桥本氏甲状腺炎合并甲状腺乳头状癌患者血清抗甲状腺球蛋白抗体、促甲状腺激素水平变化及临床意义

目的探讨桥本氏甲状腺炎(Hashimoto′sthyroiditis,HT)合并甲状腺乳头状癌(papillary thyroid carcinoma,PTC)患者血清抗甲状腺球蛋白抗体(thyroglobulin antibody,TgAb)、促甲状腺激素(thyroid-stimulating hormone,TSH)水平变化及临床意义。方法将我院2020年04月至2023年04月期间40例HT合并PTC患者作为研究组,同期HT患者40例作为对照组,其中男性患者18例,女性患者62例,年龄范围为46~60岁。使用χ^(2)检验比较两组性别、结节数量、PTC和HT家族史、病变部位,使用t检验比较两组患者年龄、体重指数、碘日常摄入量以及血清TgAb、TSH水平,并对TgAb、TSH水平诊断以及TgAb联合TSH诊断效能进行ROC曲线分析。结果研究组患者TgAb、TSH水平显著高于对照组水平(P<0.05);ROC曲线分析显示,血清TgAb、TSH水平联合诊断HT合并PTC的ROC曲线下面积(area under curve,AUC)高于单独诊断时的AUC(P<0.05)。结论TgAb、TSH在HT合并PTC患者中处于较高水平,且TgAb、TSH水平联合检测对HT合并PTC的临床预测和诊断应用价值较好。

禽蛋产品的开发与利用

:目前全世界鸡蛋消费量呈下降趋势 ,传统的禽蛋生产方式正受到冲击。研究人员正致力于禽蛋产品开发与利用的一些新途径 ,包括高 ω- 3多不饱和脂肪酸蛋、维生素强化蛋、富含矿物质的蛋、低胆固醇蛋。

Establishment of Monoclonal Antibody Competitive ELISA Using Monoclonal Antibody Against VP1 Protein of Asia 1 Type Foot-and-Mouth Disease Virus

Using the purified VP1 protein of Asia 1 type foot-and-mouth disease virus as the antigen, the purified monoclonal antibody was labeled by the sodium periodate method and the monoclonal antibody competitive ELISA was established in this study. Ten positive porcine foot-and-mouth disease serums and more than two hundreds negative serum were tested, and the results were the same as the background of samples. The sensitivity test and replicate test indicated that this method was stable and sensitive, which was suitable for monitoring Asia 1 type porcine foot-and-mouth disease virus antibody.

Development and Characterization of Monoclonal Antibody Specific to Nuclear Protein of Avian Influenza Virus Type A

Five monoclonal antibodies（Mabs） to nuclear protein of avain influenza virus（AIV） were developed by syncretizing SP 2/0 and the spleen cells from BALB of mice immuized with H9 subtype AIV. Specificity of these Mabs were identified by immunofluorescent assay（IFA） and enzyme linked immunosorbent assay （ELISA）. These five Mabs which were named as AIV-NP-2C3, AIV-NP-6A5, AIV-NP-3 H9, AIV-NP-7B4, AIV-NP-2H4 could react with all viruses of AIV-H9 strains in tests. The result of Western blotting showed that only the 60 ku protein antigen of AIV-H9 could be recognized by the Mabs but never recognized by New castle disease virus, REV and infectious bursa disease virus. The result of preliminary application showed that avian influenza viruses could be deetected bv Mabs in IFA and ELISA. All these Mabs will probably play important roles in preventing and monitoring avian influenza viruses.

Identification of Plasma Membrane Aquaporin in Guard Cells of Vicia faba and Its Role in Stomatal Movement

Water channels or aquaporins are the main pathways of water transport. Both the existence and function of aquaporins in die guard cells of Vicia faba L. were investigated both by using RD28 cDNA and RD28 antibody as probes, and by controlling stomatal movement as a parameter combined with antibody and inhibitor of aquaporins respectively. The results revealed that RD28 mRNA, encoding a plasma membrane aquaporin, expressed in ale mesophyll cells and vascular tissues of V. faba, especially in guard cells. And the location of RD28-like proteins was mainly on plasma membrane of guard cells. The addition of 25 mumol/L HgCl2, an aquaporin blocker, and antibody of RD28 as well, greatly suppressed the stomatal opening or guardcell protoplast swelling induced by fusicoccin and light, and closing induced by abscisic acid. However, 5 mmol/L, beta-mercaptoethanol, a reverse reagent of aquaporin blocker, reversed the inhibitory effect of HgCl2 Pretreatment oil stomatal opening ( i.e., HgCl2 was removed after HgCl2 pretreatment for 10 min). The results suggest that the aquaporins in V. faba are associated with stomatal movement.

