[摘 要] 目的：构建人乳头瘤病毒16型（HPV16） L1蛋白纳米抗体初级文库，通过筛选鉴定获得一株HPV16 L1特异性纳米抗体。方法：以HPV 16 L1蛋白为抗原对羊驼进行免疫，采用噬菌体展示技术构建初级抗体文库。经3轮淘选，采用ELISA法鉴定阳性克隆，将阳性反应最强克隆的VHH序列进行真核表达。经亲和纯化、凝胶过滤层析纯化、SDS‑PAGE和WB法鉴定，获得目的纳米抗体；采用表面等离子共振（SPR）技术检测纳米抗体与HPV 16 L1蛋白之间的亲和力，CCK-8法检测纳米抗体对人永生化角质细胞HaCat的毒性，荧光素酶报告基因实验检测纳米抗体对HPV 16假病毒的中和活性。结果：初级文库库容为1.304 × 1010，丰度为6.5 × 109个/mL，ELISA法鉴定获得36个阳性克隆。表达、纯化获得蛋白单体与二聚体，经鉴定为目的纳米抗体（命名为Nb）。Nb与HPV 16 L1蛋白结合的亲和力为35.41 nmol/L。Nb实验组HaCat细胞增殖活力与空白组没有显著差异（P > 0.05）。与阴性组比较，0.1和1 μmol/L Nb均能抑制假病毒感染293FT细胞（均P < 0.01）。结论：成功获得一株纯度较好、亲和力较高，对上皮细胞没有明显毒性作用、有效抑制HPV 16假病毒感染293FT细胞的纳米抗体Nb，为防治HPV 16感染提供了有效的候选抗体类药物。

Objective To investigate the related factors of early neurological deterioration in elderly patients with thrombolysis for acute occlusive stroke of large intracranial vessels. Methods Clinical materials of 480 elderly patients with thrombolysis for acute occlusive stroke of large intracranial vessels were retrospectively analyzed. The National Institutes of Health Stroke Scale (NIHSS) was used to evaluate the neurological function of patients within 72 hours after thrombectomy, and the patients were divided into deterioration group and non-deterioration group. The baseline materials were compared between the two groups, the factors of early neurological deterioration were analyzed, and the receiver operating characteristic (ROC) curve was drawn to analyze the predictive value of risk. Results Among 480 elderly patients with thrombolysis for acute occlusive stroke of large intracranial vessels, 92 cases (19.17%) had early neurological deterioration; the blood glucose level, white blood cell count, incidence of dysphagia, and incidence of coma in the deterioration group were significantly higher than those in the non-deterioration group (P < 0.05); the Logistic regression analysis showed that high level of blood glucose (95%CI, 1.203 to 1.478), high level of white blood cell count (95%CI, 1.159 to 1.408), dysphagia (95%CI, 7.049 to 31.475), and coma (95%CI, 8.375 to 43.195) were risk factors for early neurological deterioration in elderly patients with thrombolysis for acute occlusive stroke of large intracranial vessels (OR>1, P < 0.05). ROC curve results showed that blood glucose, white blood cell count, dysphagia, and coma had certain predictive value for early neurological deterioration in elderly patients with thrombolysis for acute occlusive stroke of large intracranial vessels[the area under the curve (AUC) was 0.733, 0.708, 0.707 and 0.701]; the ROC curve showed AUC was 0.934, indicating that the model was reliable and stable. Conclusion Early neurological deterioration in elderly patients with thrombolysis for acute occlusive stroke of large intracranial vessels may be related to factors such as blood glucose, white blood cell count, dysphagia, and coma. Early risk assessment and relevant intervention measures can be taken clinically to improve prognosis.

