Objective:To compare the efficiency and characteristics of different carrier proteins and signal sequences in delivering antigens into Escherichia coli outer membrane vesicles (OMVs). Methods:The fusion protein F1V, which consisted of the main protective antigen of Yersinia pestis F1 and LcrV, was expressed using the carrier proteins such as cytolysin A (ClyA), outer membrane protein A (OmpA), or β-lactamases (Bla) signal sequence as a carrier protein. The expression, localization, and content of F1V protein in OMVs were compared and analyzed. Results:All three delivery methods successfully incorporated F1V protein into OMVs and localized it on the surface of OMVs. Notably, when OmpA was used as the carrier protein, the F1V fusion protein constituted up to 30% of the total protein in OMVs. The highest yield of OMVs, reaching 4.2 mg/L, was achieved when Bla signal sequence was used as the carrier.Conclusions:There is a significant difference in the efficiency of different carrier proteins in delivering the F1V antigen into OMVs of Escherichia coli. Considering both the yield of OMVs and the proportion of antigen in the total protein of OMVs, the carrier Bla signal sequence demonstrated the highest efficiency in delivering F1V into OMVs, showing a potential for the future development of OMVs-based plague vaccines.

Objective:To compare the efficiency and characteristics of different carrier proteins and signal sequences in delivering antigens into Escherichia coli outer membrane vesicles (OMVs). Methods:The fusion protein F1V, which consisted of the main protective antigen of Yersinia pestis F1 and LcrV, was expressed using the carrier proteins such as cytolysin A (ClyA), outer membrane protein A (OmpA), or β-lactamases (Bla) signal sequence as a carrier protein. The expression, localization, and content of F1V protein in OMVs were compared and analyzed. Results:All three delivery methods successfully incorporated F1V protein into OMVs and localized it on the surface of OMVs. Notably, when OmpA was used as the carrier protein, the F1V fusion protein constituted up to 30% of the total protein in OMVs. The highest yield of OMVs, reaching 4.2 mg/L, was achieved when Bla signal sequence was used as the carrier.Conclusions:There is a significant difference in the efficiency of different carrier proteins in delivering the F1V antigen into OMVs of Escherichia coli. Considering both the yield of OMVs and the proportion of antigen in the total protein of OMVs, the carrier Bla signal sequence demonstrated the highest efficiency in delivering F1V into OMVs, showing a potential for the future development of OMVs-based plague vaccines.

ObjectiveTo explore the possible mechanisms of Shoutai Wan （寿胎丸） in treating recurrent miscarriage （RSA） from the perspective of immune tolerance under the acidic microenvironment at the maternal-fetal interface. MethodsFemale CBA/J mice were randomly divided into normal group， model group， progesterone group， and Shoutai Wan group， with 15 mice in each group. The mice in the normal group and model group were given 0.2 ml distilled water by gavage each day， the Shoutai Wan group given Shoutai Wan decoction 0.15 g/（10 g·d） by gavage， the progesterone group given progesterone tablets 0.44 mg/（10 g·d） by gavage. After gavage for 14 days， the mice were cohabited. Female CBA/J mice in the normal group were mated with male BALB/c mice at a ratio of 2∶1， and female CBA/J mice in the other groups were mated with male DBA/2 mice at a ratio of 2∶1 to establish the RSA mouse model. Vaginal smears were taken from the female mice the next morning， and the appearance of a large number of spermatozoa and the presence of a vaginal plug were considered as the first day of pregnancy. After the appearance of the plug， the mice were continued to be administered according to the previous method until the 10th day of pregnancy. On the 10th day of pregnancy， maternal-fetal interface tissues were collected from each group of mice， and lactate dehydrogenase colorimetric method was used to detect lactate （LA） content； qPCR method and Western blot method were used to detect the expression of immune-related factors interleukin-4 （IL-4）， interferon-gamma （IFN-γ）， transforming growth factor beta 1 （TGF-β1）， and forkhead box protein 3 （Foxp3） mRNA and protein； flow cytometry was used to detect the numbers of helper T lymphocyte 1 （Th1）， helper T lymphocyte 2 （Th2）， regulatory T cell （Treg）， classical macrophage （M1）， and alternative macrophage （M2）. The bivariate Pearson test was used to analyze the correlation between LA content and the numbers of Th1， Th2， Treg， M1， and M2 cells， as well as the correlation between LA content and the expression of IL-4， IFN-γ， TGF-β1， Foxp3 protein， and mRNA. ResultsOn the 10th day of pregnancy， compared with the normal group， the LA content decreased in the model group， and the expression of IL-4， TGF-β1， Foxp3 protein and mRNA in the maternal-fetal interface tissues decreased， while the expression of IFN-γ protein and mRNA increased. The numbers of Th1 and M1 cells increased， while the numbers of Th2， Treg， and M2 cells decreased （P＜0.05 or P＜0.01）. Compared with the model group， the LA content increased in the Shoutai Wan group and progesterone group. The expression of IL-4， TGF-β1， Foxp3 protein and mRNA in the maternal-fetal interface tissues increased， while the expression of IFN-γ protein and mRNA decreased. The numbers of Th1 and M1 cells decreased， while the numbers of Th2， Treg， and M2 cells increased （P＜0.05 or P＜0.01）. The LA content was positively correlated with the numbers of Th2， Treg， and M2 cells， and the expression of IL-4， TGF-β1， Foxp3 protein， and mRNA （P＜0.05 or P＜0.01）； the LA content was negatively correlated with the numbers of Th1， M1 cells， and the expression of IFN-γ protein and mRNA （P＜0.05 or P＜0.01）. ConclusionShoutai Wan may improve immune tolerance by regulating the expression of immune-related factors in the acidic microenvironment at the maternal-fetal interface of RSA model mice， thereby exerting its role in preventing miscarriage.

