AKT inhibition interferes with the expression of immune checkpoint proteins and increases NK-induced killing of HL60-AML cells

ABSTRACT Objective To determine the role of the AKT pathway in the regulating of natural Killer-induced apoptosis of acute myeloid leukemia cells and to characterize the associated molecular mechanisms. Methods BALB/c nude mice were injected with HL60 cells to induce a xenogenic model of subcutaneous leukemic tumors. Mice were treated with perifosine, and their spleens were analyzed using biometry, histopathology, and immunohistochemistry. Gene expression analysis in leukemia cells was performed by real-time PCR. Protein analysis of leukemia and natural Killer cells was performed by flow cytometry. AKT inhibition in HL60 cells, followed by co-culture with natural Killer cells was performed to assess cytotoxicity. Apoptosis rate was quantified using flow cytometry. Results Perifosine treatment caused a reduction in leukemic infiltration in the spleens of BALB/c nude mice. In vitro , AKT inhibition reduced HL60 resistance to natural Killer-induced apoptosis. AKT inhibition suppressed the immune checkpoint proteins PD-L1, galectin-9, and CD122 in HL60 cells, but did not change the expression of their co-receptors PD1, Tim3, and CD96 on the natural Killer cell surface. In addition, the death receptors DR4, TNFR1, and FAS were overexpressed by AKT inhibition, thus increasing the susceptibility of HL60 cells to the extrinsic pathway of apoptosis. Conclusion The AKT pathway is involved in resistance to natural Killer-induced apoptosis in HL60 cells by regulating the expression of immune suppressor receptors. These findings highlight the importance of AKT in contributing to immune evasion mechanisms in acute myeloid leukemia and suggests the potential of AKT inhibition as an adjunct to immunotherapy.

Impact of moderate and severe primary open-angle glaucoma on quality of life due to activity limitation

Purpose: To study the impact of moderate and severe primary open?angle glaucoma (POAG) on the quality of life (QoL) due to activity limitation using glaucoma?specific questionnaires. Methods: This cross?sectional study enrolled 122 participants, 50% (n = 61) being controls and 50% were diagnosed cases of moderate/ severe POAG. Three orally administered glaucoma?specific QoL instruments were used: Glaucoma Activity Limitation (GAL?9), Glaucoma Quality of Life (GQL?15), and Viswanathan questionnaires. The questions related to activity limitation were identified and analyzed for each questionnaire separately. Results: The mean age of the participants was 61.04 ± 9.88 years and a majority were males (64.8%, n = 79). The mean scores in controls, moderate glaucoma, and severe POAG patients for GAL?9 questionnaire were 9.77 ± 1.36 (P = 0.44), 13.75 ± 4.76 (P < 0.001), and 23.45 ± 5.62 (P < 0.001), for GQL?15, these were 16.39 ± 2.18 (P = 0.5), 22.75 ± 7.89 (P < 0.001), and 39.34 ± 9.42 (P < 0.001), respectively, while for the Viswanathan questionnaire, they were 9.49 ± 0.94 (P = 0.38), 7.91 ± 1.59 (P < 0.001), and 4.41 ± 2.20 (P < 0.001), respectively. The GQL?15 and GAL?9 questionnaires concluded that activity limitation pertaining to dark adaptation?related tasks affected the QoL the most in moderate as well as severe POAG (P < 0.001). Using the Viswanathan questionnaire, it was observed that the peripheral vision?related activity limitation was most significant for the decrease in QoL in moderate POAG while near vision?related activity limitation affected the QoL the most in severe POAG (P < 0.001). Conclusion: All three questionnaires concluded that the activity limitation due to moderate and severe glaucoma has a negative impact on the QoL. The limitation of the tasks involving dark adaptation/glare and peripheral vision has the most significant impact on the QoL in moderate glaucoma. As the disease progresses to a severe category, the limitation of activities requiring central and near vision causes the most significant worsening in QoL.

