Clinical and gene mutation characteristics of patients with hereditary ellipsocytosis: nine cases report and literature review / 中华血液学杂志

Objective: To report gene mutations in nine patients with hereditary elliptocytosis (HE) and analyze the characteristics of pathogenic gene mutations in HE. Methods: The clinical and gene mutations of nine patients clinically diagnosed with HE at Institute of Hematology & Blood Diseases Hospital from June 2018 to February 2022 were reported and verified by next-generation sequencing to analyze the relationship between gene mutations and clinical phenotypes. Results: Erythrocyte membrane protein gene mutations were detected among nine patients with HE, including six with SPTA1 mutation, one with SPTB mutation, one with EPB41 mutation, and one with chromosome 20 copy deletion. A total of 11 gene mutation sites were involved, including 6 known mutations and 5 novel mutations. The five novel mutations included SPTA1: c.1247A>C (p. K416T) in exon 9, c.1891delG (p. A631fs*17) in exon 15, E6-E12 Del; SPTB: c.154C>T (p. R52W) ; and EPB41: c.1636A>G (p. I546V) . Three of the six patients with the SPTA1 mutation were SPTA1 exon 9 mutation. Conclusion: SPTA1 is the most common mutant gene in patients with HE.

Analysis of LMP specific T cell immune response and clinical factors in stage III-IVa nasopharyngeal carcinoma / 中华放射肿瘤学杂志

Objective:To explore the Epstein-Barr virus (EBV) latent infection membrane protein (LMP) 1 or LMP2 specific T cell immune response and clinical significance in stage III-IVa nasopharyngeal carcinoma (NPC), aiming to provide ideas and evidence for immunotherapy in NPC.Methods:Fifty-nine NPC patients admitted to the Affiliated Tumor Hospital of Xinjiang Medical University from February 2018 to October 2020 for primary treatment were collected. Peripheral blood monocytes (PBMCs) were stimulated by LMP antigen. Intracellular cytokine staining and flow cytometry were applied to study the expression levels of IL-2, IL-13, interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) from CD4 + T and CD8 + T cells, and then analyzed in conjunction with clinical factors. Results:The positive rates of total PBMCs to LMP1 and LMP2 in NPC patients were different. The positive rate of LMP1 specific CD4 + T cells was statistically higher in stage T 3-T 4 NPC than that in stage T 1-T 2 (51.0% vs. 10.0%, P=0.042). There were also differences in the expression of cytokines between LMP1 and LMP2, CD4 +T cells and CD8 +T cells. Survival analysis showed the 2-year and 3-year overall survival (OS) rates were 91.5% and 88.2%, and the 2-year and 3-year progression-free survival (PFS) rates were 83.3% and 75.3%. Univariate analysis suggested that smoking history, male and LMP1 stimulated IL-13 positive expression in CD4 + T cells affected the disease progression ( P=0.026, 0.045 and 0.006); multivariate analysis showed LMP1 stimulated IL-13 positive expression in CD4 + T cells and smoking history were the independent prognostic factors affecting PFS ( P=0.017, 0.019). Conclusions:LMP1 and LMP2 generate specific T-cell immune response in PBMCs of NPC patients, with differential expression in two T-cell subsets. LMP1 and LMP2 specific T cell immune response is associated with primary tumor size and metastatic lymph node volume. LMP1 stimulated IL-13 positive expression in CD4 + T cells and smoking history affects the disease progression.

