Objective Currently, there is no commercially available quantitative detection kit for the main Felis domestic allergen (Fel d 1) in China. To establish a rapid detection method for Fel d 1, this study aims to prepare monoclonal antibodies against Fel d 1 protein. Methods The codon preference of Escherichia coli was utilized to optimize and synthesize the Fel d 1 gene. The prokaryotic expression plasmid pET-28a-Fel d 1 was constructed and used to express and purify the recombinant Fel d 1 protein. Subsequently, the recombinant protein was immunized into BALB/c mice and monoclonal antibodies (mAbs) were prepared by the hybridoma technique. An indirect ELISA was established using the recombinant Fel d 1 as the coating antigen, and hybridoma cell lines were screened for positive clones. The specificity and antigenic epitopes of the mAbs were confirmed by Western blot analysis. Finally, the selected hybridoma cells were injected into the peritoneal cavities of BALB/c mice for large-scale monoclonal antibody production. Results The recombinant plasmid pET-28a-Fel d 1 was successfully constructed, and soluble Fel d 1 protein was obtained after optimizing the expression conditions. Western blot and antibody titer assays confirmed the successful isolation of two hybridoma cell lines, 7D11 and 5H4, which stably secreted mAbs specific to Fel d 1. Antibody characterization revealed that the 5H4 mAb was of the IgG2a subtype and could recognize the amino acid region 105-163 of Fel d 1, while the 7D11 mAb was the IgG1 subtype and could recognize the amino acid region 1-59. Conclusion The high-purity recombinant Fel d 1 protein produced in this study provides a promising alternative for clinical immunotherapy of cat allergies. Furthermore, the monoclonal antibody prepared in this experiment lays a material foundation for the in-depth study of the biological function of Fel d 1 and the development of ELISA detection.
Animals ; Antibodies, Monoclonal/biosynthesis* ; Mice, Inbred BALB C ; Cats ; Mice ; Allergens/genetics* ; Glycoproteins/genetics* ; Enzyme-Linked Immunosorbent Assay ; Hybridomas/immunology* ; Recombinant Proteins/genetics* ; Female ; Antibody Specificity

Dent disease is a rare X-linked recessive inherited renal tubular disorder characterized by low molecular weight proteinuria (LMWP), hypercalciuria, nephrocalcinosis, and other clinical features, and can lead to progressive renal failure. It is primarily caused by mutations in the CLCN5 gene. This article reports the case of a 10-year-old male patient of Chinese descent who was incidentally found to have asymptomatic proteinuria during a routine health examination. Comprehensive biochemical testing and clinical evaluation revealed significant LMWP and hypercalciuria, while renal biopsy showed mesangial cell and matrix proliferation. Whole exome sequencing identified a novel deletion mutation in the CLCN5 gene (NM_001127899.4, c.1158delC, p.F387Lfs*42) causing a frameshift and premature termination, which is likely to disrupt its role in chloride/hydrogen ion exchange and endosomal acidification. Bioinformatic analysis indicated the variant is pathogenic. Genetic testing plays an important role in diagnosing rare kidney diseases. Early identification of pathogenic mutations is essential for facilitating timely intervention and appropriate management, potentially enhancing patient outcomes. This report expands the CLCN5 mutation spectrum and contributes to understanding the genetic and molecular mechanisms of Dent disease.
Humans ; Chloride Channels/genetics* ; Dent Disease/genetics* ; Male ; Child ; Computational Biology ; Mutation ; Proteinuria/genetics* ; Hypercalciuria/genetics*

