Hemoglobin, the principal protein in red blood cells, is crucial for oxygen transport in the bloodstream. The quantification of hemoglobin concentration is indispensable in medical diagnostics and health management, which encompass the diagnosis of anemia and the screening of various blood disorders. Immunological methods, based on antigen-antibody interactions, are distinguished by their high sensitivity and accuracy. Consequently, it is necessary to develop hemoglobin-specific antibodies characterized by high specificity and affinity to enhance detection accuracy. In this study, we immunized a Bactrian camel (Camelus bactrianus) with human hemoglobin and subsequently constructed a nanobody library. Utilizing a solid-phase screening method, we selected nanobodies and evaluated the binding activity of the screened nanobodies to hemoglobin. Initially, human hemoglobin was used to immunize a Bactrian camel. Following four immunization sessions, blood was withdrawn from the jugular vein, and a nanobody library with a capacity of 2.85×10⁸ colony forming units (CFU) was generated. Subsequently, ten hemoglobin-specific nanobody sequences were identified through three rounds of adsorption-elution-enrichment assays, and these nanobodies were subjected to eukaryotic expression. Finally, enzyme-linked immunosorbent assay and biolayer interferometry were employed to evaluate the stability, binding activity, and specificity of these nanobodies. The results demonstrated that the nanobodies maintained robust binding activity within the temperature range of 20-40 ℃ and exhibited the highest binding activity at pH 7.0. Furthermore, the nanobodies were capable of tolerating a 10% methanol solution. Notably, among the nanobodies tested, VHH-12 displayed the highest binding activity to hemoglobin, with a half maximal effective concentration (EC₅₀) of 10.63 nmol/L and a equilibrium dissociation constant (K_D) of 2.94×10^-7 mol/L. VHH-12 exhibited no cross-reactivity with a panel of eight proteins, such as ovalbumin and bovine serum albumin, while demonstrating partial cross-reactivity with hemoglobin derived from porcine, goat, rabbit, and bovine sources. In this study, a hemoglobin-specific high-affinity nanobody was successfully isolated, demonstrating potential applications in disease diagnosis and health monitoring.
Animals ; Camelus/immunology* ; Single-Domain Antibodies/immunology* ; Hemoglobins/immunology* ; Humans ; Peptide Library

Diabetes mellitus is the third most prevalent disease worldwide,following cardiovascular diseases and cancer.There is no obvious symptom in the early stage of diabetes mellitus.However,in the middle and late stages,diabetes mellitus may lead to severe clinical complications such as diabetic cardiomyopathy,kidney disease,retinopathy,or neuropathy,the primary causes for diabetes-related deaths.Therefore,the early diagnosis and treatment of diabetes mellitus are of practical significance.Compared with traditional diagnostic methods including fasting blood-glucose and oral glucose tolerance tests,the measurement of glycated hemoglobin serves as a gold standard for evaluating long-term blood glucose control,due to the relative stability of glycated hemoglobin under a period of dietary and daily practice.Ear-ly treatment of diabetes mellitus may significantly improve the prognosis of patients and enhance the overall therapeutic out-come.Diet control,physical exercise,medication,as well as psychological and social supports are critical for the treat-ment of diabetes mellitus.To date,the therapeutic methods for diabetes mellitus have been constantly enriching,along with promoted drug design and development.Some emerging technologies,such as therapies using antibodies or stem cells,have been applied to treat diabetes mellitus.Hereby the latest progress in the prevention and treatment of diabetes mellitus was comprehensively reviewed,and the application of antibodies was discussed,which may provide insights into the research and development of antibody drugs for chronic human diseases including diabetes mellitus.

