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1.
Biol Res ; 57(1): 24, 2024 May 06.
Article in English | MEDLINE | ID: mdl-38711133

ABSTRACT

Despite the record speed of developing vaccines and therapeutics against the SARS-CoV-2 virus, it is not a given that such success can be secured in future pandemics. In addition, COVID-19 vaccination and application of therapeutics remain low in developing countries. Rapid and low cost mass production of antiviral IgY antibodies could be an attractive alternative or complementary option for vaccine and therapeutic development. In this article, we rapidly produced SARS-CoV-2 antigens, immunized hens and purified IgY antibodies in 2 months after the SARS-CoV-2 gene sequence became public. We further demonstrated that the IgY antibodies competitively block RBD binding to ACE2, neutralize authentic SARS-CoV-2 virus and effectively protect hamsters from SARS-CoV-2 challenge by preventing weight loss and lung pathology, representing the first comprehensive study with IgY antibodies. The process of mass production can be easily implemented in most developing countries and hence could become a new vital option in our toolbox for combating viral pandemics. This study could stimulate further studies, optimization and potential applications of IgY antibodies as therapeutics and prophylactics for human and animals.


Subject(s)
Antibodies, Neutralizing , Antibodies, Viral , COVID-19 , Chickens , Egg Yolk , Immunoglobulins , SARS-CoV-2 , Animals , SARS-CoV-2/immunology , Antibodies, Neutralizing/immunology , COVID-19/prevention & control , COVID-19/immunology , Chickens/immunology , Cricetinae , Immunoglobulins/immunology , Egg Yolk/immunology , Antibodies, Viral/immunology , Female , Mesocricetus , COVID-19 Vaccines/immunology
2.
J Zoo Wildl Med ; 53(4): 832-837, 2023 Jan.
Article in English | MEDLINE | ID: mdl-36640087

ABSTRACT

Staphylococcal A and streptococcal G proteins are widely used in immunoassays when specific immunological reagents are unavailable, such as for wild animals. The affinity of bacterial proteins A and G to the immunoglobulins of seven Brazilian mammals were tested, including black-tufted marmoset (Callithrix penicillata, n = 5), golden-bellied capuchin (Sapajus xanthosternos, n = 13), woolly mouse opossum (Micoureus demerarae, n = 6), long-nosed armadillo (Dasypus novemcinctus, n = 5), collared anteater (Tamandua tetradactyla, n = 5), ocelot (Leopardus pardalis, n = 6), and vampire bat (Desmodus rotundus, n = 5). Blood samples were collected from animals that were rescued in peri-urban rainforest fragments. Sera pools of each species were tested by ELISA to determine the intensity of each bacterial protein affinity to the immunoglobulins. When comparing the affinity to both proteins, immunoglobulins from D. rotundus, S. xanthosternos, and T. tetradactyla presented a higher affinity to protein G, whereas a higher affinity to protein A was found for immunoglobulins of C. penicillata and L. pardalis. The only species that presented a very low affinity to both bacterial proteins was M. demerarae. This study can be used as a reference for further studies on the development of sensitive and specific immunodiagnostic assays to be used for the monitoring of the health of these wild mammals.


Subject(s)
Bacterial Proteins , Immunoglobulins , Mammals , Animals , Animals, Wild/immunology , Bacterial Proteins/immunology , Brazil , Immunoglobulins/immunology , Mammals/immunology
3.
Int J Mol Sci ; 23(12)2022 Jun 20.
Article in English | MEDLINE | ID: mdl-35743310

ABSTRACT

γδT cells mature in the human thymus, and mainly produce IL-17A or IFN-γ, but can also produce IL-22 and modulate a variety of immune responses. Here, we aimed to evaluate whether IgG from AD patients (AD IgG) can functionally modulate thymic nonatopic γδT cells. Thymic tissues were obtained from 12 infants who had not had an atopic history. Thymocytes were cultured in mock condition, or in the presence of either AD IgG or therapeutic intravenous IgG (IVIg). Following these treatments, intracellular cytokine production, phenotype, and microRNA expression profiles were investigated. AD IgG could downregulate α4ß7, upregulate CLA, and induce the production of IFN-γ, IL-17, and IL-22 in γδT cells. Although both AD IgG and IVIg could directly interact with γδT cell membranes, AD IgG could reduce γδT cell apoptosis. AD IgG could upregulate nine miRNAs compared to IVIg, and six when compared to the mock condition. In parallel, some miRNAs were downregulated. Target gene prediction and functional analysis indicated that some target genes were enriched in the negative regulation of cellular transcription. This study shows that AD IgG influences the production of IL-17 and IL-22 by intrathymic nonatopic γδT cells, and demonstrates epigenetic implications mediated by miRNAs.