Anafilaxia a alfa-gal: um relato de caso paraibano / Alpha-gal anaphylaxis: a case report from Paraíba, Brazil

O presente relato apresenta um caso de anafilaxia tardia ao carboidrato alfa-gal em um adolescente da cidade de Belém, na Paraíba, Brasil. O paciente desenvolveu reação tardia à ingesta de carne e vísceras de animais. Ele mora em fazenda e tem contato próximo com animais potencialmente contaminados por carrapatos. Essa causa de reação alérgica é nova, e estudos começaram a atribuí-la a casos antes ditos idiopáticos. A anafilaxia é uma reação potencialmente fatal, que deve ser prontamente diagnosticada e tratada. Sendo assim, a descoberta de seu fator desencadeante é um dos principais itens que direcionam o tratamento. No Brasil, nenhum caso de anafilaxia por alfa-gal foi antes descrito na literatura local.

This report presents a case of late anaphylaxis to alpha-gal carbohydrate in a teenager living in the city of Belém, Paraíba, Brazil. The patient developed a late reaction to eating meat and offal of animals; he lives on a farm and has close contact with animals potentially contaminated by ticks. This cause of allergic reaction is new, and studies have started to attribute it to cases previously said to be idiopathic. Anaphylaxis is a potentially fatal reaction that must be promptly diagnosed and treated. Thus, the discovery of its triggering factor is one of the main items that guide treatment. In Brazil, no case of alpha-gal anaphylaxis had been described in the local literature.

Guiqi Dingnian Prescription Delays Senescence of Mouse Mesangial Cells by Regulating JAK2/STAT Pathway Activity / 中国实验方剂学杂志

Objective:To investigate the effects of Guiqi Dingnian prescription （GDP） on the expression of related molecules in Janus tyrosine kinase 2 （JAK2）/signal transducer and activator of transcription （JAK2/STAT） signaling pathway of <italic>D</italic>-galactose （<italic>D</italic>-gal）-induced senescent mesangial cells. Method:The senescent mouse mesangial cells induced by 10 g·L^-1 <italic>D</italic>-gal were continuously treated with 40 mg·L^-1GDP for three days. The senescence of the treated cells was determined by senescence-associated （SA）-<italic>β</italic>-gal staining. The cell cycle was detected by flow cytometry. The cell viability was analyzed using the cell counting kit-8 （CCK-8）. The mRNA expression levels of tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>）， interleukin-6 （IL-6）， nuclear transcription factor-<italic>κ</italic>B （NF-<italic>κ</italic>B）， and IL-1<italic>α</italic> were detected by real-time polymerase chain reaction （Real-time PCR）. The protein expression levels of STAT1， phosphorylated STAT1 （p-STAT1）， STAT3， and p-STAT3 in the JAK2/STAT signaling pathway were determined by Western blot. Result:CCK-8 results showed that the optimal concentration of GDP was 40 mg·L^-1. Compared with the blank group， the positive rate of SA-<italic>β</italic>-gal in the model group was significantly higher（<italic>P</italic><0.01）， the percentage of cells in G₀/G₁ phase was significantly increased（<italic>P</italic><0.05）， the percentage of cells in G₂/M and S phase was significantly decreased（<italic>P</italic><0.01）. The mRNA expression levels of TNF-<italic>α</italic>，IL-6，NF-<italic>κ</italic>B and IL-1<italic>α </italic>were significantly increased（<italic>P</italic><0.01）. Compared with the model group， the model + GDP group exhibited significantly decreased SA-<italic>β</italic>-gal-positive cells （<italic>P</italic><0.05）， reduced cells in the G₀/G₁ phase （<italic>P</italic><0.05）， increased cells in the G₂/M and S phases （<italic>P</italic><0.01）， and down-regulated TNF-<italic>α</italic>， IL-6， NF-<italic>κ</italic>B， and IL-1<italic>α </italic>mRNA expression （<italic>P</italic><0.05） and STAT1， p-STAT1， STAT3， and p-STAT3 protein expression （<italic>P</italic><0.05）. Conclusion:GDP delays the senescence of mouse mesangial cells possibly by down-regulating the expression of related molecules in the JAK2/STAT pathway.