Objective:To compare the clinical efficacy and adverse events of different postoperative radiotherapy strategies (adjuvant radiotherapy versus salvage radiotherapy) and different irradiation fields (prostate bed versus prostate bed + pelvic radiation) in patients after radical prostatectomy for prostate cancer.Methods:This retrospective analysis included clinical data from 115 patients with localized or locally advanced prostate cancer who received intensity-modulated radiotherapy (IMRT) after radical prostatectomy at Beijing Hospital between March 2014 and September 2023. Among them, 40 patients received adjuvant radiotherapy, and 75 received salvage radiotherapy. And 74 patients received irradiation to both the prostate bed and pelvic (prostate bed + pelvic radiation group), while 41 patients received irradiation to the prostate bed alone (prostate bed irradiation group). Comparison was made between the adjuvant radiotherapy group and salvage radiotherapy group, as well as between prostate bed + pelvic radiation group and prostate bed irradiation group, in terms of overall survival (OS), progression-free survival (PFS), locoregional recurrence-free survival (LRRFS), and the incidence of adverse events. Clinical characteristics were compared using the chi-square test. Survival rates were calculated using the Kaplan-Meier method and compared using the log-rank test. Prognostic factors affecting survival were analyzed using Cox multivariate regression.Results:The median follow-up duration was 73.1 months. The 5-year OS, PFS and LRRFS rates for the entire cohort were 96.4%, 86.4%, and 93.2%, respectively. A total of 59 patients (51.3%) experienced grade 1-2 acute radiotherapy-related adverse events, while 43 patients (37.4%) experienced grade 1-2 late radiotherapy-related adverse events. No grade ≥ 3 late adverse events were observed. There were no statistically significant differences in OS, PFS, or LRRFS between the adjuvant and salvage radiotherapy groups ( P = 0.807, 0.996, and 0.976, respectively), or in the incidence of grade 1-2 acute or late adverse events ( P > 0.05). The OS rate in the prostate bed + pelvic radiation group was significantly lower than that in the prostate bed irradiation group ( P = 0.036), while no significant differences were found in PFS or LRRFS ( P = 0.109 and 0.190, respectively), or in the incidence of grade 1-2 acute or late adverse events ( P > 0.05). Multivariable analysis showed no statistically significant differences in OS, PFS, or LRRFS between the adjuvant and salvage radiotherapy groups, or between the prostate bed and prostate bed + pelvic irradiation groups ( P = 0.756, 0.341, 0.605; 0.938, 0.987, 0.605, respectively). Conclusions:In the era of modern IMRT, both adjuvant and salvage radiotherapy, as well as prostate bed and prostate bed + pelvic irradiation, demonstrate similar efficacy and safety profiles after radical prostatectomy for prostate cancer. Treatment outcomes were favorable, and adverse events were minimal.

Objective:To investigate whether there is an association between the total dose of breast cancer radiotherapy, the mean dose of radiation field involving the heart and its substructures, and the long-term incidence of coronary heart disease (CHD) in patients.Methods:A retrospective analysis was conducted on 1125 patients with breast cancer who received radiotherapy with radiation fields involving the hear at Beijing Hospital from January 2009 to June 2022. The heart and its substructures of 54 patients were manually delineated, trained an automatic delineation model, and applied this model to the original radiotherapy planning images to automatically extract dosimetric parameters for the heart and substructures in the original plan. Based on the follow-up results, 1125 breast cancer patients were categorized into the CHD event group ( n=19) and non-event group ( n=1106). Wilcoxon rank-sum test, Chi-square test and adjustment for confounding factors using inverse probability weighting were used to compare the mean radiation dose received by the heart and its substructures, age at presentation, history of smoking, history of alcohol consumption, history of hypertension, and history of diabetes between two groups. The influencing factors of CHD were analyzed by univariate and multivariate logistic regression models. Results:The mean heart dose ( P=0.035), mean dose of right atrium ( P=0.049), right coronary artery ( P=0.013), septum ( P=0.045), and right ventricle ( P=0.039) of the event group were higher than that of the non-event group, and the differences were statistically significant. History of alcohol consumption was an independent risk factor for long-term CHD events in the breast cancer patients ( OR=7.35,95% CI: 1.56-25.58, P=0.004). After adjusting for confounding factors using inverse probability weighting, age at presentation was an independent risk factor for long-term CHD events ( OR=1.03, 95% CI: 1.01-1.05, P=0.004). Conclusions:In the breast cancer population with traditional high-risk factors of CHD receiving radiotherapy, the possibility of CHD probably remains high even if the dose of radiation field involving the heart and its substructures is low. Compared to traditional risk factors of CHD, the mean dose to the heart and its substructures in the radiation field of breast cancer patients exerts less impact on the occurrence of CHD after radiotherapy.