Objective:To evaluate the value of deep-inhaled breath-hold (DIBH)-30s scanning with total-body PET/CT under half-dose injection mode in improving the poor alignment of thoracic and upper abdominal tumors.Methods:Forty-six patients (28 males, 18 females, age (57.3±11.4) years) who underwent half-dose 18F-FDG total-body DIBH-30s PET/CT examination because of suspect or confirmed thoracic and upper abdominal tumors in Sun Yat-Sen University Cancer Center between October 2022 and February 2023 were analyzed retrospectively. SUV, standard deviation (SD) and signal-to-noise ratio (SNR) of the liver and mediastinal blood pool in free breath (FB)-8min, FB-30s and DIBH-30s PET images were measured; SUV of lesions, metabolic tumor volume (MTV) and tumor-to-background ratio (TBR) in DIBH-30s and FB-8min images were also measured; maximum diameter of contraposition offset and offset rate in coronal, transverse and sagittal directions of lesions in DIBH-30s and FB-8min images were calculated. Five-point Likert scale was used to score the overall image quality, image noise level and diagnostic confidence of fused images. Kruskal-Wallis rank sum test, Nemenyi test or Mann-Whitney U test was used to compare the parameters of different groups. Results:Among the 46 patients, 38 successfully completed breath-holding collection, and 80 lesions were detected, including 37 in the lungs and 43 in the livers. The liver SUV max (3.40(3.15, 3.63), 3.44(3.06, 3.70)) and SD (0.36(0.32, 0.41), 0.35(0.30, 0.40)) in DIBH-30s group and FB-30s group were higher than those (SUV max: 2.73(2.45, 2.92), SD: 0.15(0.13, 0.17)) in FB-8min group ( H values: 49.79, 85.27, χ2 values: 3.26-3.65, all P<0.001). The SUV max and the SD of mediastinal blood pool in DIBH-30s group and FB-30s group were also higher ( H values: 9.31, 59.73, χ2 values: 2.13-2.75, all P<0.01), while SNR liver and SNR med in those 2 groups were lower ( H values: 87.90, 54.11, χ2 values: 3.36-5.47, all P<0.001). The image noise scores of DIBH-30s group and FB-30s group were lower than the score of FB-8min group (3(3, 3) vs 3(3, 4) vs 5(5, 5); H=93.02, χ2 values: 2.13, 2.23, all P<0.001). The overall image quality score and diagnostic confidence score of DIBH-30s group were higher than those of FB-30s group and FB-8min group ( H values: 70.13, 24.22, χ2 values: 2.11-2.48, all P<0.001). The SUV and TBR of lesions in DIBH-30s group were higher than those of FB-8min group ( Z values: from -3.82 to -2.44, all P<0.05), while the MTV, contraposition offset and offset rate were lower than those of FB-8min group ( Z values: from -6.20 to -3.18, all P<0.001). Conclusions:DIBH-30s scanning with total-body PET/CT can make the focus alignment more accurate, which is suitable for short-time collection or low drug administration activity. It has a unique value in improving the poor focus alignment of chest and upper abdomen tumors.

This paper summarized the experience of professor WANG Yongyan in differentiating and treating vascular cognitive impairment （VCI）. It is believed that the core etiology and pathogenesis of VCI are deficient qi retention and stagnation， and toxin damaging brain collaterals. The essence lies in depletion of essence and qi， and malnutrition of brain collaterals as the root cause， while phlegm， turbidity， stasis， and heat blocking brain collaterals act as the branch aspects. It reveals the pathogenesis essence of VCI as stagnation caused by deficiency and qi stagnation transforming into excess， internal generation of toxin and collateral injury. The overall therapeutic principle is to tonify deficiency， move stagnation， resolve toxin， and unblock the collaterals， guided by the three core treatment methods of nourishing， unblocking， and clearing. Clinically， according to the different characteristics of the plateau phase， fluctuation phase， and decline phase， treatment methods including replenishing qi and blood， tonifying the kidney and supplementing essence； promoting sanjiao （三焦） circulation and activating blood to dredge collaterals； clearing heat and resolving toxin， eliminating phlegm and reducing turbidity are respectively adopted. This provides clinical guidance for the treatment of VCI in traditional Chinese medicine.

OBJECTIVE To explore the changes of serum inflammatory factors and immune function indexes in the children with asthma induced by Mycoplasma pneumoniae infection so as to make a new breakthrough in diagnosis and treatment of the children with M.pneumoniae infection-induced asthma.METHODS A total of 118 children with M.pneumoniae infection who were treated in pediatrics department of The Affiliated Hospital of Binzhou Medical College from Jan.2020 to May 2023 were enrolled in the study and were divided into the asthma group with 26 cases and the no asthma group with 92 cases according to the status of induction of asthma.The levels of peripheral blood interleukin(IL)-1β,IL-18,tumor necrosis factor-α(TNF-α)and immune function indexes(CD3+,CD4+,CD8+and CD4+/CD8+)were observed and compared between the two groups.The values of the above indexes in diagnosis of the M.pneumoniae infection-induced asthma were analyzed by means of receiver op-erating characteristic(ROC)curves.RESULTS There were significant differences in the levels of inflammatory factors and immune function indexes between the asthma group and the no asthma group(P＜0.05).The levels of TNF-α,IL-1β,IL-18 and CD8+of the asthma group were(46.33±5.30)pg/ml,(50.60±6.33)pg/ml,(40.26±4.89)pg/ml and(40.11±6.85)％,respectively,higher than those of the no asthma group;the levels of CD3+,CD4+and CD4+/CD8+of the asthma group were(43.33±5.89)％,(15.62±3.61)％and(0.35±0.12),re-spectively,lower than those of the no asthma group.In the asthma group,the levels of TNF-α,IL-1β,IL-18 and CD8+of the MP-Immunoglobulin M(IgM)-positive children were higher than those of the MP-IgM-negative chil-dren(P＜0.05),while the levels of CD3+,CD4+and CD4+/CD8+of the MP-IgM-positive children were lower than those of the MP-IgM-negative children(P＜0.05).The areas under the ROC curves(AUCs)of the serum TNF-α,IL-1β,IL-18,CD3+,CD4+,CD8+and CD4+/CD8+were 0.812,0.805,0.795,0.814,0.768,0.805 and 0.841,respectively,in diagnosis of the M.pneumoniae infection-induced asthma.CONCLUSION The serum TNF-α,IL-1β,IL-18,CD3+,CD4+,CD8+and CD4+/CD8+of the children with M.pneumoniae infection can be used for prediction of the asthma.