Vitamin D regulates cell viability, migration and proliferation by suppressing galectin-3 (Gal-3) gene in ovarian cancer cells

Vitamin D deficiency is identified as a risk factor for the occurrence and recurrence of ovarian cancer.Galectin-3 (Gal-3) participates in many physiological and pathological processes. In present study, serumvitamin D level was detected using chemiluminescence enzyme immunoassay. Gal-3 expression wasexamined using real-time polymerase chain reaction (PCR), Western blot and immunocytochemistry analysis. SKOV3 cells viability was assessed by the water-soluble tetrazolium salt (WST-1) assay, the migrationof SKOV3 cells was detected using transwell assay, and the proliferation of SKOV3 cells was measured by3H-thymidine incorporation (3H-TdR). Our study demonstrated that vitamin D levels were lower in 40ovarian cancer patients: vitamin D deficiency is closely related to the pathogenesis of ovarian cancer.Treatment with vitamin D reduced the migration and proliferation of ovarian cancer cells. Gal-3 wasoverexpressed in ovarian cancer, which could induce the viability, migration and proliferation ability ofovarian cancer cells, and these effects were abrogated by vitamin D downregulating the expression of Gal-3gene. Therefore, our results support that vitamin D may suppress Gal-3-induced viability, migration andproliferation ability of ovarian cancer cells, which suggests that the use of vitamin D may have beneficialeffects in preventing and treating ovarian cancer.

Effect of ginsenoside Rg_1 in delaying premature ovarian failure induced by D-gal in mice through PI3K/Akt/mTOR autophagy pathway / 中国中药杂志

The aim of this paper was to study the role of phosphoinositide 3-kinase(PI3 K), protein kinase B(Akt) and mamma-lian target of rapamycin(mTOR) in the inhibition of premature ovarian failure induced by D-galactose(D-gal) in mice model by ginsenoside Rg_1(Rg_1). Fifty-four female SPF BALB/c mice were randomly divided into PBS group, D-gal group, and Rg_1 group. In the D-gal group, D-galactose(200 mg·kg~(-1)·d~(-1)) was injected subcutaneously into the neck and back for 42 days. In the PBS group, an equal amount of phosphate buffered saline(PBS) was injected into the neck and back for 42 days. In addition to the therapy of D-gal group, Rg_1 group was given Rg_1(20 mg·kg~(-1)·d~(-1)) through intraperitoneal injection since the 15 th day for 28 days, at the same time, the D-gal group and the PBS group were also given an equal amount of PBS through intraperitoneal injection since the 15 th day for 28 days. After the treatment, the estrous cycle changes of the mice were detected, and the ovarian SA-β-Gal staining was used to detect the changes of ovarian aging. Western blot was used to detect the changes in protein expressions of PI3 K, Akt, mTOR, S6 k, LC3-Ⅱ and P16~(INK4 a). Fluorescence quantitative PCR was used to detect the changes in mRNA expressions of PI3 K, Akt, mTOR, S6 k, LC3-Ⅱ and P16~(INK4 a). According to the findings, compared with the PBS group, the D-gal group began to show estrous cycle disorder in the 3 rd week,the ovarian SA-β-Gal staining positive granulosa cells increased in the D-gal group, the expression of senescence marker P16~(INK4 a) increased, while the expression of autophagy signaling molecule LC3-Ⅱ decreased. After treatment with Rg_1, the positive rate of ovarian SA-β-Gal staining in Rg_1 group decreased, the expression level of autophagy signaling molecule LC3-Ⅱ in Rg_1 group was higher than that in D-gal group, while the expression level of senescence marker P16~(INK4 a) was lower than that in D-gal group. Compared with the PBS group, the protein and mRNA expressions of PI3 K, Akt, mTOR and S6 k in the D-gal group were up-regulated, the protein expressions of Akt, mTOR and S6 k in the Rg_1 group were up-regulated, and the mRNA expressions of PI3 K and mTOR were up-regulated. After treatment with Rg_1, the protein expressions of PI3 K, Akt, mTOR and S6 k in the Rg_1 group were lower than those in the D-gal group, while the mRNA expressions of Akt, mTOR and S6 k in the Rg_1 group were lower than those in the D-gal group. The finding ssuggested that Rg_1 has the effect in delaying ovarian premature failure in D-gal-induced mouse models, and PI3 K/Akt/mTOR autophagy signaling pathways play an important role.