Objective:To investigate the clinical characteristics and prognosis of follicular lymphoma (FL) patients with different primary sites using the Surveillance, Epidemiology, and End Results (SEER) database.Methods:Clinical data of 7167 patients with early-stage FL (stage I-II) from the SEER database between 2000 and 2015 were respectively analyzed. Primary sites were divided into intranodal and extranodal types. Intranodal primary sites included supradiaphragmatic lymph nodes (LN), subphrenic lymph nodes and Waldeyer's ring. Extranodal primary sites consisted of skin, gastrointestinal tract, duodenum, head and neck, other sites. Prognostic factors and overall survival (OS) in patients with different primary sites were analyzed. OS rate was evaluated using Kaplan-Meier method and survival difference between primary sites was compared with log-rank test. Inverse probability treatment weighting (IPTW) and multi-variable analysis were applied to adjust for confounding factors. Multivariate Cox regression analysis of influencing factors of OS was performed.Results:The median age was 63 years old, with the median follow-up time of 63 months. There was no difference in prognosis among the intranodal groups or between the intranodal and extranodal groups. The 10-year OS rates of the supradiaphragmatic lymph LN ( n=2146), subdiaphragmatic LN ( n=2811), and the Waldeyer's ring ( n=151) groups were 70.7%, 69.9% and 73.4%, respectively ( P=0.422 for infradiaphragmatic LN vs. supradiaphragmatic LN, P=1.000 for Waldeyer's ring vs. supradiaphragmatic LN), and 70.3% and 68.9% for intranodal ( n=5108) and extranodal ( n=2059), respectively. There was no significant difference in OS between the groups ( P=0.581) after IPTW adjustment. The most common primary sites in extranodal disease were skin, gastrointestinal tract, head and neck, and duodenum. The 10-year OS for skin, gastrointestinal tract, and cutaneous was 74.2%, 74.7%, and 87.3%, respectively, significantly higher than 55.6% for other sites (duodenum vs. others sites, gastrointestinal vs. others sites, skin vs. others sites: all P<0.001). Multivariate Cox regression analysis revealed that difference in OS was not significant among the intranodal groups or between the intranodal and extranodal groups. However, different extranodal primary site was an independent prognostic factor for OS. Conclusions:Early FL patients with supradiaphragmatic LN, subdiaphragmatic LN and Waldeyer's ring, and between the intranodal and extranodal primary sites obtain similar prognosis. However, early-stage FL patients with different extranodal primary sites have prognostic differences. The prognosis of primary skin, gastrointestinal tract and duodenum is significantly better than that of other extranodal primary sites.

Diabetes mellitus is the third most prevalent disease worldwide,following cardiovascular diseases and cancer.There is no obvious symptom in the early stage of diabetes mellitus.However,in the middle and late stages,diabetes mellitus may lead to severe clinical complications such as diabetic cardiomyopathy,kidney disease,retinopathy,or neuropathy,the primary causes for diabetes-related deaths.Therefore,the early diagnosis and treatment of diabetes mellitus are of practical significance.Compared with traditional diagnostic methods including fasting blood-glucose and oral glucose tolerance tests,the measurement of glycated hemoglobin serves as a gold standard for evaluating long-term blood glucose control,due to the relative stability of glycated hemoglobin under a period of dietary and daily practice.Ear-ly treatment of diabetes mellitus may significantly improve the prognosis of patients and enhance the overall therapeutic out-come.Diet control,physical exercise,medication,as well as psychological and social supports are critical for the treat-ment of diabetes mellitus.To date,the therapeutic methods for diabetes mellitus have been constantly enriching,along with promoted drug design and development.Some emerging technologies,such as therapies using antibodies or stem cells,have been applied to treat diabetes mellitus.Hereby the latest progress in the prevention and treatment of diabetes mellitus was comprehensively reviewed,and the application of antibodies was discussed,which may provide insights into the research and development of antibody drugs for chronic human diseases including diabetes mellitus.

