A novel lectin from mushroom Phellodon melaleucus displays hemagglutination activity,and antitumor activity in a B16 melanoma mouse model

A novel lectin(termed PML)was purified from fruiting bodies of the edible mushroom Phellodon melaleucus(division Basidiomycota)by ion exchange,hydrophobic interaction,and gel filtration chromatographies,with overall titer recovery~60%and 20-fold purification.PML displayed hemagglutination activity 13319 units/mg toward rabbit erythrocytes.SDS-PAGE and gel filtration analyses revealed that PML is a homodimeric lectin with a molecular weight of 28.8 kDa.PML hemagglutination activity was not inhibited by various simple sugars or their derivatives,but was enhanced by cations Ca^(2+),Mg^(2+),Zn^(2+),and Cu^(2+).The activity was stable in pH range 6–9 and in the temperature range 20–60°C.Circular dichroism(CD)spectroscopic analysis showed that PML was composed primarily ofβ-sheets with lowα-helix content.In a B16 melanoma mouse model,PML treatment significantly inhibited tumor growth,and increased cytokine IL-10 content.Our findings suggest that PML is a potential anticancer therapeutic agent.

New model for cardiomyocyte sheet transplantation using a virus-cell fusion technique

AIM: To facilitate close contacts between transplanted cardiomyocytes and host skeletal muscle using cell fusion mediated by hemagglutinating virus of Japan envelope(HVJ-E) and tissue maceration. METHODS: Cardiomyocytes(1.5 × 106) from fetal rats were first cultured. After proliferation, some cells were used for fusion with adult muscle fibers using HVJ-E. Other cells were used to create cardiomyocyte sheets(area: about 3.5 cm2 including 2.1 × 106 cells), which were then treated with Nile blue, separated, and transplanted between the latissimus dorsi and intercostal muscles of adult rats with four combinations of HVJ-E and/or Na OH maceration: G1: HVJ-E(+), Na OH(+), Cardiomyocytes(+); G2: HVJ-E(-), NaO H(+), Cardiomyocytes(+); G3: HVJ-E(+),Na OH(-), Cardiomyocytes(+); G4: HVJ-E(-), Na OH(-), Cardiomyocytes(-). At 1 and 2 wk after transplantation, the four groups were compared by detection of beating domains, motion images using moving target analysis software, action potentials, gene expression of MLC-2v and Mesp1 by reverse transcription-polymerase chain reaction, hematoxylin-eosin staining, and immunostaining for cardiac troponin and skeletal myosin.RESULTS: In vitro cardiomyocytes were fused with skeletal muscle fibers using HVJ-E. Cardiomyocyte sheets remained in the primary transplanted sites for 2 wk. Although beating domains were detected in G1, G2, and G3 rats, G1 rats prevailed in the number, size, motion image amplitudes, and action potential compared with G2 and G3 rats. Close contacts were only found in G1 rats. At 1 wk after transplantation, the cardiomyocyte sheets showed adhesion at various points to the myoblast layer in the latissimus dorsi muscle. At 2 wk after transplantation, close contacts were seen over a broad area. Part of the skeletal muscle sarcoplasma seemed to project into the myocardiocyte plasma and some nuclei appeared to share both sarcoplasmas.CONCLUSION: The present results show that close contacts were acquired and facilitated the beating function, thereby providing a new cellular transplantation method using HVJ-E and NaO H maceration.

