Cancer remains a leading cause of global mortality, necessitating the development of advanced therapeutic strategies with enhanced efficacy and reduced systemic toxicity. Among promising bioactive agents, lactoferrin (LF)—a multifunctional iron-binding glycoprotein abundantly found in mammalian milk and exocrine secretions—has garnered significant interest for its potent and multifaceted anti-cancer properties. This review provides a comprehensive analysis of the current understanding of LF’s role in oncology, encompassing its structural biology, diverse mechanisms of action, and groundbreaking advancements in its application through nano-engineering. LF exerts anti-tumor effects through multiple pathways, including extracellular action, intracellular action, and immune regulation. It demonstrates a remarkable affinity for cancer cell membranes, binding to overexpressed anionic components such as glycosaminoglycans and sialic acids, as well as to specific receptors including the low-density lipoprotein receptor-related protein-1 (LRP-1). This selective binding facilitates targeted uptake. Upon internalization, LF orchestrates a direct assault by inducing cell-cycle arrest in phases such as G0/G1 or S phase through the modulation of key regulators including cyclins, CDKs, and p53. Furthermore, it promotes programmed cell death via apoptotic pathways, involving caspase activation and downregulation of anti-apoptotic proteins such as survivin. A more recently elucidated mechanism is the induction of ferroptosis, an iron-dependent form of cell death characterized by overwhelming lipid peroxidation. Beyond direct cytotoxicity, LF acts as a potent immunomodulator. It enhances natural killer (NK) cell activity, modulates T-lymphocyte populations, and crucially reprograms tumor-associated macrophages (TAMs) from a pro-tumor M2 state to an anti-tumor M1 state, thereby reversing the immunosuppressive tumor microenvironment (TME). The translation of LF’s potential has been significantly accelerated by nanotechnology. The inherent biocompatibility and natural tumor-targeting capabilities of LF make it an ideal platform for sophisticated drug-delivery systems. This review details various fabrication strategies for LF-based nanoparticles (NPs), including self-assembly, sol-in-oil emulsion, and electrostatic nanocomplexes, among others. Research demonstrates that nano-formulations not only protect LF from degradation but also enhance its bioactivity and anti-cancer potency. More importantly, LF NPs serve as versatile carriers for a wide array of therapeutic agents, including conventional chemotherapeutics, natural compounds, and imaging agents. These engineered systems enable synergistic therapy and facilitate site-specific delivery. Notably, the ability of LF to bind to receptors on the blood-brain barrier (BBB) has been leveraged to develop nano-systems for glioblastoma treatment. Other innovative designs utilize LF to modulate the TME—for instance, by alleviating tumor hypoxia to sensitize cells to radiotherapy and chemotherapy. Despite compelling pre-clinical evidence, the clinical translation of LF and its nano-formulations remains nascent. While early-phase trials have established a favorable safety profile for recombinant human LF, larger Phase III studies have yielded mixed results, underscoring the complexity of its action in humans. Key challenges include enhancing drug targeting, optimizing loading efficiency, ensuring batch-to-batch reproducibility, and achieving deep tumor penetration. Future research must focus on the rational design of next-generation LF-NPs. This entails developing standardized manufacturing protocols, engineering “smart” stimuli-responsive systems for targeted drug release in the TME, and constructing multi-targeting platforms. A concerted interdisciplinary effort is paramount to bridge the gap between bench and bedside. In conclusion, LF, particularly in its nano-engineered forms, represents a highly promising and versatile agent in the oncological arsenal, holding immense potential for precise and effective cancer therapy.