The effect of Brucella outer membrane protein L16, L19 on the expression of immune regulatory factors in THP-1 cells / 中华地方病学杂志

Objective:To investigate the effect of recombinant lipoproteins of Brucella outer membrane protein 16, 19 (L16 and L19) on the expression of immune regulatory factors in human monocytic leukemia cell line (THP-1 cells). Methods:THP-1 cells activated with phorbol ester (PMA) were used as an in vitro experimental cell model, and a group design was used to co-culture L16, L19 and THP-1 cells (L16 stimulated group, L19 stimulated group), respectively. THP-1 cells activated with PMA were used as the control group. When co-cultured for 4 hours, immunofluorescence staining (IFS) and Western blotting were used to detect whether L16 and L19 entered the cells, respectively; when co-cultured for 12, 24 hours, real-time fluorescent quantitative PCR was used to measure the mRNA expression levels of interferon regulatory factor 1 (IRF-1) and trans activator protein of major histocompatibility complex class Ⅱ (CⅡTA); Western blotting was used to detect the protein expression levels of T cell immunoglobulin mucin-3 (Tim-3) and γ interferon receptor 1 (IFNGR1). Results:When co-cultured for 4 hours, L16 and L19 were observed entering THP-1 cells in the L16 stimulated group and L19 stimulated group, respectively. When co-cultured for 12 hours, the expression level of IRF-1 mRNA in the L16 stimulated group (0.16 ± 0.15) was significantly lower than that in the control group (1.00 ± 0.00, P < 0.05). When co-cultured for 24 hours, the expression level of CⅡTA mRNA in the L16 stimulated group (0.17 ± 0.10) was significantly lower than that in the control group (1.00 ± 0.00, P < 0.05). When co-cultured for 12 and 24 hours, there were no statistically significant differences in the expression levels of IRF-1 and CⅡTA mRNA between the L19 stimulated group and the control group ( P > 0.05). Western blotting results showed that there were statistically significant differences in the expression levels of INFGR1 and Tim-3 protein among the control group, L16 stimulated group, and L19 stimulated group after co-cultured for 12 and 24 hours ( F = 50.92, 6.80, 148.73, 156.57, P < 0.05). Among them, when co-cultured for 12 hours, the expression level of INFGR1 protein in the L16 and L19 stimulated groups were significantly lower than that in the control group, and the L19 stimulated group was higher than the L16 stimulated group ( P < 0.05), and the expression level of Tim-3 protein in the L19 stimulation group was higher than that in the control group ( P < 0.05). When co-cultured for 24 hours, the expression level of INFGR1 protein in the L16 and L19 stimulated groups were lower than that in the control group, and the L19 stimulated group was higher than that in the L16 stimulated group ( P < 0.05); and the expression level of Tim-3 protein in the L16 stimulated group was higher than that in the control group and L19 stimulated group ( P < 0.05). Conclusions:Brucella L16 can downregulate the expression levels of IRF-1 and CⅡTA mRNA in THP-1 cells. Both L16 and L19 can downregulate IFNGR1 and upregulate Tim-3 protein expression levels.

Feasibility of outer membrane protein C as a protein presenting platform targeting antigen to surface of outer membrane vesicle / 中国生物制品学杂志

@#Abstract：Objective To investigate the feasibility of outer membrane protein C（OmpC）as a protein presenting platform targeting antigen to the surface of outer membrane vesicle（OMV）. Methods The recombinant expression plasmid containing ompC gene fragment and Staphylococcus aureus EsxA antigen gene（esxA gene）was constructed，transformed to competent E. coli BL21（DE3），inducedbyIPTG，andanalyzedforexpressedproductby 12%SDS⁃PAGE. Thetotalproteinofrecombinant strain OMV was analyzed by 12% SDS⁃PAGE，and the localization of fusion protein on the surface of OMV was detected by Western blot and Flow NanoAnalyzer. Results The recombinant expression plasmid containing ompC gene and esxA gene was constructed correctly as proved by sequencing. 12% SDS⁃PAGE showed that the fusion protein OmpC⁃EsxA had a relative molecular mass of about 57 000，which was consistent with the expected size，while the total protein of OMV showed multiple target protein bands，indicating that recombinant strain OMV was successfully extracted. The fusion protein OmpC⁃ EsxA on the surface of recombinant strain OMV specifically bound to mouse antibody against His⁃Tag，and OMVs labeled with fluorescent antibody were detected by Flow NanoAnalyzer. Conclusion OmpC may be used as a protein presenting plat⁃ form to locate antigen to OMV surface，which was expected to be applied in the development of antigen presentation vaccine. Keywords：Outer membrane protein C（OmpC）；Protein presentation；Outer membrane vesicle（OMV）