BACKGROUND: T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domains (TIGIT), an inhibitory receptor expressed on T cells, plays a dysfunctional role in antiviral infection and antitumor activity. However, it is unknown whether TIGIT expression on T cells influences the immunological effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inactivated vaccines. METHODS: Forty-five people living with HIV (PLWH) on antiretroviral therapy (ART) for more than two years and 31 healthy controls (HCs), all received a third dose of a SARS-CoV-2 inactivated vaccine, were enrolled in this study. The amounts, activation, proportion of cell subsets, and magnitude of the SARS-CoV-2-specific immune response of TIGIT + CD4 + and TIGIT + CD8 + T cells were investigated before the third dose but 6 months after the second vaccine dose (0W), 4 weeks (4W) and 12 weeks (12W) after the third dose. RESULTS: Compared to that in HCs, the frequency of TIGIT + CD8 + T cells in the peripheral blood of PLWH increased at 12W after the third dose of the inactivated vaccine, and the immune activation of TIGIT + CD8 + T cells also increased. A decrease in the ratio of both T naïve (T N ) and central memory (T CM ) cells among TIGIT + CD8 + T cells and an increase in the ratio of the effector memory (T EM ) subpopulation were observed at 12W in PLWH. Interestingly, particularly at 12W, a higher proportion of TIGIT + CD8 + T cells expressing CD137 and CD69 simultaneously was observed in HCs than in PLWH based on the activation-induced marker assay. Compared with 0W, SARS-CoV-2-specific TIGIT + CD8 + T-cell responses in PLWH were not enhanced at 12W but were enhanced in HCs. Additionally, at all time points, the SARS-CoV-2-specific responses of TIGIT + CD8 + T cells in PLWH were significantly weaker than those of TIGIT - CD8 + T cells. However, in HCs, the difference in the SARS-CoV-2-specific responses induced between TIGIT + CD8 + T cells and TIGIT - CD8 + T cells was insignificant at 4W and 12W, except at 0W. CONCLUSIONS TIGIT expression on CD8 + T cells may hinder the T-cell immune response to a booster dose of an inactivated SARS-CoV-2 vaccine, suggesting weakened resistance to SARS-CoV-2 infection, especially in PLWH. Furthermore, TIGIT may be used as a potential target to increase the production of SARS-CoV-2-specific CD8 + T cells, thereby enhancing the effectiveness of vaccination.
Humans ; Antibodies, Viral ; CD8-Positive T-Lymphocytes ; COVID-19/complications* ; COVID-19 Vaccines/immunology* ; HIV Infections/complications* ; Receptors, Immunologic ; SARS-CoV-2

Objective To explore the efficacy and prognostic independent factors of modified island flap(OIF)and urethroplasty(tip)in the treatment of hypospadias in children.Methods The 164 children with hypospadias analyzed retrospectively were treated from February,2013 to February,2021.They were divided into two groups according to the operation method.82 patients in tip group were treated with tip and 82 patients in OIF group were treated with OIF.The treatment effects of the two groups were compared.Then they were divided into two groups according to the cure condition,namely,the good prognosis group(cured by operation,151 case)and the poor prognosis group(not cured by operation,13 case).Independent factors affecting the prognosis of children were analyzed by binary logistic regression.Results The cure rate of OIF group was 96.34％,which was higher than that of the tip group(87.8％),and the incidence of postoperative complications in OIF group was 7.32％,which was lower than that of the tip group(23.17％,P＜0.05).The operation time in tip group(95.95±12.35)min,which was shorter than that of the OIF group(P＜0.05).At the same time,the binary logistic regression analysis showed that the degree of hypospadias and the classification of barcat were the prognostic factors of children with hypospadias.The degree of penis bending,the width of penis head and the method of operation were suspicious factors.Conclusion OIF and tip have good effects in the treatment of hypospadias in children.OIF has a higher success rate,tip has a shorter operation time and fewer postoperative complications.However,the independent factors affecting the prognosis of children are preoperative hypospadias classification and barcat classification.This operation method is not an independent factor affecting the prognosis.

Taking medical statistics major in Binzhou Medical University as an example, based on the outcome-based education theory, in order to enhance the innovation ability of university students, we put forward the systematic second classroom training system, including improving the personnel training system, implementing education, experiment teaching reform, and practice teaching reform. It has achieved outstanding results in the discipline competition, improved social service ability and high degree of employer satisfaction. The systematic second-class talent training system based on the outcome-based education theory can provide reference for other medical colleges and related applied majors.