Preparation of Monoclonal Antibody against P65 Protein of Mycoplasma hyopneumoniae

P65 protein, the major immunodominant protein of Mycoplasma hyopneu-moniae （Mhp） exhibiting no cross-reaction with other mycoplasmas, is general y used as a target protein for Mhp detection. In this study, BALB/c mice were immunized with prokaryotical y expressed P65 recombinant protein to prepare monoclonal anti-body. After screening with Mhp whole-cel protein and P65 protein, a specific hy-bridoma cel line, 3G12, was obtained by ELISA. Identification results indicated that the antibody secreted by 3G12 hybridoma cel s could react with P65 protein and Mhp whole-cel protein. According to indirect ELISA assay, 3G12 cel culture super-natant possessed a titer of 1∶12 800 against P65 protein and 1∶3 200 against Mhp whole-cel protein; 3G12 ascites possessed a titer of above 1∶4 000 000 against P65 protein and above 1∶20 000 against Mhp 168 whole-cel protein. After long-term in vitro culture and continuous freezing-thawing, 3G12 cel line could stably secrete antibodies. A monoclonal antibody against P65 protein and Mhp whole-cel protein was successful y obtained in the present study, which provided basis for further in-vestigating the pathogenic mechanism of Mhp and establishing diagnostic methods of Mycoplasmal pneumonia of swine （MPS）.

Gut barrier failure biomarkers are associated with poor disease outcome in patients with primary sclerosing cholangitis

To assess the prevalence of a panel of serologic markers that reflect gut barrier dysfunction in a mixed cohort of pediatric and adult primary sclerosing cholangitis (PSC) patients. METHODSSera of 67 PSC patients [median age (range): 32 (5-79) years, concomitant IBD: 67% and cirrhosis: 20%] were assayed for the presence of antibodies against to F-actin (AAA IgA/IgG) and gliadin (AGA IgA/IgG)] and for serum level of intestinal fatty acid-binding protein (I-FABP) by ELISA. Markers of lipopolysaccharide (LPS) exposure [LPS binding protein (LBP)] and various anti-microbial antibodies [anti-OMP Plus IgA and endotoxin core IgA antibody (EndoCAb)] were also determined. Poor disease outcome was defined as orthotopic liver transplantation and/or liver-related death during the follow-up [median: 99 (14-106) mo]. One hundred and fifty-three healthy subjects (HCONT) and 172 ulcerative colitis (UC) patients were the controls. RESULTSA total of 28.4%, 28.0%, 9% and 20.9% of PSC patients were positive for AAA IgA, AAA IgG, AGA IgA and AGA IgG, respectively. Frequencies of AAA IgA and AAA IgG (P < 0.001, for both) and AGA IgG (P = 0.01, for both) but not AGA IgA were significantly higher compared to both of the HCONT and the UC groups. In survival analysis, AAA IgA-positivity was revealed as an independent predictor of poor disease outcome after adjusting either for the presence of cirrhosis [HR = 5.15 (1.27-20.86), P = 0.022 or for the Mayo risk score (HR = 4.24 (0.99-18.21), P = 0.052]. AAA IgA-positivity was significantly associated with higher frequency of anti-microbial antibodies (P < 0.001 for EndoCab IgA and P = 0.012 for anti-OMP Plus IgA) and higher level of the enterocyte damage marker (median I-FABPAAA IgA posvsneg: 365 vs 166 pg/mL, P = 0.011), but not with serum LBP level. CONCLUSIONPresence of IgA type AAA identified PSC patients with progressive disease. Moreover, it is associated with enhanced mucosal immune response to various microbial antigens and enterocyte damage further highlighting the importance of the gut-liver interaction in PSC.