Cancer remains a leading cause of global mortality, necessitating the development of advanced therapeutic strategies with enhanced efficacy and reduced systemic toxicity. Among promising bioactive agents, lactoferrin (LF)—a multifunctional iron-binding glycoprotein abundantly found in mammalian milk and exocrine secretions—has garnered significant interest for its potent and multifaceted anti-cancer properties. This review provides a comprehensive analysis of the current understanding of LF’s role in oncology, encompassing its structural biology, diverse mechanisms of action, and groundbreaking advancements in its application through nano-engineering. LF exerts anti-tumor effects through multiple pathways, including extracellular action, intracellular action, and immune regulation. It demonstrates a remarkable affinity for cancer cell membranes, binding to overexpressed anionic components such as glycosaminoglycans and sialic acids, as well as to specific receptors including the low-density lipoprotein receptor-related protein-1 (LRP-1). This selective binding facilitates targeted uptake. Upon internalization, LF orchestrates a direct assault by inducing cell-cycle arrest in phases such as G0/G1 or S phase through the modulation of key regulators including cyclins, CDKs, and p53. Furthermore, it promotes programmed cell death via apoptotic pathways, involving caspase activation and downregulation of anti-apoptotic proteins such as survivin. A more recently elucidated mechanism is the induction of ferroptosis, an iron-dependent form of cell death characterized by overwhelming lipid peroxidation. Beyond direct cytotoxicity, LF acts as a potent immunomodulator. It enhances natural killer (NK) cell activity, modulates T-lymphocyte populations, and crucially reprograms tumor-associated macrophages (TAMs) from a pro-tumor M2 state to an anti-tumor M1 state, thereby reversing the immunosuppressive tumor microenvironment (TME). The translation of LF’s potential has been significantly accelerated by nanotechnology. The inherent biocompatibility and natural tumor-targeting capabilities of LF make it an ideal platform for sophisticated drug-delivery systems. This review details various fabrication strategies for LF-based nanoparticles (NPs), including self-assembly, sol-in-oil emulsion, and electrostatic nanocomplexes, among others. Research demonstrates that nano-formulations not only protect LF from degradation but also enhance its bioactivity and anti-cancer potency. More importantly, LF NPs serve as versatile carriers for a wide array of therapeutic agents, including conventional chemotherapeutics, natural compounds, and imaging agents. These engineered systems enable synergistic therapy and facilitate site-specific delivery. Notably, the ability of LF to bind to receptors on the blood-brain barrier (BBB) has been leveraged to develop nano-systems for glioblastoma treatment. Other innovative designs utilize LF to modulate the TME—for instance, by alleviating tumor hypoxia to sensitize cells to radiotherapy and chemotherapy. Despite compelling pre-clinical evidence, the clinical translation of LF and its nano-formulations remains nascent. While early-phase trials have established a favorable safety profile for recombinant human LF, larger Phase III studies have yielded mixed results, underscoring the complexity of its action in humans. Key challenges include enhancing drug targeting, optimizing loading efficiency, ensuring batch-to-batch reproducibility, and achieving deep tumor penetration. Future research must focus on the rational design of next-generation LF-NPs. This entails developing standardized manufacturing protocols, engineering “smart” stimuli-responsive systems for targeted drug release in the TME, and constructing multi-targeting platforms. A concerted interdisciplinary effort is paramount to bridge the gap between bench and bedside. In conclusion, LF, particularly in its nano-engineered forms, represents a highly promising and versatile agent in the oncological arsenal, holding immense potential for precise and effective cancer therapy.

Osteoarthritis (OA), a highly prevalent degenerative joint disease worldwide, is defined by articular cartilage degradation, abnormal bone remodeling, and persistent chronic inflammation. It severely compromises patients’ quality of life, and currently, there is no radical cure. Abnormal mechanical stress is widely regarded as a core driver of OA pathogenesis, and the exploration of mechanical signal perception and transduction mechanisms has become crucial for deciphering OA’s pathophysiological processes. Piezo1, a key mechanosensitive cation channel belonging to the Piezo protein family, has recently gained significant attention due to its pivotal role in mediating cellular responses to mechanical stimuli in joint tissues. This review systematically examines Piezo1’s expression patterns, regulatory mechanisms, and pathological functions in OA, with a particular focus on its dual roles in modulating chondrocyte homeostasis and bone metabolism disorders, while also delving into the underlying molecular signaling pathways and potential therapeutic implications. Piezo1, consisting of approximately 2 500 amino acids and forming a unique trimeric propeller-like structure, is widely expressed in chondrocytes, osteocytes, mesenchymal stem cells, and synovial cells. It exhibits permeability to cations such as Ca²⁺, K⁺, and Na⁺, and directly responds