Multi-disciplinary team(MDT)mode is regarded as the key to standardized diagnosis and treatment of malignant tumors. The model, however, encounters such roadblocks in the current form of MDT organization, as costly clinical resources and time consumption, low efficiency, poor management of participating experts in MDT, and lack of enforceability of the therapeutic decisions made. This paper summarized the practical MDT experiences of the Affiliated Cancer Hospital of Shandong First Medical University. It introduced the construction of an intranet-based MDT system covering a large proportion of newly diagnosed malignant tumor patients, and the practices and achievements of such MDT management system under hospital administrative guidance. The authors proposed to use reporting ratio as the main assessment indicator in promoting MDT, and that to define the performance, responsibilities and rights in MDT practice. These measures aim at to upgrading individual behaviors of doctors to organizational behaviors of hospitals, and providing cancer patients with more standardized, comprehensive and personalized diagnosis and treatment decisions.

Objective:To explore the influence of short-time pasteurization (62.5±0.5℃ for 5 s) on the main bioactive components and immune cells in human breast milk.Methods:Fresh breast milk was collected from 53 women whose premature infants were admitted to the neonatal intensive care unit of the Children's Hospital of Fudan University from May 2020 to October 2020. Each sample (20 ml) was divided into unsterilized, Holder pasteurized (62.5 ℃ for 30 min), or short-time pasteurized groups. The concentration of secretory immunoglobulin A (sIgA), lactoferrin (LTF), lysozyme (LZM), and insulin-like growth factor binding protein-3 (IGFBP-3) in breastmilk whey were detected by enzyme-linked immunosorbent assay and the number of viable immune cells (leukocytes, monocytes, T cells, and B cells) in breastmilk by flow cytometry.Results:(1) A total of 87 breast milk samples were collected. The levels of sIgA, LTF, and LZM were the highest in the unsterilized group, followed by the short-time and Holder pasteurized group [0.42 mg/ml (0.33-0.65 mg/ml) vs 0.40 mg/ml (0.28-0.62 mg/ml) and 0.25 mg/ml (0.17-0.37 mg/ml); (3.57±1.06) vs (3.53±1.11) and (0.85±0.58) mg/ml; 128.60 μg/ml (77.18-203.00 μg/ml) vs 121.70 μg/ml (68.66-188.20 μg/ml) and 83.40 μg/ml (47.40-151.40 μg/ml); all P<0.05]. There was no significant difference in the level of IGFBP-3 among the groups. The median retention rates of sIgA, LTF, and LZM in the Holder pasteurized group were all lower than those in the short-time pasteurized group [55.87% (46.01%-71.41%) vs 96.93% (83.03%-115.90%); 21.72% (12.54%-29.42%) vs 97.88% (88.98%-104.30%); 69.26% (49.42%-89.08%) vs 93.80% (74.85%-110.20%); all P<0.05]. No significant difference in the level of preserved IGFBP-3 was observed between the three groups ( P>0.05). (2) The number of viable leukocytes, monocytes, T cells, and B cells in the Holder pasteurized group were lower than those in the unsterilized group [leukocytes: 185.50 (87.00-356.50) vs 1 271.00 (540.50-2 283.00); monocytes: 12.00 (6.00-16.75) vs 266.00 (137.30-518.80); T cells: 1.00 (0.00-2.00) vs 47.50 (28.50-116.00); B cells: 1.00 (0.00-1.75) vs 21.00(10.00-41.50); all P<0.05]. The percentage of viable leukocyte to the total leukocyte and the viable monocytes, T cells, and B cells to the viable leukocytes were lower in the Holder pasteurized group than those in the unsterilized group [24.80%(16.00%-36.80%) vs 74.20%(63.55%-86.45%); 5.91%(4.09%-8.77%) vs 21.90%(17.40%-29.30%); 0.31%(0.00%-1.31%) vs 4.00%(2.69%-6.43%); 0.30%(0.00%-0.86%) vs 1.27%(0.57%-2.85%); all P<0.05]. A similar trend was observed between short-time pasteurization and unsterilized groups (all P<0.05). (3) The percentages of viable monocytes, T cells, and B cells in their subsets were lower in both Holder and short-time pasteurized groups than those in the unsterilized group [2.94%(1.33%-7.14%) vs 9.72%(5.77%-16.00%) and 52.60%(31.35%-68.75%); 0.00%(0.00%-1.61%) vs 0.49%(0.00%-2.53%) and 28.10%(10.55%-57.00%); 0.00%(0.00%-0.83%) vs 0.24%(0.00%-2.47%) and 13.80%(3.27%-41.00%); all P<0.05].The number and percentage of viable leukocytes in total leukocytes and viable monocytes in total monocytes [leukocytes: 279.50(116.80-548.50), 32.20%(20.70%-45.75%); monocytes: 32.00(21.00- 83.75),15.60%(11.10%-19.15%)] were higher than those in the pasteurized group (all P>0.05). The short-time pasteurized group was noted only for a higher percentage of the viable monocytes to viable leukocytes than the Holder pasteurized group (all P<0.05). Conclusions:Compared with the Holder pasteurization, sIgA, LTF, LZM level, and monocyte activity in breast milk can be better preserved by short-time pasteurization.

