scholarly journals Rhomboid protein 2 of Eimeria maxima provided partial protection against infection by homologous species

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Yufeng Chen ◽  
Di Tian ◽  
Lixin Xu ◽  
Ruofeng Yan ◽  
Xiangrui Li ◽  
...  
Keyword(s):  
Western Blot ◽  
Protective Efficacy ◽  
Cell Mediated Immunity ◽  
Igg Antibody ◽  
Western Blot Assay ◽  
Partial Protection ◽  
Eimeria Maxima ◽  
Polymerase Chain ◽  
Protection Against Infection ◽  
New Generation

AbstractRhomboid-like proteases (ROMs) are considered as new candidate antigens for developing new-generation vaccines due to their important role involved in the invasion of apicomplexan protozoa. In prior works, we obtained a ROM2 sequence of Eimeria maxima (EmROM2). This study was conducted to evaluate the immunogenicity and protective efficacy of EmROM2 recombinant protein (rEmROM2) and EmROM2 DNA (pVAX1-EmROM2) against infection by Eimeria maxima (E. maxima). Firstly, Western blot assay was conducted to analyze the immunogenicity of rEmROM2. The result showed that rEmROM2 was recognized by chicken anti-E. maxima serum. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay revealed apparent transcription and expression of EmROM2 at the injection site. qRT-PCR (quantitative real-time PCR), flow cytometry and indirect ELISA indicated that vaccination with rEmROM2 or EmROM2 DNA significantly upregulated the transcription level of cytokines (IFN-γ, IL-2, IL-4, IL-10, IL-17, TGF-β and TNF SF15), the proportion of CD8+ and CD4+ T lymphocytes and serum IgG antibody response. Ultimately, a vaccination-challenge trial was performed to evaluate the protective efficacy of rEmROM2 and pVAX1-EmROM2 against E. maxima. The result revealed that vaccination with rEmROM2 or pVAX1-EmROM2 significantly alleviated enteric lesions, weight loss, and reduced oocyst output caused by challenge infection of E. maxima, and provided anticoccidial index (ACI) of more than 160, indicating partial protection against E. maxima. In summary, vaccination with rEmROM2 or pVAX1-EmROM2 activated notable humoral and cell-mediated immunity and provided partial protection against E. maxima. These results demonstrated that EmROM2 protein and DNA are promising vaccine candidates against E. maxima infection.

scholarly journals Rhomboid Protein 2 of Eimeria Maxima Provided Partial Protection Against Infection by Homologous Species

2020 ◽  
Author(s):  
Yufeng Chen ◽  
Di Tian ◽  
Siying Chen ◽  
Wenxi Ding ◽  
Xiaoting Sun ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Western Blot ◽  
Protective Efficacy ◽  
Cell Mediated Immunity ◽  
Igg Antibody ◽  
Western Blot Assay ◽  
Partial Protection ◽  
Eimeria Maxima ◽  
Serum Igg ◽  
Protection Against Infection

Abstract Background: Rhomboid-like proteases (ROMs) are considered as a new candidate antigen for developing new-generation vaccine due to their important role involved in the invasion of apicomplexan protozoa. In prior works, we obtained a ROM2 sequence of Eimeria maxima (EmROM2) which is the homologous gene with ROM2 of Toxoplasma gondii. This study was conducted to evaluate the immunogenicity and protective efficacy of EmROM2 recombinant protein (rEmROM2) and EmROM2 DNA (pVAX1-EmROM2) against infection by Eimeria maxima (E. maxima).Methods: Western blot assay was conducted to analyze the immunogenicity of rEmROM2. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay were performed to determine the transcription and expression of pVAX1-EmROM2 recombinant plasmid. EmROM2-induced changes in transcriptional level of cytokines, T lymphocytes subsets and specific serum IgG antibody were detected through qPCR (quantitative real-time PCR), flow cytometry and indirect ELISA, respectively. Ultimately, a vaccination-challenge trial was performed to evaluate the protective efficacy of rEmROM2 and pVAX1-EmROM2 against infection with E. maxima. Results: The purified rEmROM2 was recognized with chicken anti-E. maxima serum. After vaccination with pVAX1-EmROM2, apparent transcription and translation of EmROM2 were observed in the vaccinated chickens. Vaccination with rEmROM2 and EmROM2 DNA significantly upregulated the proportion of CD8+ and CD4+ T lymphocytes, the transcription level of cytokines (IFN-γ, IL-2, IL-4, IL-10, IL-17, TGF-β and TNF SF15) and serum IgG antibody response. Meanwhile, the vaccination significantly alleviated enteric lesions, weight loss, and reduced oocyst output caused by challenge infection of E. maxima, and provided anticoccidial index (ACI) of more than 160, indicating partial protection against E. maxima.Conclusions: Vaccination with rEmROM2 and pVAX1-EmROM2 activated notable humoral and cell-mediated immunity and provided partial protection against infection by E. maxima. These results demonstrated that EmROM2 protein and DNA are promising vaccine candidates against E. maxima infection.