Subject(s)
Dermatitis, Atopic , MicroRNAs , Dermatitis, Atopic/metabolism , Epigenesis, Genetic , Humans , Immunoglobulins/immunology , Immunoglobulins, Intravenous , Infant, Newborn , Interleukin-17 , Interleukins , MicroRNAs/genetics , Receptors, Antigen, T-Cell, gamma-delta/immunology , Thymus Gland , Interleukin-22
4.
Sci Rep ; 12(1): 3890, 2022 03 10.
Article in English | MEDLINE | ID: mdl-35273234

ABSTRACT

The new outbreak of coronavirus disease 2019 (COVID-19) has infected and caused the death of millions of people worldwide. Intensive efforts are underway around the world to establish effective treatments. Immunoglobulin from immunized animals or plasma from convalescent patients might constitute a specific treatment to guarantee the neutralization of the virus in the early stages of infection, especially in patients with risk factors and a high probability of progressing to severe disease. Worldwide, a few clinical trials using anti-SARS-CoV-2 immunoglobulins from horses immunized with the entire spike protein or fragments of it in the treatment of patients with COVID-19 are underway. Here, we describe the development of an anti-SARS-CoV-2 equine F(ab')2 immunoglobulin using a newly developed SARS-CoV-2 viral antigen that was purified and inactivated by radiation. Cell-based and preclinical assays showed that the F(ab')2 immunoglobulin successfully neutralizes the virus, is safe in animal models, and reduces the severity of the disease in a hamster model of SARS-CoV-2 infection and disease.


Subject(s)
COVID-19/therapy , Immunoglobulins/therapeutic use , Receptors, Immunologic/therapeutic use , SARS-CoV-2/immunology , Animals , Enzyme-Linked Immunosorbent Assay , Female , Horses/immunology , Humans , Immunoglobulins/immunology , Immunoglobulins/isolation & purification , Male , Mesocricetus/immunology , Plasmapheresis/veterinary , Receptors, Immunologic/immunology
5.
J Immunol ; 208(2): 371-383, 2022 01 15.
Article in English | MEDLINE | ID: mdl-34965965

ABSTRACT

Monoallelic AgR gene expression underlies specific adaptive immune responses. AgR allelic exclusion is achieved by sequential initiation of V(D)J recombination between alleles and resultant protein from one allele signaling to prevent recombination of the other. The ATM kinase, a regulator of the DNA double-strand break (DSB) response, helps enforce allelic exclusion through undetermined mechanisms. ATM promotes repair of RAG1/RAG2 (RAG) endonuclease-induced DSBs and transduces signals from RAG DSBs during Igk gene rearrangement on one allele to transiently inhibit RAG1 protein expression, Igk accessibility, and RAG cleavage of the other allele. Yet, the relative contributions of ATM functions in DSB repair versus signaling to enforce AgR allelic exclusion remain undetermined. In this study, we demonstrate that inactivation in mouse pre-B cells of the NF-κB essential modulator (Nemo) protein, an effector of ATM signaling, diminishes RAG DSB-triggered repression of Rag1/Rag2 transcription and Igk accessibility but does not result in aberrant repair of RAG DSBs like ATM inactivation. We show that Nemo deficiency increases simultaneous biallelic Igk cleavage in pre-B cells and raises the frequency of B cells expressing Igκ proteins from both alleles. In contrast, the incidence of biallelic Igκ expression is not elevated by inactivation of the SpiC transcriptional repressor, which is induced by RAG DSBs in an ATM-dependent manner and suppresses Igk accessibility. Thus, we conclude that Nemo-dependent, ATM-mediated DNA damage signals enforce Igκ allelic exclusion by orchestrating transient repression of RAG expression and feedback inhibition of additional Igk rearrangements in response to RAG cleavage on one Igk allele.