to membrane tension changes induced by mechanical stimuli like fluid shear stress and mechanical overload. In OA patients and animal models, Piezo1 expression is significantly upregulated, especially in cartilage regions subjected to abnormal mechanical stress (e.g., human temporomandibular joint cartilage). This overexpression is closely associated with aggravated cartilage degeneration, increased chondrocyte apoptosis, accelerated cellular senescence, and intensified inflammatory responses. Mechanical overload and pro-inflammatory cytokines (e.g., IL-1β) are key inducers of Piezo1 upregulation: IL-1β activates the PI3K/AKT/mTOR signaling pathway to enhance Piezo1 expression, forming a pathogenic positive feedback loop that inhibits chondrocyte autophagy, promotes apoptosis, and further accelerates joint degeneration. Mechanistically, Piezo1 mediates OA progression through multiple interconnected pathways. When activated by mechanical stress, Piezo1 triggers excessive Ca²⁺ influx, leading to endoplasmic reticulum stress (ERS) and mitochondrial dysfunction, which directly induce chondrocyte apoptosis. This process involves the activation of downstream signaling cascades such as cGAS-STING and YAP-MMP13/ADAMTS5. YAP, a transcriptional regulator, upregulates the expression of matrix metalloproteinase 13 (MMP13) and aggrecanase (ADAMTS5), thereby accelerating cartilage matrix degradation. Additionally, Piezo1-driven Ca²⁺ overload promotes the accumulation of reactive oxygen species (ROS) and upregulates senescence markers (p16 and p21), accelerating chondrocyte senescence via the p38MAPK and NF-κB pathways. Senescent chondrocytes secrete senescence-associated secretory phenotype (SASP) factors (e.g., IL-6, IL-1β), further amplifying joint inflammation. In terms of bone metabolism, Piezo1 maintains joint homeostasis by promoting the differentiation of fibrocartilage stem cells into chondrocytes and balancing bone formation and resorption through regulating the FoxC1/YAP axis and RANKL/OPG ratio. Therapeutically, targeting Piezo1 shows promising potential. Preclinical studies have demonstrated that Piezo1 inhibitors (e.g., GsMTx4) can reduce joint damage and alleviate pain in OA mice. Simultaneously, siRNA-mediated co-silencing of Piezo1 and TRPV4 (another mechanosensitive channel) decreases intracellular Ca²⁺ concentration, inhibits chondrocyte apoptosis, and promotes cartilage repair. Conditional knockout of Piezo1 using Gdf5-Cre transgenic mice alleviates cartilage degeneration in post-traumatic OA models by downregulating MMP13 and ADAMTS5 expression. Despite existing challenges, such as off-target effects of inhibitors, inefficient local drug delivery, and interindividual genetic variability, strategies like developing selective Piezo1 antagonists, optimizing targeted nanocarriers, and combining Piezo1-targeted therapy with physical therapy provide viable avenues for clinical translation. The authors propose that Piezo1 serves as a critical therapeutic target for OA, and future research should focus on deciphering its context-dependent regulatory networks, developing tissue-specific intervention strategies, and validating their efficacy and safety in clinical trials to address the unmet medical needs of OA patients.

Objective: To investigate the incidence, characteristics, and influencing factors of resolution discrepancies within the minipool (MP) testing model across Chinese blood station laboratories in 2024. Methods: A nationwide, multicenter, cross-sectional study was conducted, including 334 blood station laboratories that reported nucleic acid reactive data among enzyme immunoassay non-reactive samples. Of these, 296 laboratories adopted the pool resolution model, with a total of 12 536 273 samples tested. Systematic analysis was performed on resolution data, focusing on the MP-NAT reactivity rate, the pool resolution concordance rate, and the resolution discrepancy rate. Subgroup analyses were conducted based on reagent types, viral targets, and Ct values. Potential causes were further explored through laboratory surveys and re-examination of raw amplification curves. Results: In 2024, the national average MP-NAT reactivity rate was 0.15%. The overall pool resolution concordance rate was 57.86%, which showed a gradual decline as Ct values increased across all reagents. The national average resolution discrepancy rate was 0.081‱(102/12 536 273), with 17.91%(53/296) of laboratories reporting at least one discrepancy. Nine reagent types were associated with these events, exhibiting reagent-specific patterns. For Reagent A2, the predominant discrepancy was HBV reactive pools resolving as HIV (36.36%); for Reagent D1, HBV pools frequently resolved as HCV (38.89%); and for Reagent E, the most common pattern was HIV pools resolving as HBV (48.00%). These resolution discrepancies were strongly associated with high Ct values: the median pool Ct for HBV exceeded 38, while those for HCV and HIV both exceeded 40. Investigations across 16 laboratories revealed that most discrepant samples exhibited “tailing” amplification curves, with some cases linked to cross-contamination or reagent batch-specific issues. Conclusion: While the incidence of resolution discrepancies in the MP-NAT model remains low in China, variations exist across different reagents and laboratories. These discrepancies are closely associated with low viral load, reagent performance, and laboratory operational practices.