scholarly journals Vaccine prophylaxis of abattoir-associated Q fever: eight years' experience in Australian abattoirs

1990 ◽  
Vol 104 (2) ◽  
pp. 275-287 ◽  
Author(s):  
B. P. Marmion ◽  
R. A. Ormsbee ◽  
M. Kyrkou ◽  
J. Wright ◽  
D. A. Worswick ◽  
...  
Keyword(s):  
Q Fever ◽  
Protective Efficacy ◽  
Phase 1 ◽  
South Australia ◽  
Risk Groups ◽  
The Other ◽  
Cell Mediated Immunity ◽  
Igg Antibody ◽  
Inoculation Site ◽  
Other Hand

SUMMARYDuring the period 1981–8 a clinical trial of a Q fever vaccine (Q-vax; Commonwealth Serum Laboratories, Melbourne) has been conducted in abattoir workers and other at-risk groups in South Australia. Volunteers in four abattoirs and visitors to the abattoirs were given one subcutaneous dose of 30 μg of a formalin-inactivated, highly-purifiedCoxiella burnetiicells, Henzerling strain, Phase 1 antigenic state, in a volume of 0·5 ml.During the period, over 4000 subjects have been vaccinated and the programme continues in the abattoirs and related groups. ‘Common’ reactions to the vaccine comprised tenderness and erythema, rarely oedema at the inoculation site and sometimes transient headache. Two more serious ‘uncommon’ reactions, immune abscess at the inoculation site, were observed in two subjects, and two others developed small subcutaneous lumps which gradually dispersed without intervention.Protective efficacy of the vaccine appeared to be absolute and to last for 5 years at least. Eight Q fever cases were observed in vaccinees, but all were in persons vaccinated during the incubation period of a natural attack of Q fever before vaccine-induced immunity had had time (≥ 13 days after vaccination) to develop. On the other hand, 97 Q fever cases were detected in persons working in, or visiting the same abattoir environments.Assays for antibody and cellular immunity showed an 80–82% seroconversion after vaccination, mostly IgM antibody to Phase 2 antigen, in the 3 months after vaccination. This fell to about 60%, mostly IgG antibody to Phase 1 antigen, after 20 months. On the other hand, 85–95% of vaccinees developed markers of cell mediated immunity as judged by lymphoproliferative responses withC. burnetiiantigens; these rates remained elevated for at least 5 years.The Q fever vaccine, unlike other killed rickettsial vaccines, has the property of stimulating long-lasting T lymphocyte memory and this may account for its unusual protective efficacy as a killed vaccine.

scholarly journals Enzyme Immunoassay Detection of Antigen-Specific Immunoglobulin G Antibodies in Longitudinal Serum Samples from Patients with Cryptosporidiosis

2001 ◽  
Vol 8 (2) ◽  
pp. 415-423 ◽  
Author(s):  
Jeffrey W. Priest ◽  
Anna Li ◽  
Mohamad Khan ◽  
Michael J. Arrowood ◽  
Patrick J. Lammie ◽  
...  
Keyword(s):  
Western Blot ◽  
Immunoglobulin G ◽  
Protozoan Parasite ◽  
Epidemiologic Studies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Igg Antibody ◽  
Large Format ◽  
Western Blot Assay ◽  
Wide Range