Geographically Distinct Expression Profile of Host Defense Peptides in the Skin of the Chinese Odorous Frog, Odorrana margaretae

Odorrana margaretae （Anura： Ranidae） is widely distributed in the southern provinces of China. Previously, 72 antimicrobial peptides （AMPs） belonging to 21 families were identified from the skin of O. margaretae, which were captured in the Hunan province. In the present study, five O. margaretae frogs were captured from the Guizhou province and a total of 28 cDNAs encoding 17 host defense peptides （HDPs） belonging to 14 families were cloned from the skin cDNA library of O. margaretae. Among the 17 HDPs, only one （brevinin-1-Omar5） had been characterized. The distinct HDP expression profiles for O. margaretae in the previous and present study may be attributed to the environmental differences between the sampling locations and the genetic divergence among O. margaretae populations. Besides, 11 of the 17 HDPs identified in the present study were novel for ranids. In order to understand their roles in host defense reactions, three HDPs （odorranain-H-OM1, odorranain-M-OM and ranatuerin-2-OM）, which possess low sequence similarity with the known amphibian HDPs, were selected for further chemical synthesis and functional analysis. Odorranain-H-OM1 showed direct antimicrobial activity against bacteria and fungi. Odorranain-M-OM exhibited concentration-dependent anti-oxidant activity. Ranatuerin-2-OM showed lectin-like activity and could strongly hemagglu -tinate human intact erythrocytes with or without the presence of Ca2＋. The diverse activities of HDPs implied that they may play different roles in host defense reactions of O. margaretae.

Highly Selective Photodynamic Therapy with a Short Drug-Light Interval Using a Cytotoxic Photosensitizer Porphyrus Envelope for Drug-Resistant Prostate Cancer Cells

Background: Photodynamic therapy (PDT) is a less invasive cancer treatment using photochemical reactions induced by light irradiation to a photosensitizer (PS). Highly selective PDT with fast accumulation of the PS in target site might be a promising treatment option for drug-resistant prostate cancer facing high incidence rate of elderly men who have no effective treatment options and require a minimally invasive treatment. Hemagglutinating virus of Japan envelope (HVJ-E) allows selective and fast drug delivery to the drug-resistant prostate cancer cells via rapid cell membrane fusion. PS named porphyrus envelope (PE) has been developed by insertion of lipidated protoporphyrin IX (PpIX lipid) into HVJ-E. In this study, we investigated the optimal conditions for PE preparation and laser irradiation for highly selective PDT using PE with a short drug-light interval. Materials and Methods: Human hormon refractory prostate cancer cell line PC-3 and human normal prostate epithelial cell line PNT2 were cultured. PpIX lipid uptake and cytotoxicity of PDT in the cells incubated with PE for 10 min were evaluated by measuring fluorescence intensity and by using a cell counting reagent 24 h after PDT, respectively. Results: PpIX lipid uptake and cytotoxicity of PDT were increased with PpIX lipid concentration. Cytotoxicity of PDT using PE was more than 9 times as strong as that with PpIX lipid and PpIX induced by 5-aminolevulinic acid. Much stronger cytotoxicity was induced in PC-3 cells than PNT2 cells with the ratio of cell death rate for cancer to normal cells up to 4.64 ± 0.09. Conclusions: Fast PS delivery with HVJ-E allows highly selective PDT with a short drug-light interval. Therefore, PDT using PE has a potential to shorten treatment period and reduce side effects of PDT.

A Novel Therapeutic Strategy Combining Use of Intracellular Magnetic Nanoparticles under an Alternating Magnetic Field and Bleomycin