Enhancing effect of Lupeol on migration and invasion abilities of ST3Gaim-silenced MDA-MB-231 cells / 解剖学报

Objective In this study, we expored the enhancing effect of Lupeol on migration and invasion abilities of ST3Gal III-silenced MDA-MB-231 cells. Methods Human breast cancer cell line ST3Gal III -silenced MDA-MB-231 was cultured in vitro. The cell adhesion, Transwell and woud healing test were utilized to test the effect of Lupeol on ST3GalM-silenced MDA-MB-231 cells. The expression of matrix metalloproteinase( MMP)-2, -9, phosphatidylinositol 3-kinase/protein kinase B/nuclear factor κB ( PI3K/Akt/NF-KB) signaling pathway were examined by Western blotting. Results No significant influence of the decreased expression of ST3Gal III and Lupeol ( 5μmol/L) on proliferation and apoptosis of MDA-MB-231 cells were found. Lupeol inhibited the migration and invasion of ST3Gal III -silenced MDA-MB-231 cells in vilro( P<0. 05). Furthermore, the expression of NF-κB p65, p-Akt, Akt, p85, MMP-2 and MMP-9 levels were significantly down-regulated. Conclusion These observations suggest that Lupeol may inhibit the abilities of migration and invasion of ST3Gal III-silenced MDA-MB-231 cells in vitro by inhibiting the protein expression of MMP-2, -9 and effect the signaling pathway of PI3K/Akt/NF-κB.

Recent advance in association of galectin-3 with / 上海交通大学学报（医学版）

Atrial fibrillation is a common cardiac arrhythmia encountered closely related to structural remodeling such as atrial fibrosis. Galectin-as a biomarker of fibrosis; therefore, it may be involved in atrial remodeling association of Gal-3 with atrial fibrillation.

Analysis of Tim-3/Gal-9 expression and T cell infiltration in adenocarcinoma of the esophagogastric junction / 上海交通大学学报（医学版）

Objective • To detect T cell immunoglobulin mucin 3 (Tim-3) and galectin 9 (Gal-9) expression as well as CD3+ T cells and CD8+ T cells infiltration in the tumor tissues of adenocarcinoma of the esophagogastric junction (AEG), and analyze their correlations with the patients' clinical characteristics and survival prognosis. Methods • A retrospective case study was used to collect clinical data and follow-up data of 116 AEG patients who were admitted to Renji Hospital, Shanghai Jiao Tong University School of Medicine from Dec. 2005 to Dec. 2013. Tim-3, Gal-9, CD3, and CD8 protein expressions were detected by immunohistochemistry in the tumor tissues, and the clinical characteristics and prognosis were compared among the patients with different levels of protein expression and T cells infiltration. Results • The results of immunohistochemistry showed that Tim-3 mainly expressed in the infiltrating immune cells, and Gal-9 mainly expressed in the tumor cells. The analysis on the clinical characteristics revealed that Tim-3 expression level was related to the Siewert classification (P=0.030) and CD8+ T cells infiltration level was related to the tumor TNM stage (P=0.042). The results of survival analysis showed that the patients with high level of CD8+ T cells infiltration had a better survival prognosis (P=0.047). However, there was no difference in the prognosis among the patients with different Tim-3 and/or Gal-9 expression levels or with different CD3+ T cell infiltration levels. Conclusion • AEG patients with high level of CD8+ T cells infiltration usually have earlier TNM stages and better prognosis. There is no significant difference in the prognosis of AEG patients with different Tim-3/Gal-9 expression levels.

Comparison of clinical application and modern research of Lagopsis supina used in different ethnic medicine cultures / 中草药

Lagopsis supina has been used as a traditional Chinese medicine (TCM), Tibetan medicine and Mongolian medicine for centuries in China. In this paper, we compared the same and differences of L. supina used in TCM, Tibetan medicine and Mongolian medicine, including the medicinal theories, functions and indications, prescriptions, processing methods. Furthermore, we summarized the modern research and application of L. supina from literatures. This paper provided the foundation for the clinical applications, quality control, comprehensive utilization and further development of L. supina.