Objective: To propose an improved method for constructing the internal quality control (IQC) framework for ELISA assays and validate its efficacy by statistically analyzing IQC data from nine blood center laboratories. Methods: 1) IQC data was collected from nine blood centers and analyzed using a domestic HBsAg ELISA detection kit as an example. 2) Differences between IQC values across batches within Blood Center 1 were assessed. 3) Statistical analyses were performed on batch usage, number of batches used, days of use, number of QC points, batch-specific means, and coefficients of variation (CV) across all nine centers. 4) Using the improved construction method for IQC framework, provisional and permanent frames were established for batches within Blood Center 1 and Blood Center 9, followed by outlier determination. Results: 1) Statistically significant differences were observed in IQC data between batches within Blood Center 1 (P<0.01). It is recommended that both the control material/reagents and the control chart framework be replaced simultaneously. 2) There were substantial differences among 9 blood centers regarding the control material/reagent lot numbers used, the number of QC runs per batch, and the QC values for identical lots. Therefore, individual laboratories should establish their own IQC chart frameworks. 3) The improved IQC framework construction method for ELISA assays is as follows: provisional frames are established via frame-shifting, using the pre-experimental mean and cumulative coefficient of variation (CV) from the preceding batch. For batches used >20 days with >20 QC points, permanent frames are constructed by aggregating in-control data accumulated over ≥20 days with ≥20 points to calculate cumulative mean and standard deviation. The provisional and permanent frames constructed by this method identified all 26 extreme outliers across Blood Centers 1 and 9 as out-of-control. Among the 218 general outliers, 10 were classified as normal by the provisional frames, while the remainder were designated as warnings or out-of-control. This method effectively monitors assay stability. Conclusion: Based on the statistical analysis of IQC practices across blood centers of varying scales, combined with the inherent characteristics of ELISA assays and the batch-to-batch instability of reagents/QC materials, it is recommended to reconstruct QC charts upon lot changes. The proposed method—utilizing frame-shifting for provisional frames and establishing permanent frames based on cumulative data—is applicable to blood center laboratories of differing sizes and effectively monitors the stability of the ELISA assay process.

Objective: To explore the setting of gray zone of Treponema pallidum (TP) testing by retrospective analysis of blood donors with single reagent reactive anti-TP results, so as to improve blood utilization and supply safety. Methods: Blood samples were collected from 112 blood donors previously deferred due to single reagent reactive TP antibody results between January 2020 and December 2023, and subjected to dual ELISA reagents and TPPA test. The gray zone panel analysis was performed on the two ELISA reagents currently used in our department. The detection rate at each concentration of the gray zone panle was counted, and the corresponding concentrations for C , C , and C and gray zone cut-off were calculated. Results: Among the 50 samples deferred by reagent 1, 19 were confirmed reactive and 31 non-reactive in supplementary testing. Among the 62 samples deferred by reagent 2, 12 were confirmed reactive and 50 non-reactive in supplementary testing. For reagent 1, the detection rate of was 56% for S/CO≥1 and 20% for 0.5≤S/CO<1, retrospectively. For reagent 2, the detection rate was 27% for S/CO≥1 and 12.5% for 0.5≤S/CO<1, retrospectively. The detection rate for S/CO≥1 was higher than those for 0.5≤S/CO<1 for both reagents. All the 112 samples were negative in TPPA test. The C concentration of reagent 1 was 1.51 mIU/mL, and the concentration range of C ±20% was 1.21-1.81 mIU/mL. The C concentration of reagent 2 was 1.45 mIU/mL, and the concentration range of C ±20% was 1.16-1.74 mIU/mL. The C and C concentration of both reagents were within the C ±20% range, suggesting that the gray zone cutoff for both Reagent 1 and Reagent 2 should be set at S/CO=0.8 (80% of the CO value). Conclusion: All anti-TP single reagent reactive samples with S/CO value within the gray zone was tested negative by TPPA. It is necessary to consider the rationality and necessity of establishing the gray zone, so as to ensure blood safety and improve the utilization rate of blood resources.