Purpose: The purpose of this study was to present a novel therapeutic strategy combining use of intracellular magnetic nanoparticles (MNPs) under an alternating magnetic field (AMF) and bleomycin (BLM), and to evaluate its therapeutic effect using tumor-bearing mice. Materials and Methods: MNPs (Resovist?, 1.05 mg iron) were incorporated into the hemagglutinating virus of Japan-envelope (HVJ-E) vector (~5 × 109 particles) (HVJ-E/MNPs) by centrifugation at 10,000 × g for 5 min at 4°C. Tumor-bearing mice were prepared by inoculating Colon-26 cells subcutaneously into the backs of BALB/c mice. When the tumor volume reached ~100 mm3, HVJ-E/MNPs and/or BLM were injected directly into the tumor. The AMF was applied to the mice one hour after the injection of agents (AMF treatment). The mice injected with HVJ-E/MNPs were imaged using our magnetic particle imaging (MPI) scanner immediately (13 min) before, immediately (22 min) after, and 3, 7, and 14 days after the injection of agents, and the temporal changes of the average and maximum MPI pixel values in the tumor were quantitatively evaluated. The therapeutic effect was evaluated by calculating the relative tumor volume growth (RTVG) from the tumor volumes measured each day. Transmission electron microscopic (TEM) observation of resected tumors was also performed to confirm the intracellular distribution of MNPs. Results: The AMF treatment combined with BLM significantly decreased the RTVG value compared with AMF treatment alone at 9 to 14 days, and BLM alone at 3 to 5 days after AMF treatment. The average and maximum MPI pixel values in the tumor were almost constant for 14 days. TEM observation confirmed that most of the HVJ-E/MNPs were internalized into tumor cells within one hour after injection. Conclusion: A novel therapeutic strategy with use of AMF treatment and BLM was presented, and the time-dependent change of MNPs in tumors was evaluated using MPI. The present results suggest that this novel strategy can suppress tumor volume growth over AMF treatment or BLM alone, and can be performed repeatedly with a single injection of HVJ-E/MNPs. They also suggest that HVJ-E is effective for internalizing MNPs into cancer cells and that MPI allows for longitudinal monitoring of the distribution of MNPs in tumors.

In Vitro Evaluation of Gamma Irradiation on a Gel Formulation of Cratylia Mollis: Rheological Proporties and Microbiological Control

Lectin Cramoll-1,4, obtained from Cratylia mollis seeds (beans camaratu) was structurally characterized, biologically and pharmacologically, but its use as a biopharmaceutical is not well documented. The objective of this study is to propose a biopharmaceutical formulation lectin Cramoll-1,4, test their hemagglutinating properties in vitro as well as the use of gamma radiation as a continuous process of decontamination formulation. It was made of the extraction and purification Cramoll-1,4, was developed a gel formulation using Carbopol? as a vehicle, at concentrations of 50, 100, 200, 300 and 600 μg was irradiated with 60Co gamma rays in a dose of 7.549 kGy·h–1. The proposed formulation at a concentration of 300 μg produced an increase in the hemagglutinating units Cramoll-1,4 due to the synergistic effect caused by gamma radiation. Considering the diverse use of lectins, specific molecular and structural factors, as well as changes resulting from its formulation, concentration, irradiation and route of administration is of utmost importance to continue the studies in vitro, for subsequent application in vivo to characterize the physiological and molecular processes involved in the response and cellular effects.

Immunodiagnostic efficacy of detection of Schistosoma japonicum human infections in China:a meta analysis

Objective:To assess the diagnostic efficacy of the currently most widely used indirect hemagglutination assay(IHA) and enzyme-linked immunosorbent assay(ELISA) for detection of Schistosoma japonicum human infections.Methods:A comprehensive search was undertaken from China National Knowledge Infrastructure,Wanfang Database.VIP Database,PubMed. Cochrane Library,Science Citation Index Expanded.Proquest,and the inclusion and exclusion criteria were strictly settled.The funnel plot was used to assess the publication bias.Cochran’s Q test was employed to measure the homogeneity between studies,a summary receiver operating characteristic(SROC) curve was used to compare the diagnostic accuracy between the IHA and ELISA qualitatively by means of the Weighted Least Square method,the Ordinary Least Square method and the Robust regression method,and the diagnostic odds ratio(DOR) was drawn to compare the accuracy quantitatively.Results:Out of 785 publications,19 papers were eventually selected for analysis.Literature finality assessment indicated that minor publication bias existed in studies pertaining IHA test,but no bias was found in literatures regarding ELISA test.The heterogeneity test showed a heterogeneity between studies was present(χ~2 =466.07 and 34.67. both P values【0.0001).The areas under the SROC curves of IHA were all higher than that of ELISA test using the three methods(Weighted Least Square method:0.766 vs.0.695.Ordinary Least Square method:0.826 vs.0.741.Robust regression:0.815 vs.0.715).The TPR* values for IHA and EUSA were 0.710.0.759.0.749.and 0.650.0.686 and 0.666.respectively,and OR values were 5.997.9.937.8.893.and 3.432.4.784 and 3.959.respectively.The DOR of IHA was 9.41(95% CI:4.88-18.18).and 4.78(95%CI:3.21-7.13) for ELISA.Conclusions:All above results revealed that the diagnostic performance of IHA is better than that of ELISA.However,taking into account their unsatisfactory diagnostic value in areas with low infection intensity,a search for a better diagnostic test that can be applied in field situations in China should be given high priority.