Application of VP1 Protein to Develop Monoclonal Antibody against Foot-and-mouth Disease Virus Asia1 Type

In order to develop an anti-FMDV Asial type monoclonal antibody （mAb）, BABL/c mice were immunized with recombinant FMDV VP1 protein. Three mAbs, 1B8, 5El and 5E2, were then further optimized. The result indicated that prepared anti-FMDV Asial mAbs had no cross-reactivity with Swine vesicular disease （SVD） and FMDV O, A and C type antigen. Their titers in abdomen liquor were 1：5×10^6, 1：2×10^6 and 1：5×10^6, respectively. 1B8 was found to be of IgG1 subtype, 5El and 5E2 belonged to IgG2b subtype. In this study, the prepared mAbs are specific for detecting FMDV type Asia1, and is potentially useful for pen-side diagnosis.

Association of primary biliary cirrhosis with idiopathic thrombocytopenic purpura

Although both primary biliary cirrhosis (PBC) and idiopathic thrombocytopenic purpura (ITP) are autoimmune diseases, the association of the 2 diseases is rare. Here, we report a case of ITP that developed during the follow-up of PBC in a 74-year- old man. The patient had been diagnosed with PBC 12 years previously, and had received treatment with ursodeoxycholic acid. The platelet count decreased from approximately 60 × 109/L to 8 × 109/L, and the association of decompensated liver cirrhosis (PBC) with ITP was diagnosed. Steroid and immune gamma globulin therapy were successful in increasing the platelet count. Interestingly, human leukocyte antigen genotyping detected the alleles DQB10601 and DRB10803, which are related to both PBC and ITP in Japanese patients. This case suggests common immunogenetic factors might be involved in the development of PBC and ITP.

Negative feedback regulation of cellular antiviral signaling by RBCKl-mediated degradation of IRF3

Viral infection causes host cells to produce type I interferons （IFNs）, which are critically involved in viral clearance. Previous studies have demonstrated that activation of the transcription factor interferon regulatory factor （IRF）3 is essential for virus-triggered induction of type I IFNs. Here we show that the E3 ubiquitin ligase RBCC protein interacting with PKC1 （RBCK1） catalyzes the ubiquitination and degradation of IRF3. Overexpression of RBCK1 negatively regulates Sendai virus-triggered induction of type I IFNs, while knockdown of RBCK1 has the opposite effect. Plaque assays consistently demonstrate that RBCKI negatively regulates the cellular antiviral response. Furthermore, viral infection leads to induction of RBCK1 and subsequent degradation of IRF3. These findings suggest that the cellular antiviral response is controlled by a negative feedback regulatory mechanism involving RBCKl-mediated ubiquitination and degradation of IRF3.

Subcellular distribution of nitric oxide synthase isoforms in the rat duodenum

AIM:To study the cell-type specific subcellular distribution of the three isoforms of nitric oxide synthase(NOS) in the rat duodenum.METHODS:Postembedding immunoelectronmicroscopy was performed,in which primary antibodies for neuronal NOS(nNOS),endothelial NOS(eNOS),and inducible NOS(iNOS),were visualized with protein A-gold-conjugated secondary antibodies.Stained ultrathin sections were examined and photographed with a Philips CM10 electron microscope equipped with a MEGAVIEW II camera.The specificity of the immunoreaction in all cases was assessed by omitting the primary antibodies in the labeling protocol and incubating the sections only in the protein A-gold conjugated secondary antibodies.RESULTS:Postembedding immunoelectronmicroscopy revealed the presence of nNOS,eNOS,and iNOS immunoreactivity in the myenteric neurons,the enteric smooth muscle cells,and the endothelium of capillariesrunning in the vicinity of the myenteric plexus of the rat duodenum.The cell type-specific distributions of the immunogold particles labeling the three different NOS isozymes were revealed.In the control experiments,in which the primary antiserum was omitted,virtually no postembedding gold particles were observed.CONCLUSION:This postembedding immunoelectronmicroscopic study provided the first evidence of celltype-specific differences in the subcellular distributions of NOS isoforms.