Objective To explore the genetic mutation characteristics, clinical manifestations, and treatment outcomes of catecholaminergic polymorphic ventricular tachycardia (CPVT), and to construct a quantitative scoring system for the severity of CPVT. The correlation between the mutations in different structural domains of the RyR2 gene and clinical manifestations and prognosis was analyzed. Methods By searching the PubMed and Web of Science databases for CPVT-related case reports published up to December 2024, data such as patient age, clinical manifestations, gene mutation sites, and treatment responses were collected. The quality of the literature was assessed using the CARE guidelines. The χ2 test was used to compare the severity and treatment response differences among different RyR2 structural domain mutation groups, and an innovative quantitative scoring system based on symptoms and efficacy was established. Results A total of 80 articles were included, with 102 patients in total. The quality of the literature was reliable. The age of the patients ranged from 1 to 84 years, with a higher proportion of children under 10 years old (25.5%). Female patients (54.9%) outnumbered males (45.1%). For CPVT patients, a quantitative scoring system was developed, with a total score of 2 to 10 points. Among them, 2 to 4 points were classified as mild, 5 to 7 points as moderate, and 8 to 10 points as severe. The results showed that severe patients often had a history of cardiac arrest and were resistant to treatment. Out of the 102 CPVT patients, RyR2 gene mutations accounted for 53.9% (55/102) of patients. Among them, the proportion of severe patients with N-terminal structural domain mutations was significantly higher than other regions, indicating that the RyR2 gene mutation structural domain has a significant impact on the severity of CPVT (χ2=17.530, P=0.008). The proportion of patients with mutations in the central hinge region who were ineffective with β-blockers reached 42.9% (3/7), which was significantly higher than other regions. Left cardiac sympathectomy was performed in 24 cases, and postoperative symptoms were almost completely controlled, significantly better than the drug treatment group.Conclusion Mutations in the N-terminal structural domain of the RyR2 gene are significantly correlated with the severity of CPVT. Left cardiac sympathectomy has gradually become an effective intervention for refractory cases. The scoring system proposed in this study can provide a basis for clinical stratified treatment. In the future, there is a need to expand the sample size to verify mutation-specific treatment strategies.

Objective To investigate the impact of sleep modes on the risk for diseases of the body systems.Methods Based on a subset of UK Biobank dataset comprising 87 617 participants,3 sleep dimensions including 6 sleep indicators were obtained through a wrist-worn accelerometer,that is sleep duration and onset,sleep rhythm(relative amplitude and stability),and sleep quality(sleep efficiency and number of awakenings).Latent profile analysis(LPA)was applied to identify and classify distinct sleep modes.Then their longitudinal medical records were the association between different sleep modes and the risk for 467 diseases.Results LPA identified 5 subgroups of unique sleep modes in the participants.Among the 5 subgroups,the subgroup 4 had relatively optimal levels in above sleep indicators.Compared to the subgroup 4,the other 4 subgroups exhibited variations in different sleep dimensions,with at least one indicator demonstrating an unfavorable trend.These subgroups also revealed differences in racial composition,shift work and social deprivation index.Moreover,there were notable differences in the risk of various system diseases among the subgroups(P<0.05).When compared to the subgroup 4,the other 4 subgroups exhibited an elevated risk for certain diseases(comprising a total of 126 diseases),with the diseases of the circulatory system,digestive system and musculoskeletal system most common.Among the 5 subgroups,the subgroup 2(shorter sleep duration and later sleep onset)and the subgroup 5(rhythm disorder)had the highest counts of associated risks,amounting to 85 and 91 types,respectively,but there was certain difference in their systematic composition.Conclusion There are different sleep modes within the participants,and the modes are potentially associated with an increased risk for diseases of body systems.Comprehensive interventions targeting overall sleep modes rather than single sleep indicator may yield obvious health benefits.