Subject(s)
DNA Breaks, Double-Stranded , DNA Repair/genetics , Immunoglobulins/immunology , Intracellular Signaling Peptides and Proteins/metabolism , Loss of Heterozygosity/genetics , Animals , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , Cells, Cultured , Clonal Anergy/genetics , Clonal Anergy/immunology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/immunology , Immunoglobulins/genetics , Intracellular Signaling Peptides and Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , V(D)J Recombination/genetics
6.
Breast Cancer ; 28(6): 1328-1339, 2021 Nov.
Article in English | MEDLINE | ID: mdl-34240315

ABSTRACT

PURPOSE: Dendritic cells (DCs) are the most potent antigen-presenting cells that play a major role in initiating the antitumor immune response in different types of cancer. However, the prognostic significance of the accumulation of these cells in human early breast tumors is not totally clear. The aim of this study is to evaluate the prognostic relevance of CD1a( +) and CD83( +) dendritic cells in early breast cancer patients. METHODS: We conducted immunohistochemical assays to determine the number of stromal CD1a( +) and CD83( +) DCs in primary tumors from early invasive ductal breast cancer patients, and analyzed their association with clinico-pathological characteristics. RESULTS: Patients with high CD1a( +) DC number had lower risk of bone metastatic occurrence, as well as, longer disease-free survival (DFS), bone metastasis-free survival (BMFS) and overall survival (OS). Moreover, CD1a( +) DC number was an independent prognostic factor for BMFS and OS. In contrast, we found that patients with high number of CD83( +) DCs had lower risk of mix (bone and visceral)-metastatic occurrence. Likewise, these patients presented better prognosis with longer DFS, mix-MFS and OS. Furthermore, CD83( +) DC number was an independent prognostic factor for DFS and OS. CONCLUSION: The quantification of the stromal infiltration of DCs expressing CD1a or CD83 in early invasive breast cancer patients serves to indicate the prognostic risk of developing metastasis in a specific site.


Subject(s)
Antigens, CD1/analysis , Antigens, CD/analysis , Breast Neoplasms/pathology , Immunoglobulins/analysis , Membrane Glycoproteins/analysis , Adult , Aged , Aged, 80 and over , Antigens, CD/immunology , Antigens, CD1/immunology , Biomarkers, Tumor/immunology , Dendritic Cells/immunology , Dendritic Cells/pathology , Female , Humans , Immunoglobulins/immunology , Membrane Glycoproteins/immunology , Middle Aged , Retrospective Studies , Survival Analysis , CD83 Antigen
7.
Front Immunol ; 12: 666356, 2021.
Article in English | MEDLINE | ID: mdl-34054836

ABSTRACT

Type II interferon gamma (IFNγ) is a pleiotropic cytokine capable of modulating the innate and adaptive immune responses which has been widely characterized in several teleost families. In fish, IFNγ stimulates the expression of cytokines and chemokines associated with the pro-inflammatory response and enhances the production of nitrogen and oxygen reactive species in phagocytic cells. This work studied the effect of IFNγ on the expression of cell-surface markers on splenocytes of Atlantic salmon (Salmo salar). In vitro results showed that subpopulations of mononuclear splenocytes cultured for 15 days were capable of increasing gene expression and protein availability of cell-surface markers such as CD80/86, CD83 and MHC II, after being stimulated with recombinant IFNγ. These results were observed for subpopulations with characteristics associated with monocytes (51%), and features that could be related to lymphocytes (46.3%). In addition, a decrease in the expression of zbtb46 was detected in IFNγ-stimulated splenocytes. Finally, the expression of IFNγ and cell-surface markers was assessed in Atlantic salmon under field conditions. In vivo results showed that the expression of ifnγ increased simultaneously with the up-regulation of cd80/86, cd83 and mhcii during a natural outbreak of Piscirickettsia salmonis. Overall, the results obtained in this study allow us to propose IFNγ as a candidate molecule to stimulate the phenotypic progression of a small population of immune cells, which will increase antigen presenting cells markers. Thereby, modulatory strategies using IFNγ may generate a robust and coordinated immune response in fish against pathogens that affect aquaculture.


