and scored independently using ccLS v1.0 and ccLS v2.0. Random- effects logistic regression modeling was used to generate plot receiver operating characteristic curves (ROC) to evaluate the diagnostic performance of ccLS v1.0 and ccLS v2.0 for ccRCC, and the area under curve (AUC) of these two scoring systems were compared using the DeLong's test. Weighted Kappa test was used to evaluate the interobserver agreement of the ccLS score, and differences in the weighted Kappa coefficients was compared using the Gwet consistency coefficient. RESULTS: In total, 691 patients (491 males, 200 females; mean age, 54 ± 12 years) with 700 renal masses were included in this study. The pooled accuracy, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of ccLS v1.0 for diagnosing ccRCC were 77.1%, 76.8%, 77.7%, 90.2%, and 55.7%, as compared with 80.9%, 79.3%, 85.1%, 93.4%, 60.6% with ccLS v2.0, respectively. The AUC of ccLS v2.0 was significantly higher than that of ccLS v1.0 for diagnosis of ccRCC (0.897 vs 0.859; P < 0.01). The interobserver agreement did not differ significantly between ccLS v1.0 and ccLS v2.0 (0.56 vs 0.60; P > 0.05). CONCLUSION ccLS v2.0 has better performance for diagnosing ccRCC than ccLS v1.0 and can be considered for use to assist radiologists with their routine diagnostic tasks.
Female ; Male ; Humans ; Adult ; Middle Aged ; Aged ; Carcinoma, Renal Cell/diagnosis* ; Retrospective Studies ; Kidney ; Carcinoma ; Kidney Neoplasms/diagnosis*