Research progress on vesicle-associated membrane protein 8 / 中国生物制品学杂志

@#Membrane fusion is essential for all life activities of eukaryotes and the fusion process requires the interaction of different vesicular transporters,which are highly conserved in eukaryotes,to coordinate specifically and facilitate the fusion of different biofilms. Vesicle associated membrane protein 8(VAMP8)mainly locates in vesicular as well as lysosomal membranes,and plays a vital role in the fusion of many different biofilms. This paper focused on the molecular structure,transcriptional regulation,post-translational modification and biological function of VAMP8 as well as the research progress on its correlation with human diseases,so as to provide new ideas for the treatment of related diseases and the development of effective therapeutics targeting VAMP8.

Preparation of colloidal gold immunochromatographic test strip for rapid detection of Pseudomonas aeruginosa / 中国生物制品学杂志

@#Objective To develop a colloidal gold immunochromatographic test strip for rapid and accurate detection of Pseudomonas aeruginosa(P.aeruginosa,Pa).Methods After bioinformatics analysis of Pa outer membrane protein OprF,the gene sequence with abundant antigenic determinants and high intraspecific homology was chemically synthesized,and then connected to pET-28a(+)vector to construct the expression vector pET-28a-OprF,which was transformed into E.coli BL21(DE3)and induced by IPTG. The recombinant OprF protein was purified by Ni Sepharose~(TM)6 Fast Flow and used to immunize two female BALB/c mice for 3～4 times by multi-point subcutaneous injection in the back at the first immunization and intraperitoneal injection at subsequent immunizations. The monoclonal antibodies were screened by animal cell fusion technique,and the colloidal gold immunochromatographic test strip for rapid detection of Pa was prepared by using monoclonal antibody and double antibody sandwich immunochromatography technique. The specificity,sensitivity and stability of the test strip were evaluated.Results Two monoclonal antibodies,Pa-1# and Pa-2#,were obtained with the titer of 1∶409 600,and both of them recognized OprF specifically. The prepared colloidal gold immunochromatographic test strip showed a sensitivity of 1. 0×10~6CFU/mL and had no cross reaction with 9 common respiratory pathogens with a good stability.Conclusion The prepared colloidal gold immunochromatographic test strip can detect Pa rapidly within 15 min,with high specificity and good stability.

Preliminary study on high throughput screening small molecules targeting Gram-negative bacilli outer membrane protein BamA / 中华检验医学杂志

Objective:High-throughput screening to obtain small molecular compounds against Gram-negative bacilli by targeting BamA outer membrane protein.Methods:The sybyl-X2.1 software was used to perform high-throughput virtual screening of small molecular compounds in Chemdiv compound library based on the molecular docking. The top 150 hits by high-throughput screening were re-screened through in vitro biological experiments. The top 4 small molecules with obvious antibacterial activity were selected for in-depth molecular docking analysis, and the small molecule 8308-0401 with the highest docking score was selected for further experiments. The antibacterial effect of 8308-0401 combined with rifampicin was tested by checkerboard assay. Finally, the affinity between 8308-0401 and BamA was tested by plasma surface resonance assay. Results:The docking score of the top 150 hits calculated by high-throughput virtual screening had a mean value of 5.63. In vitro biological experiments showed that small molecules 8308-0401, 8365-1335, C066-2507 and L582-0346 exhibited strong antibacterial activity. Among those molecules, 8308-0401 showed the highest molecular docking score, and synergistic antibacterial activity against both types of strains and clinical isolates when combined with rifampicin. 8308-0401 has a strong affinity to BamA with binding a constant of 182 μmol/L. Conclusion:The small molecule 8308-0401 exerts antibacterial activity against Gram negative bacilli by targeting the outer membrane protein BamA.