Objective:To investigate the diagnostic accuracy of serological indicators and evaluate the diagnostic value of a new established combined serological model on identifying the minimal hepatic encephalopathy (MHE) in patients with compensated cirrhosis.Methods:This prospective multicenter study enrolled 263 compensated cirrhotic patients from 23 hospitals in 15 provinces, autonomous regions and municipalities of China between October 2021 and August 2022. Clinical data and laboratory test results were collected, and the model for end-stage liver disease (MELD) score was calculated. Ammonia level was corrected to the upper limit of normal (AMM-ULN) by the baseline blood ammonia measurements/upper limit of the normal reference value. MHE was diagnosed by combined abnormal number connection test-A and abnormal digit symbol test as suggested by Guidelines on the management of hepatic encephalopathy in cirrhosis. The patients were randomly divided (7∶3) into training set ( n=185) and validation set ( n=78) based on caret package of R language. Logistic regression was used to establish a combined model of MHE diagnosis. The diagnostic performance was evaluated by the area under the curve (AUC) of receiver operating characteristic curve, Hosmer-Lemeshow test and calibration curve. The internal verification was carried out by the Bootstrap method ( n=200). AUC comparisons were achieved using the Delong test. Results:In the training set, prevalence of MHE was 37.8% (70/185). There were statistically significant differences in AMM-ULN, albumin, platelet, alkaline phosphatase, international normalized ratio, MELD score and education between non-MHE group and MHE group (all P<0.05). Multivariate Logistic regression analysis showed that AMM-ULN [odds ratio ( OR)=1.78, 95% confidence interval ( CI) 1.05-3.14, P=0.038] and MELD score ( OR=1.11, 95% CI 1.04-1.20, P=0.002) were independent risk factors for MHE, and the AUC for predicting MHE were 0.663, 0.625, respectively. Compared with the use of blood AMM-ULN and MELD score alone, the AUC of the combined model of AMM-ULN, MELD score and education exhibited better predictive performance in determining the presence of MHE was 0.755, the specificity and sensitivity was 85.2% and 55.7%, respectively. Hosmer-Lemeshow test and calibration curve showed that the model had good calibration ( P=0.733). The AUC for internal validation of the combined model for diagnosing MHE was 0.752. In the validation set, the AUC of the combined model for diagnosing MHE was 0.794, and Hosmer-Lemeshow test showed good calibration ( P=0.841). Conclusion:Use of the combined model including AMM-ULN, MELD score and education could improve the predictive efficiency of MHE among patients with compensated cirrhosis.