Objective To explore the effect of Omaha system continuity nursing on the degree of pain and quality of life in patients undergoing arthroscopic rotator cuff repair.Methods A total of 110 patients who underwent arthroscopic rotator cuff repair at the First Affiliated Hospital of Wenzhou Medical University from September 2022 to May 2023 were selected.They were divided into intervention group and control group according to the random number table method,with 55 cases in each group.Patients in control group were given conventional continuous nursing,while patients in intervention group were given Omaha system continuity nursing.Both groups of patients were intervened for 6 months.The range of motion of shoulder joint,shoulder joint function,degree of pain and quality of life of two groups of patients before and after the intervention were compared.Results After the intervention,the range of motion of shoulder joint in intervention group was significantly greater than that in control group,University of California,Los Angeles shoulder joint score and the 8-item short form health survey were significantly higher than those in control group,and numerical rating scale score of pain was significantly lower than that in control group(P＜0.05).Conclusion Omaha system continuity nursing can improve shoulder joint mobility and function,alleriate pain,and improve quality of life for patients undergoing arthroscopic rotator cuff repair surgery.

BACKGROUND:Postoperative adjuvant chemotherapy is a common method for the treatment of gastric cancer,but the curative effect of chemotherapy in different patients varies considerably.A new pre-clinical treatment model is needed to guide personalized drug therapy for patients with gastric cancer.OBJECTIVE:To construct organoid model based on gastric cancer tissue and investigate its application in personalized drug screening.METHODS:The tissue samples of 20 patients with gastric cancer were collected,digested and decomposed,mixed with matrix glue,and cultured with organoid medium containing epidermal growth factor and fibroblast growth factor 10.Hematoxylin-eosin staining and immunohistochemical method were used to verify the homogeneity of pathological morphology and immune molecular markers of gastric cancer organoids and original tumor tissues.The feasibility of the established gastric cancer organoid model for drug screening was evaluated through drug sensitivity screening of six drugs including carboplatin,irinotecan,fluorouracil,oxaliplatin,paclitaxel,and epirubicin.RESULTS AND CONCLUSION:Fourteen organoids of gastric cancer cases were successfully cultured.There were individual differences in morphology and growth characteristics of organoids.All organoids could be stably passed through,froze and resuscitated.Gastric cancer organoids retained the same morphological features and immunomolecular expression as primary tumor tissues.Six organoids showed different drug sensitivities to six chemotherapy drugs,which initially confirmed the feasibility of gastric cancer organoids as a drug screening model in vitro.

