To enhance the specificity of anti-TNF-α single chain Fv antibody (TNF-scFv) to inflamed site, we constructed a bispecific antibody BsDb that targets TNF-α and ED-B-containing fibronectin (B-FN) by covalently linking TNF-scFv and the anti-ED-B scFv L19 at the gene level via a flexible peptide linker deriving from human serum albumin. BsDb was successfully secreted from Pichia pastoris as functional protein, identified by immunoblotting, and purified to homogeneity with affinity chromatography. BsDb retained the immunoreactivity of its original antibodies TNF-scFv and L19, and showed a marked gain in antigen-binding affinity and in TNF-α-neutralizing ability, when compared to TNF-scFv and L19 that were produced in Escherichia coli. In the adjuvant-induced arthritis (AIA) mice model, BsDb showed selective accumulation and retention in the inflamed paws but rapid clearance from blood, resulting in high arthritic paw to blood ratios. These data indicate that BsDb is endowed with high specificity to inflamed site and low toxicity to normal tissues and holds great potential for in vivo application for the targeted therapy of RA and other chronic inflammatory diseases.
Animals ; Antibodies, Bispecific ; biosynthesis ; immunology ; Antibodies, Neutralizing ; biosynthesis ; immunology ; Escherichia coli ; Fibronectins ; chemistry ; immunology ; Humans ; Mice ; Single-Chain Antibodies ; biosynthesis ; immunology ; Tumor Necrosis Factor-alpha ; immunology

Objective To study the promotive effect of chitosan liquid on wound healing.Methods Wound model was established on the back of 60 rats,which were classified into 3 groups to be treated with topical chitosan liquid (group A),basic fibroblast growth factor (group B),and sodium chloride physiological solution (group C),respectively.The time required for the healing of wound was recorded.Regenerated tissues were resected from the rats on day 3,7 and 14 after the establishment of wound model,and observed with light microscopy.Results The time required for the healing of wound was 17.3 ± 1.35 days in group A,18.2 ± 1.15 days in group B,and 24.0 ± 1.37 days in group C.For the time required for the healing of wound,no significant difference was observed between the group A and B (P ＞ 0.05),but group C significantly differed from group A and B (both P ＜ 0.05).Chitosan liquid accelerated the generation of capillary sprouts and vascular endothelial cells at the early stage of wound healing,promoted the production of fibroblasts and collagen fibers at the middle stage,and improved the organization of collagen fibers at the late stage.Conclusion Chitosan liquid exerts a promising promotive effect on wound healing.

In order to enhance the specificity of TNF-α monoclonal antibody to inflamed site, a bispecific antibody BsDb that targets TNF-α and the extra-domain B (ED-B) of fibronectin (FN) was constructed by covalently linking the anti-TNF-α single chain Fv antibody (TNF-scFv) and the anti-ED-B scFv L19 via a flexible peptide linker deriving from human serum albumin (HSA). ED-B is an antigen specifically expressed at the inflamed site. BsDb is expressed in E. coli, identified by immunoblot, and purified with affinity chromatography. This was followed by further examination of its bioactivities and pharmacokinetics. We demonstrated that BsDb retained the immunoreactivity of its original antibodies as it could simultaneously bind to TNF-α and ED-B and neutralize the biological action of TNF-α. In the collagen-induced arthritis mice model, BsDb selectively accumulate in the inflamed joint with a maximal uptake of (12.2 ± 1.50)% ID/g in a single inflamed paw and retain in the inflamed paw for at least 72 h. In contrast, BsDb showed a short serum half-life of (0.50 ± 0.05) h and a rapid clearance from normal tissues. The findings reported herein indicate that BsDb has good specificity to the inflamed site and low toxicity to normal tissues. BsDb is therefore likely to have greater clinical applications in the treatment of rheumatoid arthritis and other autoimmune diseases. This laid a stable basis for its preclinical study.