Olfactory neuroepithelium in the middle turbinate: is there any impact on olfaction function after lateral marsupialization for concha bullosa surgery?

Abstract Introduction: The effect of the quantity of olfactory neuroepithelium in the middle turbinate on the postoperative olfactory function for middle turbinate concha bullosa patients has not yet been evaluated. Objective: The primary aim of this study was to investigate the olfactory structures in the middle turbinate by immunohistochemical analysis of the olfactory marker protein and to correlate the immunostaining results with the olfaction test results for patients with middle turbinate concha bullosa. Methods: Surgical materials of 18 middle turbinate concha bullosa patients who had undergone lateral marsupialization surgery were immunostained with olfactory marker protein antibodies. Smell diskettes olfaction test was applied to all of the study group patients both preoperatively and three months postoperatively. A visual analog scale was used to quantify the sense of nasal obstruction. Results: It was observed that the postoperative smell scores and the nasal obstruction visual analog scale values were significantly improved as compared to the preoperative values (p<0.05). In addition, there was a significant correlation between the smell score gain and the visual analog scale gain values (r = 0.682). Results also indicated no significant correlation between the olfactory marker protein staining scores and the smell scores (p > 0.05). Conclusion: This first paper demonstrated that the quantity of the olfactory mucosa in the middle turbinate was not a determining factor for the postoperative smell function degree for middle turbinate concha bullosa patients. The underlying cause of the olfactory deficit for middle turbinate concha bullosa patients seems to be obstruction related rather than the middle turbinate's olfactory mucosa containing status.

Optimizing factors for the efficient expression and purification of spao and lamb from Salmonella typhi

We optimized the expression and purification of outer membrane proteins SpaO and LamB from Salmonella typhi. We investigated various factors in the expression and purification processes, including the use of isopropyl ß-d-1 thiogalactopyranoside (IPTG), imidazole, and urea. First, PCR amplification was carried out on SpaO and LamB genes. The genes were then cloned in pTZ57R/T, and then expressed in pET28a vector and transformed into Escherichia coli BL21 (DE3). Gene insertion was confirmed by enzymatic digestion with NdeI and XhoI. Inclusion bodies expressing recombinant SpaO and LamB were induced with 200 and 400 µL 0.5 mM IPTG, respectively. The formed protein inclusion bodies were then isolated from the pellet and solubilized in IB buffer containing 8 M urea for SpaO and 6 M urea for LamB. Proteins were refolded by dialysis in 3M urea. Purified proteins with nickel-nitrilotriacetic acid affinity chromatography and eluted with buffer containing 250 mM imidazole for SpaO and 150 mM imidazole for LamB. The protein expression profiles were analyzed by SDS-PAGE, which identified the 33 and 49 kDa bands corresponding to rSpaO and rLamB. Western blotting Purification was carried out by nickel affinity resin with 250 mM and 150 mM imidazole for rSpaO and rLamB and refolded through stepwise dialysis with anti-His tag antibodies confirmed their expression. These optimized methods can be used to generate recombinant proteins for the development of future vaccines.

Antimicrobial resistance in Pseudomonas aeruginosa

ABSTRACT@#In recent years, antimicrobial resistance (AMR) has become a global public health concern. The growth of resistant bacteria is increasing dramatically, while the number of new antibiotics accessible is decreasing. This is especially true in the case of Pseudomonas aeruginosa, an important causative agent of healthcare-associated infections. The ability of P. aeruginosa to survive in different environments and on medical devices has made it more resistant to antibiotics. This causes bacteremia in hospitalized patients, ventilator-associated pneumonia, catheter-associated urinary tract infections and wound infections, particularly in patients with severe burns, bed ulcers and immunocompromised individuals. The rise in the AMR rate in both developed and developing countries may be attributed to a number of factors such as variations in the standard health care, large population, awareness about antibiotic resistance, inadequate training on rationale antibiotic usage and inadequate infection control facilities in many hospitals. The emergence of Extensive Drug Resistance (XDR) and Pan Drug Resistance (PDR) among organisms that cause various infections leads to increased treatment costs, morbidity and mortality, leaving no therapeutic options. This review highlights the different mechanisms of antibiotic resistance, including intrinsic and acquired resistance, which are frequently observed in P. aeruginosa.