Objective:To investigate the effects of different durations of antiretroviral therapy (ART) on metabolism in HIV/AIDS patients.Methods:a single center cross-sectional study, 424 HIV/AIDS were divided into four groups according to the different duration of treatment: group Ⅰ (0.5-2 years), group Ⅱ (2-4 years including 2 years), the group Ⅲ (4-6 years including 4 years), group Ⅳ (≥6 years), the differences of abnormal blood lipid, renal function, blood glucose and liver function in each group were compared.Results:incidence of hypercholesteremia: group Ⅰ 9/106 (8.5%), group Ⅱ 17/134 (12.7%), group Ⅲ 22/123 (17.9%), group Ⅳ 4/61 (6.6%), p=0.076; incidence of hypertriglyceridemia: groupⅠ 32/106 (30.2%), group Ⅱ 58/134 (43.3%), group Ⅲ 50/123 (40.7%), group Ⅳ 27/61 (44.3%), p=0.152; incidence of High LDL-C: group Ⅰ 8/106 (7.5%), group Ⅱ 17/134 (12.7%), group Ⅲ 15/123 (12.2%), group Ⅳ 3/61 (4.9%), p=0.249; Incidence of low HDL-C: groupⅠ 48/106 (45.3%), group Ⅱ 66/134 (49.3%), group Ⅲ 55/123 (44.7%), group Ⅳ 33/61 (54.1%), p=0.612; incidence of glomerular filtration rate < 90 ml/min / 1.73 m2: groupⅠ4/106 (3.8%), group Ⅱ 6/134 (4.5%), group Ⅲ 6/123 (4.9%), group Ⅳ 2/61 (3.3%), P=0.953; eGFR was negatively correlated with treatment duration (r=-0.165, P <0.001). Incidence of hypophosphatemia: group Ⅰ 7/106 (6.6%), group Ⅱ 7/134 (5.2%), group Ⅲ 12/123 (9.8%), group Ⅳ 6/61 (9.8%), P=0.478; incidence of hyperuricemia: groupⅠ26/106 (24.5%), group Ⅱ 35/134 (26.1%), group Ⅲ 28/123 (22.8%), group Ⅳ 10/61 (16.4%), P=0.508; incidence of diabetes: groupⅠ1/106 (0.9%), group Ⅱ 9/134 (6.7%), group Ⅲ 6/123 (4.9%), group Ⅳ 3/61 (4.9%), P=0.140; incidence of GGT > 60 U/L: group Ⅰ(21.7%), 23 group Ⅱof 34 (25.4%), 25 group Ⅲ (20.3%), group Ⅳ of 12 (19.7%), P=0.736; incidence of ALT > 50 U/L: groupⅠ(21.7%), 23 group Ⅱ of 33 (24.6%), group Ⅲ of 24 (19.5%), group Ⅳ of 12 (19.7%), p=0.761Conclusion:The types of dyslipidemia in HIV/AIDS patients treated for more than half a year are mainly low HDL-C and hypertriglyceridemia, and the incidence of dyslipidemia does not increase with the prolonging of treatment. It showed a decreasing trend of eGFR with the prolongation of treatment and the occurrence of hypophosphatemia and hyperuricemia should be concerned. The damage of ART drugs to liver function persisted during treatment.