OBJECTIVE:To evaluate the affordability of 3 anti-tumor targeted drugs gefitinib,trastuzumab and sunitinib in ur-ban and rural residents of Hubei province,and to provide reference for medical insurance price admission of anti-malignant tumor targeted drugs in China. METHODS:Referring to the incidence of malignant tumor stated in statistical yearbook of Hubei province and income data of urban and rural residents in Hubei province,based on the policy of reducing the price of imported drugs by 50% mentioned in the national drug price negotiations,and assume the drugs are included in the medical insurance reimbursement list,WHO/HAI standard survey method,catastrophic expenditure evaluation method and poverty effect evaluation method were ad-opted to calculate the affordability of 3 drugs. RESULTS:According to WHO/HAI standard survey method,increment of payment for 3 drugs were 64.00%-74.00% before and after 50% discount and reimbursement. According to catastrophic expenditure evalua-tion method,50% discount of gefitinib and reimbursement gefitinib,trastuzumab and sunitinib in urban area would result in cata-strophic expenditures of 20.00%、59.28% and 35.48% patients;in rural area,would result in catastrophic expenditures of 50.63%、74.72% and 75.93% patients. According to poverty effect evaluation method,50% discount of 3 drugs and reimbursement caused less than 31.95% urban and rural patients falling to poverty. CONCLUSIONS:Fifty percentage discount of 3 anti-tumor targeted drugs mentioned in the national drug price negotiations cause the economic burden more serious for rural residents than urban resi-dents. In the formulation of policies,the corresponding reimbursement ratio should be adjusted according to urban and rural areas, drug price and disease types for a balance of patients with different economic burden.

Objective: To analyze the New Cooperative Medical System ( NCMS ) funds and Individual afford-ability of anti-tumor targeted drugs under different medical insurance entry price, and to provide the basis for establis-hing the access price for medical insurance. Methods: Choosing Conmana or Kemer ( the lung cancer targeted drug) and Herceptin (breast cancer targeted drug) to analyze the Wuhan NRCMS operating status from 2012 to 2014, use tumor surveillance data from Hubei Province during the period from 2011 to 2015;consult clinical experts to form expert consensus price, refer to the Jiangsu Province Access Price and National Negotiation Price, and explore the fund bal-ance and individual affordability when the afore-mentioned two kinds of drugs can be compensated by medical insurance under different price. Results:The basic account balances of NRCMS in Wuhan from 2016 to 2018 are-11. 948 million Yuan, 2. 513 million Yuan and 82. 955 million Yuan when Kemer can be compensated by medical insurance under Na-tional Negotiation Price. Taking the compensation of Herceptin under National Price after the bargaining, the basic ac-count balances are -26. 901 million Yuan,-35. 962 million Yuan and 17. 542 million Yuan respectively. The rate of poverty caused by illness falls to 33. 40% from 45. 85% when Conmana can be compensated by Medical Insurance un-der National Negotiation Price, while this rate falls to 45. 42% from 46. 00% for Herceptin. Conclusion:The two kinds of drugs can be afforded by the Wuhan NRCMS after the medical insurance access price is negotiated by the govern-ment, but the individual affordability of Herceptin at the National Negotiation Price is worse.

The incidence of small cell lung cancer ( SCLC) is relatively low, which approximately accounts for 13％-20％ of all types of lung cancers. SCLC is characterized by high-degree malignancy and high metastasis and recurrence rates within a short period of time. Upon the confirmed diagnosis, limited-stage SCLC approximately accounted for 30％ of SCLC and merely 5％ of these patients were eligible for surgery. Over half of the patients who obtained complete remission after chemoradiotherapy presented with brain metastases. The postoperative incidence of brain metastases in patients with stageⅠ,ⅡandⅢSCLC was 6％-14％,13％-38％ and 11％-36％, respectively. Prophylactic cranial irradiation ( PCI) can enhance the overall survival rate of patients with complete remission after chemoradiotherapy and reduce the incidence of brain metastasis, which is a pivotal part of comprehensive treatment of limited-stage SCLC. However, the application of PCI remains controversial in clinical practice and the clinical efficacy for patients with surgically resected SCLC significantly varies. In this article, literature review was conducted and the research progress in this field was summarized.

Objective To establish a patient-derived xenografts (PDX) mouse model of liver cancer (LC) and to explore its role in precision medicine.Methods PDX model was established by subcutaneous implantation of tumor tissues in NCG mice.The morphological structure of tumor tissue was exaimed using HE staining.Fifteen BALB/c nude mice were subcutaneously inoculated with tumor cell suspension from the PDX models.The xenograft mice were randomly divided into 5-fluorouracil (5-FU) group, sorafenib group and negative control group.The tumor volume and body weight of the tumor-bearing mice were measured regularly, the tumor inhibition rate was calculated and the curative effect was evaluated.Results The success rate was 33.3% (6/18) in the establishment of liver cancer PDX mouse model, and the model well retained the characteristics of the primary tumor.In one case of PDX mouse model, the tumor inhibition rates of 5-FU and sorafenib group were 63.7% and 29.6%, with a statistically significant differece between them (P< 0.05), and there was no significant difference between the sorafenib group and negative control group, consistent with clinical observation.Conclusions The PDX mouse model of liver cancer can maintain the histological structure of primary tumor, and can be applied to precision medicine for patients with liver cancer.