Objective:To investigate the clinical characteristics and prognosis of follicular lymphoma (FL) patients with different primary sites using the Surveillance, Epidemiology, and End Results (SEER) database.Methods:Clinical data of 7167 patients with early-stage FL (stage I-II) from the SEER database between 2000 and 2015 were respectively analyzed. Primary sites were divided into intranodal and extranodal types. Intranodal primary sites included supradiaphragmatic lymph nodes (LN), subphrenic lymph nodes and Waldeyer's ring. Extranodal primary sites consisted of skin, gastrointestinal tract, duodenum, head and neck, other sites. Prognostic factors and overall survival (OS) in patients with different primary sites were analyzed. OS rate was evaluated using Kaplan-Meier method and survival difference between primary sites was compared with log-rank test. Inverse probability treatment weighting (IPTW) and multi-variable analysis were applied to adjust for confounding factors. Multivariate Cox regression analysis of influencing factors of OS was performed.Results:The median age was 63 years old, with the median follow-up time of 63 months. There was no difference in prognosis among the intranodal groups or between the intranodal and extranodal groups. The 10-year OS rates of the supradiaphragmatic lymph LN ( n=2146), subdiaphragmatic LN ( n=2811), and the Waldeyer's ring ( n=151) groups were 70.7%, 69.9% and 73.4%, respectively ( P=0.422 for infradiaphragmatic LN vs. supradiaphragmatic LN, P=1.000 for Waldeyer's ring vs. supradiaphragmatic LN), and 70.3% and 68.9% for intranodal ( n=5108) and extranodal ( n=2059), respectively. There was no significant difference in OS between the groups ( P=0.581) after IPTW adjustment. The most common primary sites in extranodal disease were skin, gastrointestinal tract, head and neck, and duodenum. The 10-year OS for skin, gastrointestinal tract, and cutaneous was 74.2%, 74.7%, and 87.3%, respectively, significantly higher than 55.6% for other sites (duodenum vs. others sites, gastrointestinal vs. others sites, skin vs. others sites: all P<0.001). Multivariate Cox regression analysis revealed that difference in OS was not significant among the intranodal groups or between the intranodal and extranodal groups. However, different extranodal primary site was an independent prognostic factor for OS. Conclusions:Early FL patients with supradiaphragmatic LN, subdiaphragmatic LN and Waldeyer's ring, and between the intranodal and extranodal primary sites obtain similar prognosis. However, early-stage FL patients with different extranodal primary sites have prognostic differences. The prognosis of primary skin, gastrointestinal tract and duodenum is significantly better than that of other extranodal primary sites.

Objective:To compare the clinical efficacy and adverse events of different postoperative radiotherapy strategies (adjuvant radiotherapy versus salvage radiotherapy) and different irradiation fields (prostate bed versus prostate bed + pelvic radiation) in patients after radical prostatectomy for prostate cancer.Methods:This retrospective analysis included clinical data from 115 patients with localized or locally advanced prostate cancer who received intensity-modulated radiotherapy (IMRT) after radical prostatectomy at Beijing Hospital between March 2014 and September 2023. Among them, 40 patients received adjuvant radiotherapy, and 75 received salvage radiotherapy. And 74 patients received irradiation to both the prostate bed and pelvic (prostate bed + pelvic radiation group), while 41 patients received irradiation to the prostate bed alone (prostate bed irradiation group). Comparison was made between the adjuvant radiotherapy group and salvage radiotherapy group, as well as between prostate bed + pelvic radiation group and prostate bed irradiation group, in terms of overall survival (OS), progression-free survival (PFS), locoregional recurrence-free survival (LRRFS), and the incidence of adverse events. Clinical characteristics were compared using the chi-square test. Survival rates were calculated using the Kaplan-Meier method and compared using the log-rank test. Prognostic factors affecting survival were analyzed using Cox multivariate regression.Results:The median follow-up duration was 73.1 months. The 5-year OS, PFS and LRRFS rates for the entire cohort were 96.4%, 86.4%, and 93.2%, respectively. A total of 59 patients (51.3%) experienced grade 1-2 acute radiotherapy-related adverse events, while 43 patients (37.4%) experienced grade 1-2 late radiotherapy-related adverse events. No grade ≥ 3 late adverse events were observed. There were no statistically significant differences in OS, PFS, or LRRFS between the adjuvant and salvage radiotherapy groups ( P = 0.807, 0.996, and 0.976, respectively), or in the incidence of grade 1-2 acute or late adverse events ( P > 0.05). The OS rate in the prostate bed + pelvic radiation group was significantly lower than that in the prostate bed irradiation group ( P = 0.036), while no significant differences were found in PFS or LRRFS ( P = 0.109 and 0.190, respectively), or in the incidence of grade 1-2 acute or late adverse events ( P > 0.05). Multivariable analysis showed no statistically significant differences in OS, PFS, or LRRFS between the adjuvant and salvage radiotherapy groups, or between the prostate bed and prostate bed + pelvic irradiation groups ( P = 0.756, 0.341, 0.605; 0.938, 0.987, 0.605, respectively). Conclusions:In the era of modern IMRT, both adjuvant and salvage radiotherapy, as well as prostate bed and prostate bed + pelvic irradiation, demonstrate similar efficacy and safety profiles after radical prostatectomy for prostate cancer. Treatment outcomes were favorable, and adverse events were minimal.