Objective:Development and validation of a nomogram for predicting the 4-year incidence of type-2 diabetes mellitus (T2DM) in a Chinese population was attempted.Methods:This prospective cohort study was conducted in Shijingshan District Pingguoyuan Community (Beijing, China) from December 2011 to April 2012 among adults aged≥40 years not suffering from T2DM. Finally, 8 058 adults free of T2DM were included with a median duration of follow-up of 4 years. Participants were divided into a modeling group and verification group using simple random sampling at a ratio of 7∶3. Univariate and multivariate Cox proportional risk models were applied to identify the independent risk predictors in the modeling group. A nomogram was constructed to predict the 4-year incidence of T2DM based on the results of multivariate analysis. The Concordance Index and calibration plots were used to evaluate the differentiation and calibration of the nomogram in both groups.Results:A total of 5 641 individuals were in the modeling group and 2 417 people were in the validation group, of which 265 and 106 had T2DM, respectively, at 4-year follow-up. In the modeling group, age ( HR=1.349, 95% CI 1.011-1.800), body mass index ( HR=1.347, 95% CI 1.038-1.746), hyperlipidemia ( HR=1.504, 95% CI 1.133-1.996), fasting blood glucose ( HR=4.189, 95% CI 3.010-5.830), 2-h blood glucose level according to the oral glucose tolerance test ( HR=3.005, 95% CI 2.129-4.241), level of glycosylated hemoglobin ( HR=3.162, 95% CI 2.283-4.380), and level of γ-glutamyl transferase ( HR=1.920, 95% CI 1.385-2.661) were independent risk factors for T2DM. Validation of the nomogram revealed the Concordance Index of the modeling group and validation group to be 0.906 (95% CI 0.888-0.925) and 0.844 (95% CI 0.796-0.892), respectively. Calibration plots showed good calibration in both groups. Conclusion:These data suggest that our nomogram could be a simple and reliable tool for predicting the 4-year risk of developing T2DM in a high-risk Chinese population.

Objective:To investigate the clinical, imaging, etiological and prognostic features of patients with infarctions in different locations of the medulla oblongata.Methods:Patients with acute medullary infarction hospitalized at Tianjin Huanhu Hospital from July 2017 to July 2022 were included. The risk factors, clinical manifestation, stroke mechanism and 90-day prognosis of these patients were analyzed retrospectively.Results:Among the 256 patients enrolled, 150 (58.6%) had lateral medullary infarction (LMI), 106 (41.4%) had medial medullary infarction (MMI). The most frequent clinical manifestation of patients with LMI was dizziness (84.7%,127/150). And motor disorders (83.0%,88/106) was the most frequent clinical manifestation of patients with MMI. LMI lesions were mostly located in the middle (42.7%,64/150) and MMI lesions were mostly located in the upper (60.4%,64/106) medulla oblongata, with statistically significant difference (χ 2=47.53, P<0.001). Large artery atherosclerosis (LAA) was the main stroke mechanism in LMI and MMI [57.3%(86/150) vs 56.6%(60/106)]. Early neurological deterioration was more common in MMI (25.5%,27/106) and less common in LMI (7.3%,11/150), with statistically significant difference (χ 2=16.17, P<0.001). At discharge, more patients with MMI showed poor prognosis in short term [45.3% (48/106) vs 24.0% (36/150), with statistically significant difference (χ 2=12.76, P<0.001)] and even long term at 90-day follow-up [33.0% (35/106) vs 12.7% (19/150), also with statistically significant difference (χ 2=15.48, P<0.001)] than those with LMI. A total of 10 patients (4.0%, 10/256) developed respiratory failure during hospitalization, including 7 patients with LMI (4.7%, 7/150) and 3 patients with bilateral MMI (2.8%,3/106). Early neurological deterioration ( OR=3.38, 95% CI 1.25-9.10, P=0.016) and LAA (compared with small artery occlusion) ( OR=3.08, 95% CI 1.13-8.37, P=0.028) were independent risk factors for poor prognosis in MMI. Age ( OR=1.01, 95% CI 1.01-1.17, P=0.026) and early neurological deterioration ( OR=20.19, 95% CI=2.63-155.06, P=0.004) were independently correlated with poor outcome in LMI. Conclusions:LMI and MMI had similar etiology and significant differences in clinical manifestations, early neurological deterioration and prognosis. Further classification of medullary infarction was of great significance for diagnosis, treatment and prognosis evaluation.

Rats ; Animals ; Sciatic Neuropathy ; Muscle, Skeletal ; Sciatic Nerve/physiology* ; Sequence Analysis, RNA ; Nerve Regeneration/physiology*

Lymphocytic hypophysitis(LYH) is a rare autoimmune disease, which is more common in women and is closely associated with pregnancy. For patients with lymphocytic hypophysitis, the clinical manifestations mainly depend on the speed of disease progression, the extent of lesions, the severity and stage of disease, which can present as mass effect, decreased anterior pituitary hormone function, and central diabetes insipidus, etc. Due to physical changes in pituitary anatomy and hormone levels, as well as restriction of imaging examinations during pregnancy, it poses greater challenge to establish diagnosis in perinatal patients, which demands a comprehensive assessment of the patient′s clinical symptoms and biochemical tests. For treatment, patients with hypopituitarism during pregnancy should be given hormone replacement therapy. Moreover, patients need to adopt optimized tailored therapy according to gestational age and various clinical manifestations. At present, there is no report on the adverse effects of LYH on pregnancy outcome and re-pregnancy. This review discusses the diagnosis, treatment and prognosis of patients with LYH during pregnancy, and provides reference for the clinical management of this condition.