Objective To evaluate the olfactory function and its influence factors by using Sniffin ’ Sticks test, and to compare the quality of Parkinson ’s disease (PD) recognition between Sniffin’ Sticks and 16 kinds of odor identification in Sniffin ’ Sticks(SS-16) tests.Methods The Sniffin’Sticks test was used to assess the olfactory function of 68 PD patients and 76 healthy volunteers , and the relationship between smell and age, disease duration, Unified Parkinson’ s Disease Rating Scale score, Hoehn-Yahr (H-Y) rating, and cognitive function level (Montreal Cognitive Assessment) was analyzed.Results (1)The prevalence of olfactory dysfunction in PD group (83.3%) was significantly higher than that in control group (21.2%).The Sniffin’ Sticks test showed that the odor threshold score (6.6 ±3.2, P=0.000), odor discrimination score (6.6 ±3.3, P=0.000), 16 kinds of odor identification score (6.8 ±2.4, P=0.000) in PD group were significantly lower than those in control group.( 2 ) When comparing the PD cases and healthy controls in recognition , the sensitivity and the specificity of the Sniffin ’ Sticks test were 0.897 and 0.737, respectively, similar to the SS-16 test.However, the Sniffin’ Sticks test showed advantage compared with odor threshold and odor discrimination.( 3 ) The olfactory score in PD group was positively correlated with cognitive function (r=0.243, P=0.046), and was unrelated with age, gender, disease duration, and disease severity.The olfactory score in control group was negatively correlated with age (r=-0.270, P=0.018), but positively correlated with cognitive function (r=0.281, P=0.014).Conclusions There is a higher incidence of olfactory dysfunction in PD patients than in control group.Sniffin’ Sticks test is superior to SS-16 test in quantitative and qualitative analysis of olfactory function in PD patients.Two tests both have high sensitivity and specificity in the recognition of PD .

Objective:To construct a palliative care quality evaluation index system in ICU, and to provide guidance for implementing high-quality palliative care in ICU.Methods:The questionnaire was designed by literature review, qualitative interview and questionnaire survey, and 20 experts were inquired in two rounds by Delphi method from September to November 2020.Results:After two rounds of letter consultation, experts′ response rates were 80% and 100% respectively, experts′ authority coefficient was 0.873, and coordination coefficient of experts′ opinion were 0.198 and 0.176 respectively. The quality evaluation index system for palliative care in ICU included 6 first-level indicators, 22 second-level indicators and 95 third-level indicators.Conclution:The constructed evaluation system for palliative care is scientific and reliable,which can provide guidance for the implementation and development of ICU high-quality palliative care in China.

Objective To investigate the effects of hyperfractionated radiotherapy versus hypofractionated radiotherapy combined with concurrent chemotherapy on the prognosis of limited-stage small-cell lung cancer (SCLC).Methods A total of 188 patients with limited-stage SCLC were enrolled in this study and divided into hyperfractionated group (n=92) and hypofractionated group (n=96).The hyperfractionated group received thoracic radiotherapy at 45 Gy in 30 fractions twice a day, while the hypofractionated group received 55 Gy in 22 fractions once a day.The Kaplan-Meier method was used to calculate survival rates, and the Cox model was used for multivariate prognostic analysis.Results There were not significant differences in 1-, 2-, and 5-year progression-free survival (PFS) rates and 1-, 2-, and 5-year overall survival (OS) rates between the hyperfractionated group and the hypofractionated group (82% vs.85%, 61% vs.69%, 59% vs.69%, P=0.27;85% vs.77%, 41% vs.34%, 27% vs.27%, P=0.37).The multivariate analysis showed that the time from the initiation of chemotherapy to the initiation of thoracic radiotherapy ≤43 days was favorable prognostic factor for PFS (P=0.005).The time from the initiation of chemotherapy to the end of thoracic radiotherapy ≤63 days and prophylactic cranial irradiation were favorable prognostic factors for OS (P=0.044;P=0.000).There were significant differences in incidence rates of grade 2 and 3 acute radiation esophagitis between the two groups (28% vs.16%, 9% vs.2%, P=0.009).Conclusions Both hyperfractionated radiotherapy and hypofractionated radiotherapy combined with chemotherapy can improve the PFS and OS of patients with limited-stage SCLC.The time from the initiation of chemotherapy to the initiation of thoracic radiotherapy ≤43 days and the time from the initiation of chemotherapy to the end of thoracic radiotherapy ≤63 days are favorable prognostic factors for PFS and OS, respectively.However, the hyperfractionated group has significantly higher incidence rates of grade 2 and 3 acute radiation esophagitis than the hypofractionated group.