We strive to explore a student-centered evaluation system focused on improving students' learning efficiency and learning effect. Through classroom questioning, discussion of difficult cases, new progress report, question bank test, writing of clerkship notes, simulated inquiry between students with bedside inquiry, physical examination between students with bedside physical examination, operation training of basic clinical skills, direct observation and evaluation of clinical skills, mini-clinical exercise evaluation, graduation examination and other measures, an evaluation system for promoting learning by class evaluation, clerkship evaluation and practice evaluation has been established to cultivate the connotation construction of medical moral quality and clinical practice ability of medical students, and further promote the mastery of basic theoretical knowledge and clinical operation skills. By comparing the results of the phased examination for medical practitioners, the passing rate of 2018 is significantly higher than that of 2017, with 23.31% increased passing rate of theoretical results, and 0.79% increased passing rate of skills operation results. It can be seen that the evaluation system for promoting learning has played a guiding role in the teaching practice of clinical courses in our hospital.

Objective To investigate the changes in neutrophil immunophenotypes in neonates with late-onset sepsis and their clinical significance.Methods A total of 42 neonates with late-onset sepsis were enrolled prospectively as sepsis group from Children's Hospital of Fudan University from January 2015 to October 2016,which included 26 preterm infants and 16 term infants.Another 33 neonates without infectious diseases consisting of 20 preterm infants and 13 term infants were selected as control group.According to the severity of sepsis,neonates in the sepsis group were further divided into severe (n=11)and mild sepsis (n=31) subgroups.Expression of CD16 and CD62L on neutrophils was measured by flow cytometry to determine the distribution of neutrophil subsets in neonatal peripheral blood.Differences in the distribution of neutrophil subsets between the two groups and two subgroups were compared using Wilcoxon rank-sum test.Multivariate logistic regression analysis was used to investigate the relationship between neutrophil subsets and the severity of neonatal sepsis.Receiver operating characteristic (ROC) curves were used to assess the diagnostic value of neutrophil subsets for the severity of sepsis.Results Four neutrophil subsets were identified in neonatal peripheral blood,including early-stage neutrophils (CD16-/CD62L-),immature neutrophils (CD16-/CD62L+),mature neutrophils (CD16+/CD62L+) and activated neutrophils (CD16+/CD62L-).Activated [preterm:61.1％ (20.2％-79.4％),term:47.6％ (15.2％-70.1％)] and mature neutrophils [preterm:35.7％ (19.9％-75.8％),term:52.0％ (25.6％-82.8％)] were the dominant subsets in the control group.In preterm infants,the proportion of early-stage [3.5％ (1.7％-9.4％) vs 1.9％ (0.6％-4.0％),Z=-2.501,P=0.012] and immature neutrophils [6.3％ (0.7％-45.5％) vs 0.4％ (0.3％-0.7％),Z=-3.878,P＜0.001] were higher,but that of activated neutrophils [8.3％ (2.3％-49.2％) vs 61.1％ (20.2％-79.4％),Z=2.991,P=0.002] were lower in the sepsis group than those in the control group;same differences were found in the absolute counts of each neutrophil subsets.Among term infants,more immature neutrophils were found in the sepsis group than those in the control group [49 (18-200) vs 13 (5-36)/μl,Z=-2.193,P=0.028].The proportion and the absolute counts of early-stage and immature neutrophils in the severe sepsis subgroup were all higher than those in the mild cases [early-stage neutrophils:5.8％ (3.4％-17.8％) vs 3.0％ (1.4％-7.3％),304 (137-1478) vs 158 (53-321)/μl;immature neutrophils:23.0％ (6.3％-47.0％) vs 0.9％ (0.5％-6.8％),1003 (487-2818) vs 85 (18-275)/μl;all P＜0.05].Multivariate logistic regression analysis showed that the proportion of early-stage neutrophils was associated with the severity of sepsis (OR=1.2,95％CI:1.0-1.4,P=0.012).In addition,the diagnostic value of the proportion of early-stage neutrophils for severe sepsis was the highest when the cut-off value was 3.3％,with the area under the ROC curve was 0.7 (95％CI:0.6-0.9),sensitivity of 81.8％ (95％CI:48.2％-97.7％) and specificity of 62.3％ (95％CI:42.2％-78.2％).Conclusions There are four neutrophil subsets in the peripheral blood of neonates and autoactivation of neutrophils may exist.With the onset of sepsis,neutrophil subsets react differently between preterm and term infants.The proportion of early-stage neutrophils may be correlated with the severity of neonatal sepsis,which may have a predictive value for severe sepsis.