Objective:To investigate the effects of a eukaryotic expression plasmid for IL-6 and B-cell activating factor (BAFF) fusion protein on the histopathological changes in salivary and lacrimal glands of non-obese diabetic (NOD) mice with Sj?gren′s syndrome and to elucidate the possible therapeutic mechanism of IL-6/BAFF fusion protein eukaryotic expression plasmid in NOD mice.Methods:The eukaryotic expression plasmid for IL-6/BAFF fusion protein was constructed. After transfecting CHO cells with the plasmid, the expression of IL-6/BAFF fusion protein was detected by Western blot. BALB/c mice were injected with the plasmid every two weeks for three times and the titers of anti-IL-6 and anti-BAFF antibodies were measured by ELISA. Twenty-one NOD mice were randomly divided into three groups (control group, empty vector group and therapy group) by numerical table method. The mice in the therapy group were injected with the IL-6/BAFF fusion protein eukaryotic expression plasmid once a week for six times and the mice in the empty vector group were injected with empty plasmid. The levels of anti-IL-6 and anti-BAFF antibodies as well as cytokines (IL-6, BAFF, INF-γ, IL-10 and IL-17A) in mouse serum samples were detected by ELISA. The proportions of Th17, Treg, Th1 and Th2 cells in mouse splenocytes were measured by flow cytometry. Focal lymphocyte infiltration and pathological changes in the lacrimal and salivary glands of mice were observed under light microscopy after HE staining.Results:The eukaryotic expression plasmid for IL-6/BAFF fusion protein increased the levels of anti-IL-6 and anti-BAFF antibodies in the serum of BALB/c mice ( P<0.05). The levels of anti-IL-6 and anti-BAFF antibodies in the serum of NOD mice in the therapy group increased ( P<0.01), while the expression of IL-6, BAFF, INF-γ, IL-10 and IL-17A in NOD mice in the therapy group was lower than that in the control group and the empty vector group ( P<0.05). The percentages of Treg and Th2 cells in the splenocytes of NOD mice increased after treatment ( P<0.05). Moreover, the eukaryotic expression plasmid for IL-6/BAFF fusion protein significantly improved the irregular size and morphology of glandular vesicles in the lacrimal and salivary glands, reduced the ductal dilatation and decreased the focal lymphocyte infiltration in NOD mice. Conclusions:The eukaryotic expression plasmid for IL-6/BAFF fusion protein induced the production of anti-IL-6 and anti-BAFF antibodies, decreased the expression of inflammatory cytokines, regulated the balance of Th17/Treg and Th1/Th2 cells, improved the irregular alveolar structure and ductal dilation in the lacrimal and salivary glands and reduced the focal lymphocyte infiltration in NOD mice. This study showed that eukaryotic expression plasmid for IL-6/BAFF fusion protein might serve as a potential target for therapeutic targeting of T and B cells.

Objective:To analyze the clinical characteristics of fulminant type 1 diabetes (FT1DM) in China.Methods:Clinical data of 279 cases related to FT1DM in Chinese Database from January 2005 to December 2018 were collected, and other 20 patients from our hospital were included in the present study.Results:(1) There has been a progressive increasing in the number of reported cases every year in China, and the number in the southern region were significantly more than that in the northern region. (2) The median age of the onset of FT1DM patients in China was 32.5 years old, without significant gender difference. Moreover, 36.5% (54/148) of the female patients caught the disease during their prenatal period, most of them were onset in the second or third trimesters of pregnancy and 2 weeks after delivery (37/40), and the prognosis of the fetus was extremely poor. (3) Compared with new-onset type 1 diabetes, FT1DM patients were younger, and with higher blood glucose [(39.7±15.3) vs (21.2 ± 9.9) mmol/L], higher serum creatinine [(188.4±115.9) vs (51.8 ±23.1) μmol/L], and higher amylase levels [245.5 (26.0-5 137.0) vs 54.7 (14.0-404.9) U/L]. FT1DM patients were with more severe acidosis, and lower HbA 1C level [(6.6 ±0.8)% vs (12.9 ± 2.5)%, P<0.01]. (4) FT1DM patients may combine with multiple organ dysfunction or severe metabolic disorders, electrolyte disorders, as well as liver and kidney dysfunctions, and elevation of amylase and muscle enzymes. Conclusion:FT1DM are with some clinical characteristics different from classic new-onset type 1 diabetes, including adult-onset, frequent in the southern China. Pregnancy may be a predisposing factor for female patients. Significant metabolic disorders and multiple organ involvements are common in the patients with FT1DM.