Objective:To evaluate the feasibility of training physicians from secondary comprehensive hospitals in the clinical assessment of brain death and to provide recommendations for nationwide implementation.Methods:This prospective cohort study enrolled physicians who completed standardized training in clinical brain death determination at five pilot hospitals between June and December 2023. Participants were from internal medicine, neurology, critical care, emergency, or anesthesiology departments of secondary comprehensive hospitals and had ≥5 years of clinical experience. Organ donation coordinators and surgeons involved in organ donation or transplantation were excluded. The training program comprised four modules: didactic lectures, bedside demonstrations, simulation-based practice, and written theoretical assessment with review. The theoretical assessment was considered qualified if the score was 60 or above. Participants were categorized into ≥80 and <80 groups based on assessment scores. Between-group comparisons were conducted using rank-sum or chi-square tests.Results:A total of 191 physicians from 74 secondary comprehensive hospitals were enrolled. Most held a bachelor's degree [89.5%(171/191)] and had intermediate [47.1%(90/191)] or associate senior [36.1%(69/191)] professional titles; [59.7%(114/191)] were from non-neurology specialties. The overall pass rate was 99.5% (190/191), with a mean score of 82.4±7.1. Compared with those scoring<80 (56 participants), physicians scoring ≥80 (135 participants) differed significantly by professional title, province, and department ( P=0.014, 0.019 and 0.039). The proportion scoring<80 was higher among junior/intermediate versus senior titles [38.0%(41/108) vs 18.1%(15/83), P=0.003), and among non-neurology/critical care departments (emergency, internal medicine, anesthesiology) versus neurology/critical care [39.7%(31/78) vs 22.1%(25/113), P=0.009]. Only 2.09%(4/191) achieved a perfect score. Across all test items, the overall error rate was 14.99%(700/4 670). The five knowledge points with the highest error rates were mistriggering of mechanical ventilation [96.97%(32/33)], corneal reflex [42.25%(30/71)], spinal reflexes [24.25%(65/268)], documentation of the determination [21.21%(7/33)], and the apnea test procedure [20.73%(57/275)]. Conclusions:The pilot hospitals can effectively deliver clinical training for brain death determination, supporting nationwide promotion. However, physicians' theoretical grounding in neurology at secondary comprehensive hospitals appears relatively weak. Training curricula should be optimized to further improve training quality.

Objective:To explore the effects and possible mechanism of intestinal microbiota on lung injury in mice with acute necrotizing pancreatitis (ANP).Methods:The experimental mice were randomly assigned to normal control group (CON group), ANP model group (ANP group) and intestinal germ-free group (ABX group), with 6 mice in each group. The ANP mouse model was constructed by intraperitoneal injection of caerulein (100 μg/kg, for 10 times) at an interval of 1 hour each time, followed by 10 mg/kg lipopolysaccharide injection. Mice in ABX group were treated by Abx solution (0.5 g/L vancomycin, 1 g/L neomycin, 1 g/L metronidazole, and 1 g/L ampicillin), 1 ml/100 g gavage for 28 days before preparation of the ANP model. The CON group was injected intraperitoneally with an equal volume of PBS. Histopathologic examination of the pancreas, lungs, and terminal ileum was routinely performed. Serum amylase levels were measured using enzymatic kinetic chemistry, and serum diamine oxidase (DAO) and lung tissue myeloperoxidase (MPO) activities were measured using ELISA assay. Expression of inflammatory factors, pyroptosis-related molecules in lung tissue and intestinal epithelial tight junction proteins was detected by fluorescence quantitative PCR. Western blotting was used to detect the expression of the pyroptosis molecules caspase-1 and GSDMD in lung tissue, and intestinal epithelial tight junction proteins. Changes of bacterial distribution in lung tissue were measured by fluorescence in situ hybridization. Results:The pathological scores of pancreatic tissue of CON, ANP, and ABX group were (0.67±0.26), (7.33±0.82), and (5.67±0.81); the pathological scores of lung tissue were (1.67±0.41), (5.67±0.41), and (3.58±0.58); the pathological scores of ileal tissue were (0.58±0.52), (3.83±0.75), and (4.33±0.82); the serum amylase levels were (403.95±93.11), (1037.24±126.77), and (647.32±145.90)U/L; the MPO levels in lung tissue were (0.23±0.03), (0.63±0.09), and (0.48±0.05)U/g. ABX group had significantly lower scores in pancreatic and lung tissues, serum amylase levels, and MPO levels in lung tissue compared to ANP group, and all the differences were statistically significant (all P value <0.05). The expression level in pancreatis tissue from CON, ANP and ABX group of IL-1β mRNA was 1.84±0.90, 36.26±5.56 and 16.65±6.43, IL-6 mRNA was 1.07±0.15, 2.90±0.42 and 1.34±0.62, TNF-α mRNA was 0.47±0.11, 0.76±0.11 and 0.46±0.07, HMGB1 mRNA was 0.38±0.02, 0.72±0.22 and 0.44±0.08, caspase-1 mRNA was 1.07±0.18, 2.04±0.31 and 0.85±0.54, ASC mRNA was 1.24±0.19, 5.68±0.41 and 3.89±1.47, GSDMD mRNA was 0.79±0.17, 0.94±0.14 and 0.61±0.08, IL-18 mRNA was 0.83±0.27, 4.17±0.79 and 3.57±0.03, respectively. The expression of IL-1β, IL-6, TNF-α, HMGB1, caspase-1, ASC, and IL-18 mRNA in lung tissue was significantly increased in ANP group compared to the CON group; conversely, ABX group showed a significant decrease in the expression of these markers compared to ANP group; and all the differences were statistically significant (all P values <0.05). The protein level of caspase-1 in lung tissue of CON, ANP and ABX group was 1.59±0.51, 2.28±0.13, 1.38±0.47, and that of GSDMD was 1.90±0.09, 2.20±0.07 and 1.76±0.27, respectively, which in ANP group were significantly higher than in CON group, but in ABX group was significantly lower than in ANP group, and all the differences were statistically significant (all P values <0.05). The serum DAO levels of CON, ANP, and ABX group were (0.06±0.15), (0.52±0.11) and (0.58±0.11) ng/ml; the expression level of ileum tissue of claudin1 mRNA and protein was 0.98±0.26, 0.42±0.18, 0.32±0.24 and 1.05±0.08, 0.82±0.09, 0.19±0.04; occludin mRNA and protein was 0.91±0.07, 0.31±0.05, 0.32±0.14 and 1.03±0.07, 0.61±0.04, 0.64±0.11; ZO-1 mRNA and protein was 1.01±0.08, 0.80±0.28, 0.60±0.28, and 0.86±0.10, 0.99±0.30, 0.62±0.30. The serum DAO level was significantly elevated in both ANP and ABX groups compared to the CON group. The mRNA and protein expression of claudin-1 and occludin in both ANP and ABX groups were significantly lower than those in CON group; the expression of claudin-1 in ABX group was significantly downregulated compared to ANP group; and all the differences were statistically significant (all P values <0.05). The relative fluorescence intensities of lung tissue in CON, ANP, and ABX groups were 0.03±0.01, 0.06±0.01, and 0.04±0.01, respectively, which in ANP group was significantly higher compared to CON group, but in ABX group was significantly lower than ANP group; all the differences were statistically significant (all P values <0.05). Conclusions:Intestinal microbiota may attenuate acute pancreatitis-associated acute lung injury by inhibiting the pyroptosis pathway in lung tissue.