ABSTRACT Cryptosporidium parvum is a protozoan parasite that causes diarrheal illness in a wide range of mammalian hosts, including humans. Characteristic serum immunoglobulin G (IgG) antibody responses to antigens in the 27- and 17-kDa size ranges have been shown to develop after infection, and several enzyme-linked immunosorbent assay (ELISA) and Western blot assay formats have been used to measure these IgG levels in human serum. Using a collection of serial samples from laboratory-confirmed cryptosporidiosis patients, we compared the results obtained by using two new ELISAs with those obtained with two different Western blot assays. When assayed with the large-format Western blot, 97% of the 67 patients had a demonstrable antibody response on at least one occasion. The Cp23 ELISA correctly identified 93% of the samples that had a 27-kDa response by Western blot and 100% of the negative samples. The Triton antigen ELISA detected 77% of the samples that had a 17-kDa response by Western blot and 88% of the negative samples. The sensitivity of the Triton antigen assay was higher for samples collected between 16 and 92 days after the onset of symptoms (96%). The minigel-format Western blot did not compare favorably with the large-format blot for the detection of antibodies to the 27-kDa antigen (71% sensitivity). A half-life of about 12 weeks was estimated for antibodies to both the 27- and 17-kDa antigens. We believe the Cp23 and Triton antigen ELISAs will be useful in epidemiologic studies of the prevalence ofCryptosporidium infection in the population.

scholarly journals Effectiveness Regarding Hantavirus Detection in Rodent Tissue Samples and Urine

2021 ◽  
Author(s):  
Mónika Madai ◽  
Győző Horváth ◽  
Róbert Herczeg ◽  
Balázs Somogyi ◽  
Brigitta Zana ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Western Blot ◽  
Urine Samples ◽  
Igg Antibodies ◽  
Chain Reaction ◽  
Western Blot Assay ◽  
Tissue Samples ◽  
Natural Hosts ◽  
Polymerase Chain

The natural hosts regarding Orthohantaviruses are rodents, soricomorphs and bats, and it is well known they may cause serious or even fatal diseases among humans worldwide. The virus is persistent among animals and it is shed via urine, saliva and feces, throughout the entirety of their lives. We aim to identify the effectiveness regarding hantavirus detection from rodent tissue samples and urine originating from naturally infected rodents. Initially, animals were trapped at five distinct locations throughout the Transdanubian region in Hungary. Lung, liver, kidney and urine samples were obtained from 163 perished animals. All organs and urine were tested using nested reverse transcriptase-polymerase chain reaction (nRT-PCR). Furthermore, sera were examined for IgG antibodies against DOBV and PUUV viruses by Western Blot assay. IgG antibodies against hantaviruses and/or nucleic acid were detected in 25 (15.3%) cases. Among Apodemus, Myodes, and Microtus rodent species, DOBV, PUUV, TULV were all clearly identified. The virus nucleic acid was detected most effectively from the kidney (100%), while only 55% of screened lung tissues were positive. Interestingly, only 3 out of 20 rodent urine samples were positive regarding nRT-PCR. Moreover, five rodents were seropositive without detectable virus nucleic acid from any of the tested organs.

scholarly journals Detection of Bovine Immunodeficiency Virus Antibodies in Cattle by Western Blot Assay with Recombinant Gag Protein

1997 ◽  
Vol 9 (4) ◽  
pp. 347-351 ◽  
Author(s):  
Shucheng Zhang ◽  
Wenzhi Xue ◽  
Charles Wood ◽  
Qi-Min Chen ◽  
Sanjay Kapil ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Western Blot ◽  
Bovine Immunodeficiency Virus ◽  
Test Results ◽  
Chain Reaction ◽  
Serum Samples ◽  
Western Blot Assay ◽  
Gag Protein ◽  
Immunodeficiency Virus ◽  
Polymerase Chain

A western blot assay using purified recombinant bovine immunodeficiency virus gag protein has been developed for detection of bovine immunodeficiency virus antibodies in bovine serum samples. The test was standardized with known bovine immunodeficiency virus positive and negative bovine serum samples and the monoclonal antibody to gag protein. Both naturally and experimentally infected cattle sera demonstrated positive test results. The result of western blot assay was compared with polymerase chain reaction test results in 134 blood samples collected from Kansas. Twenty-six samples tested positive for bovine immunodeficiency virus DNA with polymerase chain reaction (18.7%) and 25 were positive for the antibody to gag protein by western blot analysis (17.9%). Of 26 cattle testing positive using the polymerase chain reaction assay, 24 were antibody-positive by western blot assay, thus establishing a strong correlation between the two tests. The sensitivity and specificity of western blot relative to polymerase chain reaction are 0.92 and 0.99, respectively. The western blot assay proved to be a specific and sensitive test.