Subject(s)
Antigens, CD/metabolism , B7-1 Antigen/metabolism , B7-2 Antigen/metabolism , Histocompatibility Antigens Class II/metabolism , Immunoglobulins/metabolism , Interferon-gamma/immunology , Membrane Glycoproteins/metabolism , Salmo salar/immunology , Spleen/immunology , Animals , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , Antigens, CD/genetics , Antigens, CD/immunology , B7-1 Antigen/genetics , B7-1 Antigen/immunology , B7-2 Antigen/genetics , B7-2 Antigen/immunology , Biomarkers/metabolism , Fish Diseases/immunology , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/immunology , Immunoglobulins/genetics , Immunoglobulins/immunology , Interferon-gamma/pharmacology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/immunology , Piscirickettsia , Piscirickettsiaceae Infections/immunology , Piscirickettsiaceae Infections/veterinary , Transcription Factors/genetics , Transcription Factors/immunology , Transcription Factors/metabolism , CD83 Antigen
8.
Int J Mol Sci ; 21(22)2020 Nov 20.
Article in English | MEDLINE | ID: mdl-33233764

ABSTRACT

Class-I Restricted T Cell-Associated Molecule (CRTAM) is a protein that is expressed after T cell activation. The interaction of CRTAM with its ligand, nectin-like 2 (Necl2), is required for the efficient production of IL-17, IL-22, and IFNγ by murine CD4 T cells, and it plays a role in optimal CD8 T and NK cell cytotoxicity. CRTAM promotes the pro-inflammatory cytokine profile; therefore, it may take part in the immunopathology of autoimmune diseases such as diabetes type 1 or colitis. Thus, antibodies that block the interaction between CRTAM and Necl2 would be useful for controlling the production of these inflammatory cytokines. In this work, using bioinformatics predictions, we identified three short disordered epitopes (sDE1-3) that are located in the Ig-like domains of murine CRTAM and are conserved in mammalian species. We performed a structural analysis by molecular dynamics simulations of sDE1 (QHPALKSSKY, Ig-like V), sDE2 (QRNGEKSVVK, Ig-like C1), and sDE3 (CSTERSKKPPPQI, Ig-like C1). sDE1, which is located within a loop of the contact interface of the heterotypic interaction with Nectl2, undergoes an order-disorder transition. On the contrary, even though sDE2 and sDE3 are flexible and also located within loops, they do not undergo order-disorder transitions. We evaluated the immunogenicity of sDE1 and sDE3 through the expression of these epitopes in chimeric L1 virus-like particles. We confirmed that sDE1 induces polyclonal antibodies that recognize the native folding of CRTAM expressed in activated murine CD4 T cells. In contrast, sDE3 induces polyclonal antibodies that recognize the recombinant protein hCRTAM-Fc, but not the native CRTAM. Thus, in this study, an exposed disordered epitope in the Ig-like V domain of CRTAM was identified as a potential site for therapeutic antibodies.


Subject(s)
Antibodies, Blocking/metabolism , Cell Adhesion Molecule-1/metabolism , Epitopes/immunology , Immunoglobulins/immunology , Animals , Antibodies/immunology , CD4-Positive T-Lymphocytes/immunology , Cells, Cultured , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Protein Binding , Rabbits
9.
Cell Immunol ; 358: 104235, 2020 12.
Article in English | MEDLINE | ID: mdl-33137645

ABSTRACT

Antibodies, T cell receptors and major histocompatibility complex molecules are members of the immunoglobulin superfamily and have pivotal roles in the immune system. The fine interrelation between them regulates several immune functions. Here, we describe lesser-known functions ascribed to these molecules in generating and maintaining immune response. Particularly, we outline the contribution of antibody- and T cell receptor-derived complementarity-determining region neoantigens, antigenized antibodies, as well as major histocompatibility complex class I molecules-derived epitopes to the induction of protective/therapeutic immune responses against pathogens and cancer. We discuss findings of our own and other studies describing protective mechanisms, based on immunogenic properties of immunoglobulin superfamily members, and evaluate the perspectives of application of this class of immunogens in molecular vaccines design.