Effect of Gualou Xiebai Banxiatang on Expression of Gal-3 in Rats After Myocardial Infarction / 中国实验方剂学杂志

Objective:To observe the effect of Gualou Xiebai Banxiatang（GXBT） on cardiac function and myocardial fibrosis in rats after myocardial infarction. Method:The 36 male SD rats were randomly divided into blank group, sham group and surgical group, and 6 males in blank group and sham operation group. The model of phlegm obstruction in myocardial infarction of rats was replicated by ligation of left anterior descending coronary artery and high fat diet, and the successful rats were randomly divided into 3 groups: model group, GXBT group and acertil group. In the sham group, only the threading was not ligated. The blank group and the sham group and the model group were given 10 mL·kg-1·d-1 of normal saline, and 2.68 g·kg-1·d-1 of the GXBT group were given intragastric administration，and 0.36 mg·kg-1·d-1 was given intragastrically in acertil group. After 4 weeks of model, the heart function was detected by heart ultrasound to verify the success of the model. After 8 weeks, the heart function of the heart of the rat was detected by heart ultrasound again, and then the samples were sacrificed. The pathological changes of the myocardial cells of the rats were observed with hematoxylin-eosin(HE) staining， and the degree of myocardial fibrosis in the rats was observed by Masson staining. The changes of serum B-type natriuretic peptide (BNP) and galectin-3 (Gal-3) in rat serum were detected by enzyme-linked immunosorbent assay（ELISA） method, and the expression of Gal-3, Collagen Ⅰ (Col-Ⅰ) and Collagen Ⅲ (Col-Ⅲ) was detected by Western blot. Result:Compared with blank group and sham group, the left ventricular ejection fraction (EF) and short-axis shortening rate (FS) of model group were significantly decreased (P<0.01)， the infiltration of inflammatory cells, the increase of the myocardial collagen fibers, the contents of BNP and Gal-3 in the serum were increased (P<0.05). The expression of Gal-3,Col-Ⅰ and Col-Ⅲ in the myocardial tissue increased significantly (P<0.01). Compared with the model group, EF and FS of GXPD were significantly increased (P<0.05), the morphological structure of myocardial cells was improved, the collagen fiber was decreased. The expression of BNP and Gal-3 in serum decreased significantly (P<0.05), and the content of Gal-3, Col-Ⅰ and Col-Ⅲ in myocardial tissue was decreased (P<0.05). Conclusion:Gualou Xiebai Banxiatang can improve cardiac function, reduce myocardial fibrosis and slow down the process of heart failure after myocardial infarction in rats with myocardial infarction. Its mechanism may be related to the decrease of Gal-3 expression.

Laboratory study on chain-killing efficacy of four gel baits / 上海预防医学

Objective To study the first-time killing efficacy and the chain-killing efficacy of four gel baits against Blattella germanica: 1% chlorpyrifos, 0.05% fipronil, 2.15% imidacloprid, and 0.5% dinotefuran and provide a basis for drug selection in controlling Blattella germanica. Methods Laboratory killing efficacy test was conducted according to the national standard GB/T 13917.7-2009 and the chain-killing efficacy test was conducted for three rounds.The first round of chain efficacy test was conducted by feeding the cockroaches killed in the laboratory efficacy test, and each next round by feeding the cockroaches killed in the last round.Median lethal time (LT50), 95% confidence limit, and toxicological regression equation of each test were calculated by software DPS V9.01. Results The LT50 of the efficacy test with 1% chlorpyrifos gel bait was 0.745 5 (0.603 4-0.890 3) d.The LT50 of the first, second and third chain experiments increased by 3.30, 2.18 and 2.76 times, respectively.The LT50 of the efficacy test with 0.05% fipronil gel bait was 0.846 5(0.464 7-1.228 0)d, and increased by 5.42, 2.09 and 1.48 times, respectively, in the first, second and third chain experiments.The LT50 of the efficacy test with 2.15% imidacloprid gel bait was 3.192 1(2.865 0-3.506 0)d, and increased by 1.13, 1.65 and 1.15 times, respectively in the first, second and third chain experiments.The LT50 of the efficacy test with 0.5% dinotefuran gel bait was 0.997 1(0.805 8-1.191 6) d, and increased by 3.85, 1.37 and 1.78 times, respectively in the first, second and third chain experiments. Conclusion In the laboratory killing efficacy test, 1% chlorpyrifos, 0.05% fipronil, and 0.5% dinotefuran gel baits are better than 2.15% imidacloprid gel bait.In the chain-killing efficacy test, 2.15% imidacloprid and 0.5% dinotefuran gel baits are better than 1% chlorpyrifos and 0.05% fipronil gel baits.Based on our results, we recommend the use of 0.5% dinotefuran gel bait for comprehensive and sustained killing effect.