Construction of single chain Fv antibody against transferrin receptor and its protein fusion with alkaline phosphatase

AIM: To construct fusion protein of a single-chain antibody (scFv) against transferrin receptor (TfR) with alkaline phosphatase(AP). METHODS: The VH-linker-VL,namely scFv gene,was prepared by amplifying the VH and VL genes from plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension polymerase chain reaction (SOE PCR). After the ScFv gene was modified by 5/71 and Not I,it was subcloned into the secretory expression vector pUC19/119, and then was transformed into E.coli TG1.The positive colonies were screened by colony PCR and their expressions were induced by IPTG.ScFv gene was gained by digesting ScFv expression vector pUC19/119 with 5/71 and NotI restriction enzymes, then subcloned into expression vector pDAP2, followed by transformation in E.coli TG1.The positive colonies were selected by bacterial colony PCR.The expression of fusion protein (scFv-AP) was induced by IPTG.Its activity was detected by enzyme immunoassay. The molecular weights of scFv and scFv-AP were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The product of SOE PCR formed a band of 700 bp in agarose gel electrophoresis. SDS-PAGE demonstrated the molecular weight of scFv was 27 ku.Immunofluorescent assay (IFA) demonstrated its reactivity with TfR.The molecular weight of scFv-AP was 75 ku.Enzyme immunoassay showed that scFv-AP could specifically bind to human TfR and play AP activity. CONCLUSION: We have successfully prepared the anti-human TfR scFv and constructed the fusion protein of scFv and AP.It is promising for immunological experiments.

MyD88-independent activation of a novel actin-Cdc42/Rac pathway is required for Toll-like receptor-stimulated phagocytosis

Phagocytosis and subsequent degradation of pathogens by macrophages play a pivotal role in host innate immune responses to microbial infection. Recent studies have shown that Toll-like receptors （TLRs） play an important role in promoting the clearance of bacteria by up-regulating the phagocytic activity of macrophages. However, information regarding the signaling mechanism of TLR-mediated phagocytosis is still limited. Here, we provide evidence that the stimulation of TLR4 with LPS leads to activation of multiple signaling pathways including MAP kinases, phosphatidylinositide 3-kinase （PI3K）, and small GTPases in the murine macrophage-like cell line RAW264.7. Specific inhibition of Cdc42/Rac or p38 MAP kinase, but not PI3K, reduced TLR4-induced phagocytosis of bacteria. Moreover, we have found that either inhibition of actin polymerization by cytochalasin D or the knockdown of actin by RNAi markedly reduced the activation of Cdc42 and Rac by LPS. TLR4-induced activation of Cdc42 and Rac appears to be independent of MyD88. Taken together, our results described a novel actin-Cdc42/Rac pathway through which TLRs can specifically provoke phagocytosis.

Inhibition of hepatitis B virus replication by pokeweed antiviral protein in vitro

AIM: To explore the inhibitory effects of pokeweed antiviral protein seed (PAP-S) and PAP encoded by a eukaryotic expression plasmid on hepatitis B virus (HBV) replication in vitro. METHODS: HepG2 2.2.15 cells in cultured medium were treated with different concentrations of PAP-S. HBsAg, HBeAg and HBV DNA in supernatants were determined by ELISA and fluorescent quantitative PCR respectively. MTT method was used to assay for cytotoxicity. HepG2 were cotransfected with various amounts of PAP encoded by a eukaryotic expression plasmid and replication competent wild-type HBV 1.3 fold over- length plasmid. On d 3 after transfection, HBsAg and HBeAg were determined by using ELISA. Levels of HBV core-associated DNA and RNA were detected by using Southern and Northern blot, respectively. RESULTS: The inhibitory effects of PAP-S on HBsAg, HBeAg and HBV DNA were gradually enhanced with the increase of PAP concentration. When the concentration of PAP-S was 10 μg/mL, the inhibition rates of HBsAg, HBeAg and HBV DNA were 20.9%, 30.2% and 50%, respectively. After transfection of 1.0 μg and 2.0 μg plasmid pXF3H-PAP, the levels of HBV nucleocapside- associated DNA were reduced by 38.0% and 74.0% respectively, the levels of HBsAg in the media by 76.8% and 99.7% respectively, and the levels of HBeAg by 72.7% and 99.3% respectively as compared with controls. Transfection with 2 μg plasmid pXF3H-PAP reduced the levels of HBV nucleocapside-associated RNA by 69.0%.CONCLUSION: Both PAP-S and PAP encoded by a eukaryotic expression plasmid could effectively inhibit HBV replication and antigen expression in vitro, and the inhibitory effects were dose-dependent.