Salmonella typhi and gallbladder cancer:report from an endemic region

BACKGROUND:Evidence exists of a link between chronic infection by Salmonella typhi(S.typhi) and the development of gallbladder cancer(GBC),but several studies from endemic regions contradict its role in the etiopathogenesis of GBC.This study used various tools to assess the prevalence of S.typhi in patients with GBC and gallstone disease(GSD) in this region with a high incidence of GBC.METHODS:S.typhi was detected in tissue and bile by PCR and culture and in serum by the Widal test and indirect hemagglutination assay(IHA).PCR with two pairs of S.typhi specific primers(flagellin gene H1d and SOP E gene) could detect 0.6 ng of S.typhi DNA.Fifty-four patients with GBC(cases) were matched with 54 patients with GSD(controls).RESULTS:Of the 54 cases,24(44.44%) were positive on the Widal test and 12(22.22%) on IHA,compared to 13(24.07%) and 5(9.26%) respectively in the controls.Eighteen(33.33%) cases showed a positive result on PCR(tissue) and 2 on PCR(bile) vs.none in the controls.Bile culture revealed no Salmonella colonies in either cases or controls.Only 3 cases were positive for Salmonella on tissue culture compared to none in the controls.The sensitivity of PCR(tissue) relative to the Widal test,IHA,culture(bile and tissue) and PCR(bile) was 100% vs.66.67%,11.11%,and 11.11%,and the specificity was 83.33% vs.100%,100%,and 100%,respectively.CONCLUSIONS:S.typhi is significantly associated with GBC compared to GSD(33% vs.0%).PCR appears to be the most specific diagnostic tool,the gold standard for S.typhi in tissue samples.

Identification and Characterization of Peptide Mimics of Blood Group A Antigen

In order to investigate peptide mimics of carbohydrate blood group A antigen, a phage display 12-mer peptide library was screened with a monoclonal antibody against blood group A antigen, NaM87-1F6. The antibody-binding properties of the selected phage peptides were evaluated by phage ELISA and phage capture assay. The peptides were co-expressed as glutathione S-transferase （GST） fusion proteins. RBC agglutination inhibition assay was performed to assess the natural blood group A antigen-mimicking ability of the fusion proteins. The results showed that seven phage clones selected bound to NaM87-1F6 specifically, among which, 6 clones bore the same peptide sequence, EYWYCGMNRTGC and another harbored a different one QIWYERTLPFTF. The two peptides were successfully expressed at the N terminal of GST protein. Both of the fusion proteins inhibited the RBC agglutination mediated by anti-A serum in a concentration-dependent manner. These results suggested that the fusion proteins based on the selected peptides could mimic the blood group A antigen and might be used as anti-A antibody-adsorbing materials when immunoabsorption was applied in ABO incompatible transplantation.