Subject(s)
Antibodies/immunology , Major Histocompatibility Complex/immunology , Receptors, Antigen, T-Cell/immunology , Amino Acid Sequence/genetics , Animals , Antibody Formation , Epitopes/immunology , Histocompatibility Antigens Class I/immunology , Humans , Immunity/immunology , Immunoglobulin Heavy Chains , Immunoglobulin Variable Region , Immunoglobulins/immunology , Immunoglobulins/metabolism
10.
Vet Microbiol ; 247: 108780, 2020 Aug.
Article in English | MEDLINE | ID: mdl-32768226

ABSTRACT

Artificial insemination and in vitro embryo production are increasingly used to improve the reproductive efficiency of herds, however success of these techniques depends on the sanitary quality of the semen. Insemination centers commonly use antibiotics in their routine procedure, but they are not able against viruses. In this paper, we demonstrate a new approach for disinfecting virus in bovine semen using photoimmunoinactivation, an adaptation of the photodynamic inactivation (PDI) methodology. The photosensitizers (PSs), hematoporphyrin (HP) and zinc tetracarboxy-phthalocyanine (ZnPc) were conjugated to Immunoglobulin Y (IgY) anti-bovine alphaherpesvirus 1 (BoHV-1) and used for PDI against the BoHV-1 viruses in cell culture and compared to the unconjugated PSs. Both treatments proved to be efficient, but a significant decrease in the irradiation time required to completely eliminate the virus was observed in the samples treated with the immunoconjugates. Photophysical measurements help us to understand the coupling between PSs and IgY and the evaluated production of singlet oxygen. Following the cell culture test, the same approach was applied in semen artificially infected with BoHV-1. The immunoconjugates were also efficient for complete virus inactivation up to 5 min of irradiation and proved to be safe using several parameters of sperm viability, demonstrating the feasibility of our strategy for disinfection viruses in semen.


Subject(s)
Cattle Diseases/prevention & control , Herpesvirus 1, Bovine/radiation effects , Immunoconjugates/pharmacology , Photosensitizing Agents/pharmacology , Semen/virology , Virus Inactivation , Animals , Cattle , Cattle Diseases/virology , Chickens , Female , Hematoporphyrins/pharmacology , Herpesvirus 1, Bovine/physiology , Immunoglobulins/immunology , Indoles/pharmacology , Isoindoles , Male , Organometallic Compounds/pharmacology , Photochemical Processes , Spermatozoa/drug effects , Virus Inactivation/drug effects , Virus Inactivation/radiation effects , Zinc Compounds
11.
Protein J ; 39(3): 224-231, 2020 06.
Article in English | MEDLINE | ID: mdl-32300914

ABSTRACT

Class-I restricted T cell-associated molecule (CRTAM) is a member of the immunoglobulin superfamily, and it is closely related to nectin-like protein. CRTAM is expressed in activated CD8 T cells, NKT cells, NK cells and in a subpopulation CD4 T cells. In this study, we produce as recombinant proteins, the Ig-domains of CRTAM (IgV-IgC), the IgV, and the IgC. These proteins were successfully purified in the soluble fraction only if the stalk region was included. The recombinant CRTAM recognizes its ligand nectin-like 2 in a cell-free system. We also demonstrate that the IgC domain of CRTAM is recognized by the anti-hCRTAM monoclonal antibody C8 with a 0.62 nM affinity. In conclusion, the stalk region of CRTAM provides solubility for the expression of its Ig-domains as recombinant proteins.