Application of GP5 Protein to Develop Monoclonal Antibody against Porcine Reproductive and Respiratory Syndrome Virus

In this study, a panel of monoclonal antibodies （mAbs） against Porcine reproductive and respiratory syndrome virus（PRRSV）, named as 8C9 and4B4, were produced by fusing SP2/0 myeloma cells and spleen cells of BALB/c mice immunized with the PRRSV （TCID50=5.5）, screened by the indirect ELISA and subjected to several limiting dilutions, mAbs were then identified by biological characterization. Among the two fusion cell strains, 8C9 belonged to the IgG1 subclass and 4B4 belonged to the IgG2a subclass. The titers in cell culture supematant and abdomen liquor reached to 1：104and 1：105, respectively. The specificity test indicated that the two cells had specific reactions for the PRRSV and GP5 protein respectively, and no reaction with Classical swine fever virus （CSFV） or Swine vesicular disease virus （SVDV）. The molecular weights of the heavy chain and light chain were about 45.0 kDa and 25.0 kDa, respectively. In neutralization activity tests, the results showed that the prepared mAb 4B4 can protect 50% of cells with no CPE in dilution up to 1：512, but mAB 8C9 has no neutralization activities to PRRSV.

Incidence of toxoplasmosis in patients with cirrhosis

AIM:It is known that toxoplasmosis rarely leads to various liver pathologies,most common of which is granulomatose hepatitis in patients having normal immune systems.Patients who have cirrhosis of the liver are subject to a variety of cellular as well as humoral immunity disorders.Therefore,it may be considered that toxoplasmosis can cause more frequent and more severe diseases in patients with cirrhosis and is capable of changing the course of the disease.The aim of this study was to investigate the frequency of toxoplasmosis in patients with cirrhosis. METHODS:Serum samples were taken from 108 patients with cirrhosis under observation in the Hepatology Polyclinic of the Gastroenterology Clinic,and a control group made up of 50 healthy blood donors.IFAT and ELISA methods were used to investigate the IgG and IgM antibodies,which had developed from these sera. RESULTS:Toxoplasma IgG and IgN antibody positivity was found in 74 (68.5%) of the 108 cirrhotic patients and 24 (48%) of the 50 people in the control group.The difference between them was significant (P<0.05). CONCLUSION:In conclusion,it was found that the toxopiasma sero-prevalence in the cirrhotic patients in this study was higher.Cirrhotic patients are likely to form a toxoplasma risk group.More detailed studies are needed on this subject.

Relationship and significance between anti-b2-glycoproteinⅠantibodies and platelet activation state in patients with ulcerative colitis

AIM： To study the relationship between anti-β2- glycoprotein Ⅰ （aβ2GPⅠ） antibodies and platelet activation state in patients with ulcerative colitis （UC） and its significance. METHODS： Peripheral blood samples were collected from 56 UC patients （34 males and 22 females, aged 43.5 years, range 21-66 years）, including 36 at active stage and 20 at remission stage, and 25 sex-and age-matched controls. The level of aβ2GP Ⅰ was measured by ELISA. The platelet activation markers, platelet activation complex- Ⅰ （PAC- Ⅰ ） and P-selectin （CD62P） were detected by flow cytometry. RESULTS： The A value for IgG aβ2GP Ⅰ in the active UC group was 0.61 ± 0.13, significantly higher than that in the remittent UC and control groups （0.50 ± 0.13 and 0.22 ± 0.14, P 〈 0.01）. There was a significant difference between the two groups （P 〈 0.01）. The A value for IgM aβ2GP Ⅰ in the active and remittent UC groups was 0.43 ± 0.13 and 0.38 ± 0.12, significantly higher than that in the control group （0.20 ± 0.12, P 〈 0.01）. However, there was no significant difference between the two groups （P 〉 0.05）. The PAC- Ⅰ positive rate for the active and remittent UC groups was 30.6% ± 7.6% and 19.6% ± 7.8% respectively, significantly higher than that for the control group （6.3% ± 1.7%,P 〈 0.01）. There was a significant difference between the two groups （P 〈 0.01）. The CD62P positive rate for the active and remittent UC groups was 45.0% ± 8.8% and 31.9% ± 7.8% respectively, significantly higher than that for the control group （9.2% ± 2.7%, P 〈 0.01）. There was a significant difference between the two groups （P 〈 0.01）. In the active UC group, the more severe the state of illness was, the higher the A value for IgG aβ2GP Ⅰ was, and the positive rate for PAC-Ⅰ and CD62P was positively correlated with the state of illness （Faβ2GP Ⅰ = 3.679, P 〈 0.05; FPAC-Ⅰ （%） = 5.346, P 〈 0.01; and FCD62P （%） = 5. 418, P 〈 0.01）. Meanwhile, in the same state of illness, the A value for IgG aβ2GP Ⅰ was positively correlated to the positive rates for PAC-Ⅰ and CD62P. CONCLUSION： aβ2GP Ⅰ level, platelet activation state and their relationship of them are closely correlated with the pathogenesis and development of UC.