Binary Ethylenimine Inactivated Japanese Encephalitis Virus Antigen Reveals Hemagglutination

Severe climate change and global warming may impact significantly on vector-borne disease including Japanese encephalitis (JE) infection in human and animals. Thus, veterinary authority requires large quantity of diagnostic tools to survey vector-borne diseases. New producing method having a relation with JE antigen is needed to substitute conventional sucrose-acetone extraction method using suckling mouse. So, we developed new manufacturing method using polyethylene glycol (PEG) precipitation. Japanese encephalitis virus (JEV) was propagated in roller bottle containing Vero cell and inactivated with two kinds of inactivating reagents. Viability of the supernatant of bulk containing antigen was checked using Vero cell after inactivation. The supernatant did not show hemagglutination (HA) activity with goose erythrocytes. The antigen inactivated by binary ethylenimine (BEI) and concentrated by PEG precipitation method was found to be 2048 HA, but the antigen inactivated by 0.3% formaldehyde solution and concentrated by PEG precipitation method did not show HA titer. The antigen prepared from mice brain using sucrose-acetone extraction method showed 256 HA titer. This BEI inactivation method does not evoke animal welfare problem and can replace the conventional method that required biological hazardous reagents and suckling mice in preparing HA antigen. This new BEI inactivation method was safe in producing HA antigen against JEV in laboratory and can reduce environmental contamination of acetone.

Correlation between Hemagglutination Inhibition Titer and Protection against Infectious Bronchitis Virus Challenge in SPF Layers

[ Objective] To study the correlation between HI titer and protection against IBV challenge in SPF layers. [ Method ] SPF layers were randomly divided into four groups, namely group A1, A2, B1 and B2. The group A1 was immunized with H120 live vaccine. The group A2 was first immunized with H120 live vaccine and later boosted with ND-IB-EDS trivalent inactivated vaccine. The group B1 was used as unimmunized chal- lenge control. The group B2 was kept as unimmunized unchallenged control. The blood samples were taken prior and post-vaccination at intervals and HI tests were conducted. At the laying peak, the group A1, A2 and B1 were challenged with IBV M4t virulent strain. The clinical features and egg production of layers were monitored and recorded. [Result] After 30 d post vaccination with H120 live vaccine, the HI titer reached 4.45 log2; after 30 days boosting with ND-IB-EDS trivalent inactivated vaccine, the HI titer reached to 7.35 log2. Before challenge, HI antibody titer in group A1, A2, B1 and B2 were respectively 4.24 log2, 7.40 Iog2, 2.10 log2 and 2.10 log2. After challenge, chickens in unimmunized challenge control group B1 showed respiratory symptoms, egg production dropped by 30.9%, and they produced more soft-shelled, no-shelled or abnormal eggs. In the group A1, some chickens had light respiratory symptoms and egg production dropped by 11.7%. In the group A2, the egg production of all chickens was as normal as the group B2. [ Conclusion] When the HI titer was over 6 log2, challenge by virulent virus had no impact on egg produc- tion; when the HI titer was 5 log2, 4 log2 and less 3 log2, egg production dropped by 6.0%, 11.3% and 29.6%, respectively. Thus, the HI anti- body level in chickens has close correlation with protection against IBV challenge.

Antibodies to Newcastle Disease Virus in Egg Yolks of Great Cormorant (<i>Phalacrocorax carbo</i>) at Qinghai Lake

Newcastle disease virus (NDV) is not considered as cause of serious disease in humans. But, recent data make it clear that, under particular circumstances, it is indeed possible for NDV to cause severe human respiratory disease. Newcastle Disease infection has been reported in many bird species. Cormorants that inhabit at the Qinghai-Tibet Plateau are mainly represented by Great Cormorant (Phalacrocorax carbo sinensis), which is distributed mainly on the Qinghai Lake area. Cormorants are considered as one of the main NDV-reservoir. We conducted the study for the presence of antibodies to Newcastle disease virus by hemagglutination inhibition test in yolks. We got 50% of seropositive yolks to Newcastle disease virus. These results show that NDV circulates in the Qinghai Lake population of cormorants. We first used the technique of detection of antibodies to Newcastle disease virus in the egg yolk for study the circulation of the virus in cormorants and demonstrated its effectiveness. We should carefully monitor cases of pneumonia in the population of people living around the lake and assess the causes of the disease.