Expression of CK19, CD56, and Galectin-3 Tumor Markers in Different Types of Thyroid Carcinoma

Background: Thyroid disorders represent a major problem in Saudi Arabia, particularly in the northern part of the country. The objective of the current study was to investigate the immunohistochemical expression of CK19, CD56, and Gal-3 in a series of Saudi patients with thyroid carcinoma in Northern Saudi Arabia (Hail) region. Methodology: This is a retrospective study, investigated 50 formalin-fixed paraffin wax tissue blocks with a confirmed diagnosis of thyroid carcinoma from Northern Saudi Arabia. Immunohistochemistry demonstration was for CK19, CD56, and Gal-3 markers applying Avidin-Biotin method. Results: Out of the 50 patients, 45(90%) were females and 5(10%) were males, aged 13-70 years old with a mean age of 43 years. CK19 was positively expressed in 74% of the thyroid carcinoma. Positive CD56 expression was demonstrated in 45.7%, 58.3%, and 100% of the papillary, follicular and undifferentiated thyroid carcinomas, correspondingly. Positive Galectin-3 expression was demonstrated in 71.4%, 58.3%, and 100% of the papillary, follicular and undifferentiated thyroid carcinomas, correspondingly. Conclusion: Papillary thyroid carcinoma is the most common thyroid cancer in Northern Saudi Arabia. Females represent more than 90% of the cases of the thyroid carcinoma in Northern Saudi Arabia. CK19, CD56, and Gal-3 are useful for the assessment of thyroid carcinoma.

Preliminary assessment of anti-α-Gal IgG and IgM levels in patients with patent Plasmodium vivax infection

Anti-α-Gal responses may exert a protective effect in falciparum malaria. However, the biological role of such antibodies is still unknown during Plasmodium vivax infections. We investigated IgG and IgM responses to α-Gal in individuals with vivax malaria. Anti-α-Gal IgG and IgM levels were higher in these patients than in controls, but no significant correlation was found between parasitaemia and anti-α-Gal response, nor between this response and ABO blood group status. This is the first study to investigate anti-α-Gal antibodies in P. vivax-infected patients; a larger survey is necessary to achieve a better understanding of host immune response during vivax malaria.

Study on the expression of serum NO and Gal-9 and its correlation with apoptosis in elderly patients with cervical cancer / 国际检验医学杂志

Objective To investigate and analyze the expression of serum NO and Gal-9and its correlation with apoptosis in elderly patients with cervical cancer.Methods 76cases of elderly patients with cervical cancer in our hospital as the cervical cancer group, 50cases of cervical epithelial dysplasia of uterus in elderly (CIN) were taken as CIN group and 50healthy elderly people as healthy controls were detected by flow cytometry in three subjects tissue apoptosis index (AI) and the detection of serum NO and Gal-9levels in three groups of subjects, difference analysis and correlation of the indicators of the three groups of subjects, and analyze the relationship between the serum of patients with cervical cancer NO, Gal-9levels and pathological features.Results There was a significant difference in the tissue apoptosis index (AI) between the three groups (P<0.05) , and the AIindex in the cervical cancer group was significantly higher than that in the healthy control group (P<0.05) , but it was significantly lower than that in the CIN group (P<0.05) .The serum NO and Gal-9levels of the three groups were statistically different (P<0.05) , and the levels of NO and Gal-9in the cervical cancer group were significantly higher than those in the healthy control group and CIN group (P<0.05) .The serum levels of NO and Gal-9in patients with cervical cancer were positively correlated with the value of AI (r=0.813, 0.872, P<0.05) .The serum NO and Gal-9levels in patients with stageⅢandⅣof cervical cancer were significantly higher than those inⅠandⅡpatients (P<0.05) .The serum Gal-9level in patients with poorly differentiated tumors was significantly higher than that in patients with high or intermediate differentiation (P<0.05) .Conclusion Serum NO and Gal-9play an important role in the occurrence and development of cervical cancer in elderly patients, and play a certain role in the apoptosis of cervical cancer tissues.The specific mechanisms of their effects need further research and analysis.