Objective To investigate the transgenerational effects of paternal PM2.5 exposure on offspring growth and development,and to preliminarily elucidate the role of sperm DNA methylation modifications in mediating these effects.Methods Eight-week-old male C57BL/6 mice were randomly divided into filtered air(FA),unfiltered air(UA),and concentrated PM2.5(CAP)groups,with 10 animals in each group.The exposure was conducted from November 2019 to April 2020,and then,these male mice were mated with unexposed females to generate F1 offspring,which were bred successively to produce F2 and F3 generations.All the offspring were living in PM2.5-free environment.The birth body weight,birth number,and sex ratio of the offspring were recorded,body weight growth was monitored,and organ coefficients of the heart,liver,lung,and brain were calculated.Whole-genome methylation sequencing was performed on the sperm DNA of the CAP group,FA group,and their F1 generation offspring to screen for differentially methylated regions,and the genes and pathways associated with these regions were analyzed.Results When compared with the F1～F3 offspring of the FA group,the CAP group had significantly reduced birth body weight in the F1 generation(P<0.05),no statistical differences were observed in the birth body weight in the F2 and F3 generations(P>0.05),or either in the sex ratio and birth number among the F1,F2 and F3 generations.Compared with the FA group offspring,the F1～F3 offspring of CAP group exhibited delayed body weight gain,especially in the males(P<0.05),the CAP-F1 male generation had obviously elevated liver organ coefficient(P<0.01),but no statistical changes were observed in the heart,lung,or brain coefficients among the F1～F3 generations.Between the FA group and the CAP group,37 997 differentially methylated regions were detected,with a reduction of approximately 50%in the number of differentially methylated regions in the F1 generation.Differentially methylated genes in F0 and F1 sperm were potentially related to developmental processes,including imprinting genes(Gnas,Igf2)and metabolic genes(Ppard,Rps6kb1).Conclusion Paternal exposure to PM2.5 leads to reduced birth weight and intergenerational growth retardation in offspring.Its impact on phenotypic effects is gradually weakened during intergenerational transmission.Changes in the methylation of development-related genes in sperm may be one of the mechanisms mediating this intergenerational effect.

Objective To analyze the diagnostic process and clinical characteristics of autoimmune gastritis(AIG)in order to improve the awareness and diagnostic proficiency of this disease.Methods A retrospective cohort study was conducted on 114 patients diagnosed with AIG in Army Medical Center of PLA between January 2021 and June 2024.Comprehensive statistical analysis was performed on clinical data,including demographic characteristics(age,sex),clinical symptoms,comorbidities,diagnostic process,Helicobacter pylori(H.pylori)infection and treatment history,laboratory indicators[results of routine blood test,anemia-related indices,thyroid function,anti-parietal cell antibody(APCA),intrinsic factor antibody(IFA)],and gastrointestinal endoscopic findings(frequency and endoscopic features).Results Among the 114 patients,males accounted for 28.1%(32/114)and females for 71.9%(82/114),and they were at a mean age of 56.3±8.4 years.Predominant symptoms included epigastric/upper abdominal pain(47.4%,54/114)and postprandial fullness(43.0%,49/114),while 24.6%(28/114)reported acid reflux or heartburn.Diagnostic delay occurred in 76.4%(87/114)of patients,with a median delay duration of 11.5 months.Primary diagnostic clues were endoscopic reverse gradient atrophy(significantly more severe mucosal atrophy in the gastric corpus/fundus versus antrum;53.5%,61/114)and repeated H.pylori eradication failure(≥2 attempts;22.8%,26/114).Positivity rate of thyroid peroxidase antibody(TPOAb)and thyroglobulin antibody(TgAb)was 56.9%(33/58)and 36.2%(21/58),respectively.APCA positive rate was 98.8%(81/82),IFA positive rate was 34.1%(28/82),and dual-antibody rate was 32.9%(27/82).Anemia was present in 25.7%(26/101)of the patients.Gastric neuroendocrine tumors(NET)were found in 12.2%(14/114),intraepithelial neoplasia in 5.3%(6/114),and gastric adenocarcinoma in 0.9%(1/114).Among colonoscopy-examined patients,tubular adenomas occurred in 25.0%(13/52)and colorectal malignancies in 3.4%(2/58).There were 18.4%(21/114)patients having gallbladder-related diseases,7.9%(9/114)having diabetes mellitus,and 1.8%(2/114)of subacute combined degeneration of the spinal cord.Conclusion AIG is frequently associated with diagnostic delay.The reverse pattern of atrophy on endoscopy serves as a critical diagnostic clue,necessitating enhanced recognition in endoscopists.Patients with recurrent H.pylori eradication failure(≥2 attempts)should be evaluated for AIG.