The Anti-tumor Activity and Mechanisms of rLj-RGD3 on Human Laryngeal Squamous Carcinoma Hep2 Cells

2020 ◽  
Vol 19 (17) ◽  
pp. 2108-2119
Author(s):  
Yang Jin ◽  
Li Lv ◽  
Shu-Xiang Ning ◽  
Ji-Hong Wang ◽  
Rong Xiao
Keyword(s):  
Wound Healing ◽  
Western Blot ◽  
Morphological Changes ◽  
In Vivo Studies ◽  
Migration And Invasion ◽  
Tunel Staining ◽  
Dna Ladder ◽  
Western Blot Assay

Background: Laryngeal Squamous Cell Carcinoma (LSCC) is a malignant epithelial tumor with poor prognosis and its incidence rate increased recently. rLj-RGD3, a recombinant protein cloned from the buccal gland of Lampetra japonica, contains three RGD motifs that could bind to integrins on the tumor cells. Methods: MTT assay was used to detect the inhibitory rate of viability. Giemsa’s staining assay was used to observe the morphological changes of cells. Hoechst 33258 and TUNEL staining assay, DNA ladder assay were used to examine the apoptotic. Western blot assay was applied to detect the change of the integrin signal pathway. Wound-healing assay, migration, and invasion assay were used to detect the mobility of Hep2 cells. H&E staining assay was used to show the arrangement of the Hep2 cells in the solid tumor tissues. Results: In the present study, rLj-RGD3 was shown to inhibit the viability of LSCC Hep2 cells in vitro by inducing apoptosis with an IC50 of 1.23µM. Western blot showed that the apoptosis of Hep2 cells induced by rLj- RGD3 was dependent on the integrin-FAK-Akt pathway. Wound healing, transwells, and western blot assays in vitro showed that rLj-RGD3 suppressed the migration and invasion of Hep2 cells by integrin-FAKpaxillin/ PLC pathway which could also affect the cytoskeleton arrangement in Hep2 cells. In in vivo studies, rLj-RGD3 inhibited the growth, tumor volume, and weight, as well as disturbed the tissue structure of the solid tumors in xenograft models of BALB/c nude mice without reducing their body weights. Conclusion: hese results suggested that rLj-RGD3 is an effective and safe suppressor on the growth and metastasis of LSCC Hep2 cells from both in vitro and in vivo experiments. rLj-RGD3 might be expected to become a novel anti-tumor drug to treat LSCC patients in the near future.

scholarly journals Salidroside promoted osteogenic differentiation of adipose-derived stromal cells through Wnt/β-catenin signaling pathway

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Xiao-hua Li ◽  
Fu-ling Chen ◽  
Hong-lin Shen
Keyword(s):  
Western Blot ◽  
Osteogenic Differentiation ◽  
Signaling Pathway ◽  
Differentially Expressed Genes ◽  
Stromal Cells ◽  
Differentially Expressed ◽  
Calcium Deposition ◽  
Western Blot Assay ◽  
Adipose Derived Stromal Cells ◽  
Interfering Rna

Abstract Background Bone disease causes short-term or long-term physical pain and disability. It is necessary to explore new drug for bone-related disease. This study aimed to explore the role and mechanism of Salidroside in promoting osteogenic differentiation of adipose-derived stromal cells (ADSCs). Methods ADSCs were isolated and treated with different dose of Salidroside. Cell count kit-8 (CCK-8) assay was performed to assess the cell viability of ADSCs. Then, ALP and ARS staining were conducted to assess the early and late osteogenic capacity of ADSCs, respectively. Then, differentially expressed genes were obtained by R software. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the differentially expressed genes were further analyzed. The expression of OCN, COL1A1, RUNX2, WNT3A, and β-catenin were measured by real-time PCR and Western blot analysis. Last, β-catenin was silenced by small interfering RNA. Results Salidroside significantly increased the ADSCs viability at a dose-response manner. Moreover, Salidroside enhanced osteogenic capacity of ADSCs, which are identified by enhanced ALP activity and calcium deposition. A total of 543 differentially expressed genes were identified between normal and Salidroside-treated ADSCs. Among these differentially expressed genes, 345 genes were upregulated and 198 genes were downregulated. Differentially expressed genes enriched in the Wnt/β-catenin signaling pathway. Western blot assay indicated that Salidroside enhanced the WNT3A and β-catenin expression. Silencing β-catenin partially reversed the promotion effects of Salidroside. PCR and Western blot results further confirmed these results. Conclusion Salidroside promoted osteogenic differentiation of ADSCs through Wnt/β-catenin signaling pathway.