OBJECTIVE To explore the changes of serum inflammatory factors and immune function indexes in the children with asthma induced by Mycoplasma pneumoniae infection so as to make a new breakthrough in diagnosis and treatment of the children with M.pneumoniae infection-induced asthma.METHODS A total of 118 children with M.pneumoniae infection who were treated in pediatrics department of The Affiliated Hospital of Binzhou Medical College from Jan.2020 to May 2023 were enrolled in the study and were divided into the asthma group with 26 cases and the no asthma group with 92 cases according to the status of induction of asthma.The levels of peripheral blood interleukin(IL)-1β,IL-18,tumor necrosis factor-α(TNF-α)and immune function indexes(CD3+,CD4+,CD8+and CD4+/CD8+)were observed and compared between the two groups.The values of the above indexes in diagnosis of the M.pneumoniae infection-induced asthma were analyzed by means of receiver op-erating characteristic(ROC)curves.RESULTS There were significant differences in the levels of inflammatory factors and immune function indexes between the asthma group and the no asthma group(P＜0.05).The levels of TNF-α,IL-1β,IL-18 and CD8+of the asthma group were(46.33±5.30)pg/ml,(50.60±6.33)pg/ml,(40.26±4.89)pg/ml and(40.11±6.85)％,respectively,higher than those of the no asthma group;the levels of CD3+,CD4+and CD4+/CD8+of the asthma group were(43.33±5.89)％,(15.62±3.61)％and(0.35±0.12),re-spectively,lower than those of the no asthma group.In the asthma group,the levels of TNF-α,IL-1β,IL-18 and CD8+of the MP-Immunoglobulin M(IgM)-positive children were higher than those of the MP-IgM-negative chil-dren(P＜0.05),while the levels of CD3+,CD4+and CD4+/CD8+of the MP-IgM-positive children were lower than those of the MP-IgM-negative children(P＜0.05).The areas under the ROC curves(AUCs)of the serum TNF-α,IL-1β,IL-18,CD3+,CD4+,CD8+and CD4+/CD8+were 0.812,0.805,0.795,0.814,0.768,0.805 and 0.841,respectively,in diagnosis of the M.pneumoniae infection-induced asthma.CONCLUSION The serum TNF-α,IL-1β,IL-18,CD3+,CD4+,CD8+and CD4+/CD8+of the children with M.pneumoniae infection can be used for prediction of the asthma.