A case of mitochondrial membrane protein-associated neurodegeneration caused by C19orf12 p.Gly58Ser mutation / 中华神经科杂志

Neurodegeneration with brain iron accumulation (NBIA) is a group of rare genetic diseases of nervous system. NBIA is characterized by varying degrees of abnormal iron metabolism and excessive iron deposition in brain tissue. The most common symptoms of NBIA are extrapyramidal symptoms. NBIA can also be associated with varying degrees of dysfunction of the pyramidal tract, cerebellum, peripheral nervous system, autonomic nervous system, mental cognition and vision functions. A patient with NBIA admitted to the Department of Neurology of Xijing Hospital in December 2020 was collected and analyzed for clinical features. Whole exome sequencing (WES) was employed to gene mutation screening, and pathogenicity analysis was performed according to the American College of Medical Genetics and Genomics (ACMG) guideline. The patient was a 13-year-old male with a chronic course of disease that began at the age of 4. The first symptom was spastic gait. With the progress of the disease, the patient developed mental retardation, arrhythmia, coughing from drinking water and loss of vision. Magnetic resonance imaging of the head showed atrophy of the optic nerve and hypointensity signal in bilateral substantia nigra and globus pallidus on T 2WI, fluid attenuated inversion recovery sequency, diffusion weighted imaging and susceptibility weighted imaging without "tiger eye sign" which was commonly found in pantothenate kinase associated neurodegeneration. The homozygous mutation c.172G>A (p.Gly58Ser) was found through WES. The proband′s father and mother are cousins (inbreeding), carried heterozygous variation of this locus. This novel mutation was not reported in mutation database. According to ACMG guideline, C19orf12 gene c.172G>A (p.Gly58Ser) was identified for possible pathogenic mutations. The conservative prediction of this locus suggests high conservatism. The final diagnosis of the patient was mitochondrial membrane protein-associated neurodegeneration (MPAN,NBIA type 4). This finding enriched the known mutation database of MPAN and provided a basis for further study of the disease.

Regulatory effect of interleukin-18 on natural killer cell activity in patients with alopecia areata / 中华皮肤科杂志