A panel of monoclonal antibodies against the prion protein proves that there is no prion protein in human pancreatic ductal epithelial cells

Prion diseases are a group of neurodegenerative diseases that are fatal. The study of these unique diseases in China is hampered by a lack of resources. Amongst the most important resources for biological study are monoclonal antibodies. Here, we characterize a panel of monoclonal antibodies specific for cellular prion protein by enzyme-linked immunosorbent assay(ELISA), immunofluorescent staining, flow cytometry, and western blotting. We identify several antibodies that can be used for specific applications and we demonstrate that there is no prion protein expression in human pancreatic ductal epithelial cells(HPDC).

Anti-microbial antibodies in celiac disease:Trick or treat?

AIM： To determine the prevalence of a new set ot anti-glycan and anti-outer membrane protein （anti- OMP） antibodies in a Hungarian cohort of adult Celiac disease （CD） patients. METHODS： 190 consecutive CD patients [M/F： 71/119, age：39.9 （SD：14.1） years], 100 healthy, and 48 gastrointestinal controls were tested for glycan anti-Saccharomyces cerevisiae （gASCA）, anti-laminaribioside （ALCA）, anti-chitobioside, anti-mannobioside, anti-OMP antibodies and major NOD2/CARD15 mutations. Thirty out of 82 CD patients enrolled at the time of diagnosis were re-evaluated for the same antibodies after longstanding gluten-free diet （GFD）. RESULTS： 65.9% of the CD patients were positive for at least one of the tested antibodies at the time of the diagnosis. Except anti-OMP and ALCA, antimicrobial antibodies were exclusively seen in untreated CD; however, the overall sensitivity was low. Any glycan positivity （LR＋： 3.13; 95% CI： 2.08-4.73） was associated with an increased likelihood ratio for diagnosing CD. Significant correlation was found between the levels of anti-glycan and anti-endomysial or anti-transglutaminase antibodies. Anti-glycan positivity was lost after longstanding GFD. Anti-glycan antibody titers were associated with symptoms at presentation, but not the presence of NOD2/CARD15 mutations. Patients with severe malabsorption more frequently had multiple antibodies at diagnosis （P = 0.019）. CONCLUSION： The presence of anti-glycan antibodies in CD seems to be secondary to the impaired small bowel mucosa which can lead to increased antigen presentation. Furthermore, anti-glycan positivity may be considered an additional marker of CD and dietary adherence.

Isolation and Identification of Canine Parvovirus Serotype 2a and Its VP2 Protein Expression in Transgenic Tobacco

A strain of canine parvovirus(CPV)was isolated from feces of an ill puppy in an animal hospital in Wuhan, China. It was designated as CPV/WH02/06. This isolate was identified as serotype CPV-2a by the hemagglutination test, CPV Ag detection strip, electron microscopy, and PCR. The vp2 gene was cloned and sequenced and assigned GenBank accession number EU377537. A 1242 bp segment of the 5' region of the vp2 gene was cloned and inserted into the binary vector pBI121 and used for Agrobacterium-mediated tobacco transformation. Transgenic tobacco plants were selected on MS medium supplemented with 100 μg/mL kanamycin and 100 μg/mL timentin. Integration of the vp2 gene into the tobacco genome was confirmed by PCR using T1 progeny plants, and the expression of the VP2 protein was confirmed by Western blotting.