Association of in-stent restenosis with serum Gal-3 and MMP-9 levels / 西安交通大学学报(医学版)

Objective To investigate the relationship of galectin-3 (Gal-3)and matrix metalloproteinase-9 (MMP-9)with in-stent restenosis (ISR).Methods We consecutively recruited 434 patients who had undergone successful drug eluting stent (DES)implantation.Then we divided them into ISR group (n=41)and NO-ISR group (n=393)according to the results of coronary angiography review.Independent risk factors for ISR were found out by multivariate analysis and the two groups were matched for these factors except for Gal-3 and MMP-9 .After elim-ination of the influence of confounders,serum Gal-3 and MMP-9 were compared between the groups and their rela-tions with the severity of ISR were analyzed.Patients were grouped based on Gal-3 and MMP-9 concentrations and major adverse cardiac events (MACEs)were compared between the two groups.Results After elimination of the influence of confounders,the results showed that serum levels of Gal-3 and MMP-9 were significantly higher in ISR group than in NO-ISR group (P＜0.001).Serum levels of Gal-3 and MMP-9 increased with the increased grade of classification.Serum levels of Gal-3 and MMP-9 were obviously higher in classes Ⅲ and Ⅳ ISR than in class Ⅰ (P＜0.05).Patients with higher levels of Gal-3 and MMP-9 had a higher incidence of MACEs (P＜0.01).ISR group had a higher incidence of MACEs than NO-ISR group (P＜0.05).Conclusion Serum levels of Gal-3 and MMP-9 are correlated with ISR and its severity,and they are independent risk factors for ISR.The rate of MACEs during follow-up period was increased with the increased levels of Gal-3 and MMP-9 .

A novel Australian tick Ixodes (Endopalpiger) australiensis inducing mammalian meat allergy after tick bite

Tick-induced mammalian meat allergy has become an emergent allergy world-wide after van Nunen et al. first described the association between tick bites and the development of mammalian meat allergy in 2007. Cases of mammalian meat allergy have now been reported on all 6 continents where humans are bitten by ticks, in 17 countries

Characterization of N-glycosylation in an anti-EGFR monoclonal antibody produced by different expression systems / 生物工程学报

The use of mammalian expression systems results in a remarkable heterogeneity of mAb products, generally due to post-translational modifications, and glycosylation is a critical post-translation modification because it has a profound impact on the safety and efficacy of mAbs. The present study was designed to explore the impact of a different expression system on mAb N-glycosylation. The detailed structures of individual glycans between anti-EGFR monoclonal antibodies produced by different expression systems were successfully characterized at the level of free oligosaccharides using liquid chromatography electrospray ionization quadrupole time-of-fight mass spectrometry (LC-ESI-QTof MS). An alternating low and elevated collision energy scan, in source collision-induced dissociation and MS/MS in combination with exoglycosidase digestion method was also adopted. The combined data revealed that the Fab region of anti-EGFR antibody produced by CHO cell expression system had a pattern of glycosylation differing from that of the SP2/0 cell expression system whereas the Fc region remained basically unchanged. We confirmed that anti-EGFR antibody produced by SP2/0 cell expression system had a much more diverse mixture of glycans with α-Gal and an undesired, aberrant form of sialylation N-glycolylneuraminic acid (NGNA). The α-Gal was absent in mAb produced by CHO cell expression system containing sialic acid predominantly N-acetyl neuraminic acid (NANA) which is the desired, normal human-type sialylation. This study theoretically predicts that anti-EGFR antibody produced by CHO cell expression system may show better clinical tolerance, and very low potential for active hypersensitivity reactions, CHO cell lines can be the preferred expression system for producing anti-EGFR biobetter.