Genomic Recombination Enhances Pathogenic Factors in the Periodontopathogenic Bacterium <i>Eikenella corrodens</i>

We reported previously that plasmid-mediated genomic recombination at the pilin gene locus increased hemagglutination activity, growth rate, biofilm formation, hemolytic activity, and adherence to epithelial cells in Eikenella corrodens 23834. To determine whether these enhancements were common in this bacterium, we introduced the recombinase gene ORF4 into seven clinically isolated strains. Genomic recombination at the type IV pilin gene locus was observed in strains 1080, L9B6, L8Ao3, and RV2 (group A), but not in strains 261-2, 612-L, and 257-4 (group B). Similarly, group A strains displayed changed colony morphology following loss of type IV pili, which was not observed in group B. Group A strains showed also enhanced hemagglutination activity, growth rate, hemolytic, activity and biofilm formation. These results suggest that ORF4-induced genomic recombination at the pilin gene locus is a general phenomenon in a part of E. corrodens, which likely stimulates patho-genicity and virulence.

Evaluation of antimicrobial activity of a lectin isolated and purified from <i>Indigofera heterantha</i>

Indigofera heterantha commonly called indigo bush is a member of leguminoseae family found in the Himalayan region of Kashmir. A lectin has been isolated from the seeds of Indigofera heterantha by the purification procedure involving anion exchange chromatography on DEAE-cellulose followed by gel filtration chromatography on Sephadex G 100. Molecular characterization of the lectin was done by gel filtration and SDS-PAGE. Activity of the lectin was checked by hemagglutination assay and the sugar specificity by sugar inhibition tests. The antimicrobial activity of the purified lectin was carried out by Agar disc diffusion using appropriate standards. On the ion exchange column, the bound protein when eluted with 0-0.5 M NaCl gradient emerged as three peaks—peak I, peak II and peak III out of which only peak II showed the hemagglutinating activity. The lectin further resolved into two peaks G1 and G2 on gel filtration, with the lectin activity residing in G1, corresponding to a molecular weight of 70 KDa. The purified lectin named as Indigofera heterantha Lectin (IHL) produced a single band on SDS PAGE (18 KDa), revealing the tetrameric nature of the lectin. It agglutinated human erythrocytes (A, B, AB, and O). Hemagglutination was inhibited by D-galactose, Dmannose and D-arabinose. The lectin is reasonably thermostable showing full activity within a temperature range of 30&degC to 90&degC. pH stability of the lectin falls in the range of 2-9. IHL demonstrated a remarkable antibacterial activity against the pathogenic bacteria Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli, and Bacillus subtilis. IHL also inhibited the growth of phytopathogenic fungi Aspergillus niger, Aspergillus oryzae and Fusarium oxysporum.

Evidence of Synergistic Activity of Medicinal Plant Extracts against Neuraminidase Inhibitor Resistant Strains of Influenza Viruses

The frequent emergence of drug resistant influenza viral strains emphasizes the urgent and continual need to develop new antiviral drugs. Given the encouraging findings of previous studies on antiviral compounds from plant sources, this study focused on medicinal plants from Borneo that were traditionally used to treat symptoms of influenza infection. Following the promising results of earlier investigations, four plant extracts that demonstrated multiple modes of viral inhibition were studied against wild-type and neuraminidase (NA) inhibitor-resistant strains of Types A and B influenza viruses. The extracts exhibited more pronounced activities against the wild-type viruses than the NA inhibitor-resistant strains. Variations in the antiviral potential of the extracts collected from different parts of the same plant were also evidenced in the in vitro micro-inhibition assays. Even though all plant extracts affected NA activity of all viruses, only two extracts demonstrated hemagglutination inhibitory (HI) activities against Type A pandemic H1N1 and Type B viruses. Furthermore, Receptor Destroying Enzyme (RDE) treatments of extracts exhibiting HI activities indicated the presence of sialic acid (SA)-like component(s) that may be responsible for HI activity. Since the antiviral potential of extracts was not completely suppressed by RDE, the possibility of non SA-like antiviral components cannot be ruled out. Therefore, synergistic activity between SA-like and non SA-like components contained in the plant extracts may be responsible for the demonstrated antiviral potential. The results also indicated the presence of non SA-like components that may act against other viral proteins apart from hemagglutinin (HA) and NA. Hence, this study supports the presence of multiple antiviral components that act against different viral proteins or interfere with different stages of viral replication. Our results suggest that these plant extracts have the potential to be developed as therapeutic agents for the treatment of influenza and could be a solution to the global occurrence of viral strains resistant to NA inhibitors.