Objective:To investigate changes of natural killer (NK) cell subsets and interleukin-18 (IL-18) level in peripheral blood of patients with alopecia areata, and to assess the regulatory effect of IL-18 on NK cell activity.Methods:A total of 67 patients with alopecia areata (alopecia areata group) and 25 healthy volunteers (control group) were collected from Shanxi Provincial People′s Hospital between December 2019 and January 2021. Peripheral blood mononuclear cells (PBMCs) and plasma were isolated. The percentage of NK cell subsets was investigated by flow cytometry, and plasma IL-18 level was measured by enzyme-linked immunosorbent assay. PBMCs were stimulated with recombinant human IL-18, and co-culture systems of PBMCs with 721.221 cells, K562 cells and P815-Ab cells were established separately. NK cell function was assessed by determining the percentage of CD107a-expressing NK cells and fluorescence intensity of CD16 + NK cells. Comparisons between groups were performed using t test or paired t test. Results:Compared with the control group, the alopecia areata group showed significantly decreased percentage of CD56 +CD16 - NK cells (8.12% ± 3.14% vs. 10.78% ± 4.08%, t = 3.33, P = 0.001) , but significantly increased percentage of CD56 +CD16 + NK cells (46.08% ± 15.21% vs. 32.14% ± 10.45%, t = 4.22, P < 0.001) , and there was no significant difference in the percentage of CD56 -CD16 + NK cells between the alopecia areata group and control group (28.81% ± 8.65% vs. 27.09% ± 7.62%, t = 0.88, P = 0.383) . The plasma IL-18 level was significantly higher in the alopecia areata group than in the control group (112.0 ± 23.72 pg/ml vs. 99.34 ± 15.15 pg/ml, t = 2.48, P = 0.015) . After co-culture with 721.221 cells, K562 cells and P815-Ab cells, the percentage of CD107a-expressing NK cells was significantly higher in NK cells from the alopecia areata group (9.53% ± 1.70%, 5.15% ± 1.35%, 6.50% ± 1.64%, respectively) than in those from the control group (5.00% ± 1.17%, 4.40% ± 1.09%, 5.13% ± 1.36%, respectively, all P < 0.05) . After the stimulation with P815-Ab cells, the alopecia areata group showed significantly decreased fluorescence intensity of CD16 + NK cells (151.10% ± 59.30%) compared with the control group (221.90% ± 93.56%, t = 4.31, P < 0.001) . After IL-18 stimulation, the percentage of CD107a-expressing NK cells significantly increased in the co-culture system of NK cells with 721.221 cells compared with the unstimulated co-culture system (14.47% ± 2.67% vs. 9.93% ± 1.94%, t = 6.00, P < 0.001) , while there was no significant difference between the IL-8-stimulated co-culture system of NK cells with K562 cells or P815-Ab cells and the unstimulated co-culture systems (both P > 0.05) . Conclusion:IL-18 could enhance NK cell activity in patients with alopecia areata, likely by promoting natural cytotoxicity receptor-mediated cytotoxicity.

SARS-CoV-2 membrane protein is incompetent at inducing antibody-dependent enhancement though Fc receptor pathway / 中华微生物学和免疫学杂志

Objective:To investigate the immune characteristics of SARS-CoV-2 membrane (M) protein, especially the possibility of inducing antibody-dependent enhancement effect (ADE).Methods:Full-length SARS-CoV-2 M protein was prepared by prokaryotic expression system and purified. BALB/c mice were immunized subcutaneously three times (on day 1, day 14 and day 21) by purified M protein. Serum samples were collected before immunization and after each immunization. The specificity of immune sera against M protein was identified by Western blot, and the antibody titers were detected by ELISA and neutralization test. In the presence of anti-M protein serum, the proliferation of SARS-CoV-2 in dendritic cells, nature killer cells, T and B cells was detected in vitro. Results:The immune sera from BALB/c mice immunized with purified full-length M protein of SARS-CoV-2 specifically recognized viral M protein. The titer of anti-whole virus antibody in immune sera was about 1∶400, but the antibody could not neutralize live virus. Moreover, the antibody could not help the virus to infect and proliferate in the various types of immune cells with Fc receptor (FcR).Conclusions:Non-neutralizing antibody induced by M protein could not cause ADE through FcR pathway.

Multi-disciplinary team of the treatment of heart transplantation for Danon disease / 器官移植

Objective To improve the understanding of Danon disease and the efficacy of heart transplantation by multi-disciplinary team (MDT) pattern. Methods Prior to heart transplantation, MDT consultation was performed on one case of Danon disease, an extremely rare X-chromosome dominant genetic disease. The content of consultation included surgical indication, preoperative preparation and postoperative precaution, as well as the role of MDT in the treatment of Danon disease was summarized. Results Preoperative echocardiography showed that the patient presented withdilated-phase of hypertrophic cardiomyopathy complicated with heart failure, which was considered as Danon disease with end-stage heart failure. After MDT consultation, the patient received the preoperative treatment including anti-heart failure, reduction of pulmonary artery pressure, intra-aortic balloon pump (IABP) assistance, liver protection, strengthening nutritional support, etc. The patient underwent orthotopic heart transplantation after a suitable donor was matched. The patient developed muscle weakness in the limbs 5 days after operation, which was gradually mitigated after reducing the dose of glucocorticoid. At postoperative 48 days, the patient was discharged in good condition and continually treated with triple immunosuppressive regimen after discharge. Reexamination at postoperative 6 months revealed that the patient was in good health without any abnormality in electrocardiogram and echocardiography images. Conclusions Danon disease is extremely rare and MDT pattern may enhance the efficacy and safety of treatment by heart transplantation.