Detection methods of non-Gal xenoantibody in human serum / 器官移植

Objective To investigate the optimal condition for the detection of anti-non-galactose (Gal) xenoantigen and antibody in human serum.Mehtods Peripheral blood mononuclear cell (PBMC) obtained from Wuzhishan miniature pig models with α-1,3-galactosyltransferase gene knockout (GTKO) were used as target cells,mixed and incubated with healthy human serum of different concentrations (4.8％,16.7％ and 100％) for 0.5,1.0,2.0,3.0 and 6.0 h,respectively.The abilities of PBMC to bind with IgM and IgG were detected by flow cytometry.Results At the serum concentration of 16.7％,the ability ofnon-Gal IgM to bind with PBMC was significantly enhanced from 0.5 h to 3.0 h incubation (P＜0.01),whereas no statistical significance was noted in terms of IgG (P＞0.05).Increasing serum concentration could also enhance the ability of non-Gal IgM to bind with PBMC.At the serum concentration of 100％ and incubation for 3 h,the ability of IgM to bind with PBMC was the highest among all groups (P＜0.01).At the serum concentration of 100％ and incubation for 6 h,the ability of IgG to bind with PBMC was significantly enhanced (P＜0.05).Prolonging incubation time and increasing serum concentration did not affect the activity of PBMC.Conclusions The optimal condition for detection of anti-non-Gal xenoantigen and antibody is determined.A quantity of 1×105 PBMC from pig should be incubated with 100％ human serum for 3 h for detection of IgM level,or incubated with 100％ human serum for 6 h for measurement of IgG level.This optimized condition contributes to screening the donor pigs which lowly express non-Gal antigen.

Senescence induced by D-galactose and its biological mechanism in rat bone marrow stromal cells / 基础医学与临床

Objective To establish an aging model of rat bone marrow stromal cells (BMSCs) in vitro and in vivo, in order to study the senescence biology of aging BMSCs .Methods The control cell group ( in vitro):isolating, puri-fying and culturing BMSCs from healthy male SD rats .collecting the third generation ( P3) of BMSCs for analysis . The aging model group (in vitro):the P3 BMSCs were incubated with D-Galactose (D-Gal, 30 g/L) for 48 hours. The aging rat model group ( in vivo): the rats were given 120 mg D-Gal by the way of daily neck subcutaneous injection for 42 consecutive days .The control rat group ( in vivo):the rats were administrated with the same volume of saline for the same times .On the second day after the aging model was established , the BMSCs were collecting and culturing for study.1)The proliferative potency was detected by cell counting Kit-8(CCK-8);the distribution of cell cycle and apoptosis was detected by flow cytometry (FCM);2)the ratio of aging BMSCs was examined by the senescence-associated β-Galactosidase(SA-β-Gal) staining;3)malonaldehyde(MDA) content and total super-oxide dismutase(SOD) was examined activity by enzymatic assay; the level of reactive oxygen species (ROS) by DCFH-DA fluorescent staining was counted with FCM;4 ) the expression level of senescence-related signaling was proteins of P16 , P21 , P53 , CDK2 and cyclin D by Western blot .Results Compared with the matched control group, the BMSCs of aging model group displayed a significant decrease in proliferation; the BMSCs were held in G1 phase arrest as the proportion of the cells in G 1 phase increased , while that decreased in S phase ( P<0.05 );and the positive ratio of SA-β-Gal stained BMSCs also significantly increased ( P <0.05 ); BMSCs in the aging model group showed an increasing level of ROS and MDA , meanwhile a decline in total SOD activity was decreased (P<0.05);P16,P21 and P53 protein expression in aging BMSCs was obviously enhanced (P<0.05), at the same time the expression of CDK2 and cyclin D was also decreased ( P<0.05 ) .Conclusions D-Gal can be used to develope an aging model of BMSCs .It acts through up-regulation of expressions of aging-related proteins and in-hibition of oxidative stress injury and chronic inflammation .