Ginkgolic acid induces apoptosis and autophagy of endometrial carcinoma cells via inhibiting PI3K/Akt/mTOR pathway in vivo and in vitro

2021 ◽  
pp. 096032712110237
Author(s):  
L Zhou ◽  
S Li ◽  
J Sun
Keyword(s):  
Endometrial Cancer ◽  
B Cells ◽  
Western Blot ◽  
Signal Pathway ◽  
Mtor Pathway ◽  
Related Protein ◽  
Western Blot Assay ◽  
Ginkgolic Acid

Endometrial cancer (EC) is the fourth most common malignancy in women in developed countries. The prognosis of EC is extremely poor, and it is an important factor that contributes to the death of patients. Therefore, studying EC pathogenesis and therapeutic targets, and exploring effective drugs are the primary tasks to improve the prognosis of EC. In the present study, we aimed to explore the function of ginkgolic acid (GA) in EC cell apoptosis and autophagy through PI3K/Akt/mTOR signal pathway in vitro and in vivo. Firstly, MTT assay and clone formation assay were employed to analyze the Ishikawa and HEC-1-B cell viabilities and proliferation after treatment with GA. The results showed that GA inhibited endometrial cancer cell survival. Flow cytometry assay and western blot assay were applied to examine the apoptosis and apoptosis related protein Bcl-2, Bax, Cleaved caspase-3 expression levels of Ishikawa and HEC-1-B cells after treatment with GA. Next, we applied western blot assay to analyze the autophagy associated proteins LC3I, LC3II, p62 and Beclin-1 in GA treated Ishikawa and HEC-1-B cells. We found that GA promoted apoptosis and induced autophagy of endometrial cancer cells. Meanwhile, western blot assay was also used to determine the expression levels of the PI3K/Akt/mTOR signal pathway related protein and the results revealed that GA inhibited the activity of PI3K/Akt/mTOR pathway. Finally, we found that GA inhibited tumor growth in vivo through immunohistochemistry assay. In conclusion, GA induces apoptosis and autophagy of EC cells via inhibiting PI3K/Akt/mTOR pathway in vivo and vitro.

scholarly journals Gambogic acid protects LPS-induced apoptosis and inflammation in a cell model of neonatal pneumonia through the regulation of TrkA/Akt signaling pathway

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Xu Gao ◽  
Jingya Dai ◽  
Guifang Li ◽  
Xinya Dai
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Cell Model ◽  
Akt Signaling ◽  
Gambogic Acid ◽  
Akt Signaling Pathway ◽  
Western Blot Assay ◽  
A Cell ◽  
Induced Apoptosis ◽  
Apoptosis And Inflammation

Abstract Objective In this work, we investigated the effects of gambogic acid (GA) on lipopolysaccharide (LPS)-induced apoptosis and inflammation in a cell model of neonatal pneumonia. Method Human WI-38 cells were maintained in vitro and incubated with various concentrations of GA to examine WI-38 survival. GA-preincubated WI-38 cells were then treated with LPS to investigate the protective effects of GA on LPS-induced death, apoptosis and inflammation. Western blot assay was utilized to analyze the effect of GA on tropomyosin receptor kinase A (TrkA) signaling pathway in LPS-treated WI-38 cells. In addition, human AKT serine/threonine kinase 1 (Akt) gene was knocked down in WI-38 cells to further investigate the associated genetic mechanisms of GA in protecting LPS-induced inflammation and apoptosis. Results Pre-incubating WI-38 cells with low and medium concentrations GA protected LPS-induced cell death, apoptosis and inflammatory protein productions of IL-6 and MCP-1. Using western blot assay, it was demonstrated that GA promoted TrkA phosphorylation and Akt activation in LPS-treated WI-38 cells. Knocking down Akt gene in WI-38 cells showed that GA-associated protections against LPS-induced apoptosis and inflammation were significantly reduced. Conclusions GA protected LPS-induced apoptosis and inflammation, possibly through the activations of TrkA and Akt signaling pathway. This work may broaden our understanding on the molecular mechanisms of human neonatal pneumonia.

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