Evaluation of Usefulness of Three Serological Tests Using Native Crude Antigen in Diagnosis of Hepatic Cystic Echinococcosis Patients

Objective: To evaluate three different serological tests [Indirect Hemaglutination (IHA), Enzyme Linked Immunosorbent Assay (ELISA) and Western Blotting (WB)] using native crude antigen for diagnosis of hepatic cystic echinococcosis (HCE) patients. Materials and Methods: Sheep hydatid fluid (HF) was collected from fertile cysts obtained from a slaughterhouse and used as an antigen. Forty patients who were attended the Dr. Ersin Arslan Training and Research Hospital in Gaziantep, Turkey, were investigated. Serum samples were obtained from surgically confirmed CE patients. Healthy Turkish people and 16 patients with other helminthic infections were included as a control group. Results: Of the 40 analyzed patients, 10 (25%) were men and 30 (75%) were female. The average age was 46.97 years (s.d.;18.95). The majority of the patients had a single cystic lesion situated in one lobe of the liver (usually in the right lobe) (55%), 32.5% of patients had two cystic lesions and 12.5% of patients had multiple cyst formations with various numbers. In all cases, ultrasound (US) examinations were positive and the size of cysts was between 2.1 - 12.7 cm. Twenty-three patients of the total 40 patients were classified according to the WHO classification system based on US findings. According to the results of WB analysis, molecular weights of 8 kDa (80%), 12 kDa (80%), 22 - 24 kDa (97.5%), 26 kDa (97.5%), 34 kDa (100%), 36 - 38 kDa (90%), 45 - 50 - 55 kDa (97.5%), and 60 - 75 kDa (97.5%) bands were identified. But 34, 50, and 55 kDa bands were also found in other helminthic diseases. Conclusion: The specificity and sensitivity of three serological tests (IHA, ELISA and WB) using crude antigen were compared by diagnosing hepatic cystic echinococcosis patients. IHA and ELISA showed high sensitivity but low specificity. Western blotting showed low sensitivity but high specificity.

The Functional Characterization of Bat and Human P[3] Rotavirus VP8*s

P[3]rotavirus(RV)has been identified in many species,including human,simian,dog,and bat.Several glycans,including sialic acid,histo-blood group antigens(HBGAs)are reported as RV attachment factors.The glycan binding specificity of different P[3]RV VP8*s were investigated in this study.Human HCR3 A and dog P[3]RV VP8*s recognized glycans with terminal sialic acid and hemagglutinated the red blood cells,while bat P[3]VP8*showed neither binding to glycans nor hemagglutination.However,the bat P[3]VP8*mutant of C189 Y obtained the ability to hemagglutinate the red blood cells,while human P[3]HCR3 A/M2-102 mutants of Y189 C lost the ability.Sequence alignment and structural analysis indicated that residue 189 played an important role in the ligand recognition and may contribute to the cross-species transmission.Structural superimposition exhibited that bat P[3]VP8*model was quite different from the simian P[3]Rhesus rotavirus(RRV)P[3]VP8*,indicating that bat P[3]RV was relatively distinct and partially contributed to the no binding to tested glycans.These results promote our understanding of P[3]VP8*/glycans interactions and the potential transmission of bat/human P[3]RVs,offering more insight into the RV infection and prevalence.