Advances and application of enrichment technology in SH2 superbinder-based tyrosine phosphoproteomics / 生物工程学报

Tyrosine phosphorylation is one of the important protein phosphorylations in eukaryotes responsible for a variety of biological processes including cell signaling transduction, cell migration, and apoptosis. In the study of phosphoproteomics, due to the low stoichiometry of tyrosine phosphorylation (pTyr) proteins and sometimes limited initial sample, traditional phosphoproteomics enrichment technology is inefficient for the enrichment of pTyr peptides. Here, we review the substantial progress in tyrosine phosphoproteomics by preparation of limited amount sample and the newly introduced SH2 superbinder.

Advancements in stabilization technologies for membrane protein and its application in drug screening / 药学学报

Membrane proteins are the main undertakers of biofilm function, and also the most important target group for innovative drug discovery and research. About 60% of drugs targets are membrane proteins. Due to the obvious aggregation and denaturation tendency of membrane proteins in aqueous solution, it is difficult to simulate the membrane like environment to maintain the correct conformation of membrane proteins in vitro, which results in the slower-growing research on the structure and function of membrane proteins and related ligand drugs than that of water-soluble proteins. Membrane protein stabilization technology is the premise of establishing high specificity, high sensitivity and high throughput drug screening methods for membrane protein ligands, which is of great significance. In this paper, some techniques for stable separation and purification of membrane proteins are reviewed, including detergents, artificial membranes, polymers, lentiviral particles and so on, as well as their specific applications in drug screening.

Toxic effect of Brucella outer membrane protein 16 on osteoblasts / 中华地方病学杂志

Objective:This study is designed to investigate the toxicity of lipoprotein (L16) and non-lipoprotein (U16) of Brucella outer membrane protein (OMP) 16 on osteoblasts. Methods:Recombinant L16 and U16 proteins were prepared by using prokaryotic expression system of Escherichia coli ( E. coli) BL21 (DE3) and purified by Ni column. Using group design, mouse osteoblasts (MC3T3 cells) were co-incubated with L16 and U16, respectively. Brucella lipopolysaccharide (LPS) stimulus was used as the positive control, and cells without any stimulation were used as the negative control. Incubation time was 24 h. The activity of co-incubated MC3T3 cells were detected by CCK-8; the supernatant of cultured cells was collected and the release rate of lactate dehydrogenase (LDH) in the supernatant was detected by bioluminescence, and the virulence of L16 and U16 on MC3T3 cells was evaluated. Annexin Ⅴ-PE/7-AAD double staining flow cytometry was further used to analyze the apoptosis rate of MC3T3 cells, and the activation level of apoptosis executive protein Caspase-3 was detected by Western blotting (WB). Results:The activity of MC3T3 cells in L16 group [(56.16±1.63)%] was significantly lower than that in U16 and LPS groups [(97.02±1.44)%, (98.64±0.90)%, P < 0.01], the LDH release rate [(84.64±0.96)%] was significantly higher than that in U16 and LPS groups [(34.82±3.41)%, (26.75±1.95)%, P < 0.01]. Annexin Ⅴ-PE/7-AAD double staining results showed that the apoptosis rate was (46.45±2.19)% in L16 group, while the remaining groups were all less than 1%. WB results showed that activated Caspase-3 (cleaved-Caspase-3) existed in L16 stimulated cells, but not in U16 stimulated cells and LPS control cells. Conclusion:L16 can induce the apoptosis of osteoblasts and inhibit the proliferation of osteoblasts, but U16 has no obvious effect indicating that Brucella L16 with complete lipid structure is necessary for virulence effect.