Objective:To investigate whether there is an association between the total dose of breast cancer radiotherapy, the mean dose of radiation field involving the heart and its substructures, and the long-term incidence of coronary heart disease (CHD) in patients.Methods:A retrospective analysis was conducted on 1125 patients with breast cancer who received radiotherapy with radiation fields involving the hear at Beijing Hospital from January 2009 to June 2022. The heart and its substructures of 54 patients were manually delineated, trained an automatic delineation model, and applied this model to the original radiotherapy planning images to automatically extract dosimetric parameters for the heart and substructures in the original plan. Based on the follow-up results, 1125 breast cancer patients were categorized into the CHD event group ( n=19) and non-event group ( n=1106). Wilcoxon rank-sum test, Chi-square test and adjustment for confounding factors using inverse probability weighting were used to compare the mean radiation dose received by the heart and its substructures, age at presentation, history of smoking, history of alcohol consumption, history of hypertension, and history of diabetes between two groups. The influencing factors of CHD were analyzed by univariate and multivariate logistic regression models. Results:The mean heart dose ( P=0.035), mean dose of right atrium ( P=0.049), right coronary artery ( P=0.013), septum ( P=0.045), and right ventricle ( P=0.039) of the event group were higher than that of the non-event group, and the differences were statistically significant. History of alcohol consumption was an independent risk factor for long-term CHD events in the breast cancer patients ( OR=7.35,95% CI: 1.56-25.58, P=0.004). After adjusting for confounding factors using inverse probability weighting, age at presentation was an independent risk factor for long-term CHD events ( OR=1.03, 95% CI: 1.01-1.05, P=0.004). Conclusions:In the breast cancer population with traditional high-risk factors of CHD receiving radiotherapy, the possibility of CHD probably remains high even if the dose of radiation field involving the heart and its substructures is low. Compared to traditional risk factors of CHD, the mean dose to the heart and its substructures in the radiation field of breast cancer patients exerts less impact on the occurrence of CHD after radiotherapy.

BACKGROUND:Electrical stimulation is a physical method that can be used to induce various cellular activities such as cell proliferation,differentiation,and apoptosis.The induction of osteogenic differentiation of stem cells will be beneficial in the field of bone regeneration. OBJECTIVE:To observe whether micro-current field can promote the proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells. METHODS:The fresh human umbilical cord tissue was cut to obtain umbilical cord mesenchymal stem cells,which were inoculated into a 6-well plate after cell culture and passage to the third generation.After 24 hours,the cells were cultured under a stimulation of 0,50,and 100 mV/mm micro-electric field,at a frequency of 1 hour per day for 3 continuous days.The growth and morphological changes of human umbilical cord mesenchymal stem cells were observed by a microscope.The cell proliferation was detected by CCK-8 assay and EdU staining.Alizarin red staining was used to detect the osteogenic differentiation ability of cells.Western blot assay was used to determine the expression of ERK signal pathway proteins. RESULTS AND CONCLUSION:(1)The optical density value and the number of proliferating cells in 50 and 100 mV/mm groups were significantly higher than those of the unstimulated group(P<0.05).(2)Human umbilical cord mesenchymal stem cells could be induced to differentiate into osteocytes before and after micro-electric field stimulation,but the differentiation rate of 50 and 100 mV/mm groups was faster than that of unstimulated groups.(3)The protein expression of p-ERK1/2 in the 50 and 100 mV/mm groups was higher than that in the unstimulated group,and significant difference was detected between the 100 mV/mm group and the unstimulated group(P<0.05).(4)Micro-electric field can promote the proliferation of human umbilical cord mesenchymal stem cells,and the mechanism may be achieved by promoting the phosphorylation of ERK.