Feasibility Study of Aluminum Hydroxide Sol as Adjuvant to Prepare Stable Vaccine Using Newcastle Disease Virus as a Model

Aluminum hydroxide sol （consisting of aluminum hydroxide nano-particles） prepared by a hydrothermal method was used as nano-aluminum adjuvant adsorbed the inactived Newcastle disease virus （NDV）. The zeta potential of aluminum hydroxide colloidal particles,the inactivated NDV and the nano-aluminum adjuvant NDV are ＋48.16 mV,？13.8 mV and ＋39.97 mV,respec-tively. The nano-aluminum adjuvant vaccine was transparent and stable without precipitate,whereas the conventional aluminum ad-juvant vaccine was turbid and a white precipitate was visible soon. The hemagglutination inhibition （HI） titers of the nano-aluminum adjuvant group were generally higher than those of the conventional aluminum group except the titer on day 29. The results show that the nano-aluminum adjuvant has better stability and higher levels of antibodies for a longer time than the conventional aluminum adjuvant.

Accelerated Evolution of H7N9 Subtype Influenza Virus under Vaccination Pressure

No avian H7 N9 outbreaks have occurred since the introduction of H7 N9 inactivated vaccine in the fall of 2017.However,H7 N9 is still prevalent in poultry.To surveil the prevalence,genetic characteristics,and antigenic changes of H7 N9,over7000 oropharyngeal and cloaca swab specimens were collected from live poultry markets and farms in 15 provinces of China from 2017 to 2019.A total of 85 influenza virus subtype H7 N9 strains were isolated and 20 representative strains were selected for genetic analysis and antigenicity evaluation.Results indicated the decreased prevalence of low-pathogenic H7 N9 strains while highly-pathogenic H7 N9 strains became dominated since the introduction of vaccine.Phylogenetic analysis showed that strains from 2019 formed an independent small branch and were genetically distant to strains isolated in 2013–2018.Analysis of key amino acid sites showed that the virus strains may adapt to the host environment evolutionally through mutation.Our analysis predicted additional potential glycosylation sites for HA and NA genes in the 2019 strains.Sequence analysis of HA gene in strains isolated from 2018 to 2019 showed that there were an increased nucleotide substitution rate and an increased mutation rate in the first and second nucleotides of coding codons within the open reading frame.The hemagglutination inhibition(HI)assay showed that H7-Re1 and H7-Re2 exhibited a lower HI titer for isolates from 2019,while H7-Re3 and r LN79 showed a high HI titer.The protective effect of the vaccine decreased after15 months of use.Overall,under vaccination pressure,the evolution of influenza virus subtype H7 N9 has accelerated.

The identification of a CD47-blocking“hotspot”and design of a CD47/PD-L1 dual-specific antibody with limited hemagglutination

Dear Editor,By targeting the programmed cell death 1(PD-1)pathway with monoclonal antibodies(mAbs),immune checkpoint therapy(ICT)has achieved unprecedented clinical success in the treatment of multiple tumors.1,2 Cancer cells evade the host immune system via both the tolerance of T cells and functional suppression of innate immune cells.3 CD47 provides a“do not eat me”signal by binding to signal regulatory protein alpha(SIRPα)to prevent innate immune cells from attacking host cells.4 Recently,macrophages were found to restore antitumor reactivity by blocking the interaction between upregulated CD47 on tumor cells and SIRPαon innate immune cells.5 However,it remains unknown whether there are blocking“hotspots”on CD47 for mAb-based anti-CD47 therapy or additional blocking hotspot regions within CD47 for therapeutic mAb development.Although it is overexpressed on tumor cells,CD47 is also expressed in many normal cells,including red blood cells and platelets.6 Some validated CD47-blocking mAbs under clinical investigation induce hemagglutination and anemia.7 Thus,designing an engineered CD47-blocking antibody to exert a therapeutic effect with limited hemagglutination is needed.