Targeting EZH2 depletes LMP1-induced activated regulatory T cells enhancing antitumor immunity in nasopharyngeal carcinoma

Objective: Regulatory T cells (Tregs) are critical factors that impair antitumor immunity. Epstein–Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is one of the most pathogenic factors in nasopharyngeal carcinoma (NPC). However, the role of EBV-encoded LMP1 in regulating Treg generation in NPC remains unclear. Materials and Methods: The in vitro stability of activated Tregs (aTregs) influenced by LMP1 was analyzed by flow cytometry. The inhibitory effects of LMP1-HONE1 antigen-induced aTregs on tumor-associated antigen (TAA)-specific T cells were analyzed in vitro and in vivo. Finally, the expression of LMP1, Foxp3, and enhancer of zeste homolog 2 (EZH2) were analyzed in samples from 86 NPC patients by immunohistochemistry and immunofluorescence. Results: LMP1 upregulated the expression of EZH2, which increased the stability of aTregs in vitro. EZH2 inhibitor, DZnep, depleted LMP1-HONE1 antigen-induced aTregs in vitro and led to potent TAA-specific T cell antitumor immunity in vivo. In NPC tissues, LMP1 expression level was positively correlated with the number of EZH2+ Tregs, which was positively correlated with clinical stage and overall survival. Conclusions: EZH2 is essential for maintaining the stability and inhibitory functions of aTregs that are induced by EBV-encoded LMP1 in NPC

Frequency of Epstein Barr Virus in Non-Hodgkin s Lymphoma Determined by Latent Membrane Protein 1 Expression.

Non-Hodgkin’s Lymphoma is a haematological malignancy with various etiological factors and one among them is Epstein Barr Virus. The expression of Epstein-Barr virus in Non-Hodgkin’s lymphoma can be identified by immunohistochemistry for detection of Epstein-Barr virus latent membrane protein (LMP-1). In our study, we are trying to clarify the extent of expression LMP-1 in our population. This can be used as a prognostic marker and for therapeutic interventions targeting EBV encoded proteins. We wanted to determine the proportion of LMP1 (EBV marker) expression in Non-Hodgkin’s Lymphoma and evaluate the association of LMP1 expression in B cell and T cell type of NHL.METHODSThis is a cross sectional analytical study conducted at Department of Pathology, Govt. Medical College, Kottayam, from December 2017 to May 2019. A total of 67 cases were studied. All of them were histopathologically diagnosed Non-Hodgkin’s Lymphoma specimens received in the Department of Pathology, Govt. Medical College, Kottayam, during the study period of 18 months. NHL was subtyped into B cell type and T cell type. LMP-1 immunohistochemistry was done on all cases to assess its expression. Then analysis was done using SPSS software and strength of association between LMP 1 positivity and cell type was calculated using Chi square test and Fisher’s exact test.RESULTSMean age of Non-Hodgkin’s lymphoma is 57.82 +/- 7.4 in this study. Minimum age is 2 years and maximum age is 89 years. The present study had 10.4 % LMP 1 positive cases. Of which there were 6% moderately stained positive cases, 3% of weakly stained cases and 1.5% cases of intensely stained cases. Among NHL 86.6% cases were B cell lymphoma and 13.4% cases of T cell lymphoma. And they had a LMP 1 positivity of 10.3% and 11.1 % respectively. But there was no significant association between LMP 1 positivity and cell type according to our study.CONCLUSIONSThe present study was done to determine the proportion of LMP 1 expression in Non-Hodgkin’s Lymphoma and to find out whether there is any association between LMP1 expression in B cell and T cell type of NHL. LMP 1 was positive in 10.4% of NHL and there was no association between LMP 1 positivity in B cell and T cell Lymphoma. This suggest that EBV might play a role in pathogenesis of NHL.