Subject(s)
Cell Adhesion Molecule-1/genetics , Cell-Free System/chemistry , Immunoglobulin Domains/genetics , Immunoglobulins/genetics , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/chemistry , Binding Sites , Cell Adhesion Molecule-1/immunology , Cell Adhesion Molecule-1/metabolism , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Female , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Hybridomas/chemistry , Immunoglobulins/immunology , Immunoglobulins/metabolism , Jurkat Cells , Mice , Mice, Inbred BALB C , Protein Binding , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism
12.
Electron. j. biotechnol ; Electron. j. biotechnol;44: 6-13, Mar. 2020. tab, graf, ilus
Article in English | LILACS | ID: biblio-1087627

ABSTRACT

BACKGROUND: Hot start can greatly improve specificity, sensitivity and yield of PCR. Non-specific amplification can occur in PCR when reaction mixture is prepared at room temperature, because Taq DNA polymerase is active and the primers can hybridize non-specifically. Hot start Taq DNA polymerases remain inactive at room temperature and are activated after heating at 95°C preventing non-specific amplification. Monoclonal antibodies against Taq DNA polymerase is the first line of reagents used for turn on regular Taq DNA polymerase into Hot start one. The goal of this research was to produce and evaluate Hot Start antibodies derived from chicken eggs. RESULTS: We performed affinity purification of yolk immunoglobulin (IgY) and obtained polyclonal Hot Start antibodies. The yield of specific antibodies was 0.36 mg per egg or 0.2% of total yolk antibodies. The protocol for real time measurement and Hot start IgY activity assessment was developed. We found that Hot start IgY can reversibly block Taq DNA polymerase activity at 50°C and have no negative impact neither on the Taq DNA polymerase activity after denaturation nor on the reverse transcriptase. We estimated that 1.0 µg of Hot start IgY effectively blocks 5 U activity of Taq DNA polymerase. CONCLUSIONS: Egg derived Hot Start polyclonal antibodies are the cheapest source of Hot start antibodies, from one immune egg we can isolate 0.36 mg IgY, this quantity is enough for producing 1800 U activity of Hot start Taq DNA Polymerase.


Subject(s)
Egg Yolk/metabolism , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/immunology , Temperature , Immunoglobulins/isolation & purification , Immunoglobulins/biosynthesis , Immunoglobulins/immunology , Enzyme-Linked Immunosorbent Assay , Polymerase Chain Reaction , Taq Polymerase , Egg Yolk/immunology , Antibodies, Monoclonal/isolation & purification
13.
J Pharm Biomed Anal ; 175: 112778, 2019 Oct 25.
Article in English | MEDLINE | ID: mdl-31352171

ABSTRACT

Toxoplasma gondii is an intracellular protozoan parasite responsible for toxoplasmosis, which affects humans and animals. Serologic detection of anti-T. gondii immunoglobulins plays a crucial role in the clinical diagnosis of toxoplasmosis. In this work, a novel electrochemical immunosensor for detecting anti-T. gondii immunoglobulins is reported, based on immobilization of an in silico predicted peptide (PepB3), obtained from membrane protein of T. gondii, on the graphite electrode modified with poly(3-hydroxybenzoic acid). Indirect ELISA confirmed infection and binding specificity of peptide PepB3. Molecular modelling and simulations show this peptide binds to the T. gondii human Fab antibody in the surface antigen 1 (SAG1) binding site, remaining a stable complex during the molecular dynamic simulations, especially by hydrogen bonds and hydrophobic interactions. This electrochemical immunosensor was able to discriminate different periods of infection, using infected mouse serum samples, showing selectivity and discriminating infected and uninfected mouse serum.


Subject(s)
Antibodies, Protozoan/immunology , Immunoglobulins/immunology , Peptides/immunology , Toxoplasma/immunology , Toxoplasmosis/immunology , Animals , Antigens, Protozoan/immunology , Enzyme-Linked Immunosorbent Assay/methods , Female , Mice , Protozoan Proteins/immunology , Sensitivity and Specificity
14.
Parasitol Int ; 72: 101933, 2019 Oct.
Article in English | MEDLINE | ID: mdl-31128257

ABSTRACT

Due to the epidemiological problem of the neglected condition of human strongyloidiasis, rapid and effective diagnosis is extremely important, with the development of new diagnostic tools being essential to reduce infections and chronic cases. Avian immunoglobulin Y (IgY) technology is an alternative for antibody production that has high specificity and profitability. This study aimed to produce and fractionate IgY antibodies from the egg yolks of hens that were immunized with the total antigenic extracts of Strongyloides venezuelensis infectious filariform larvae (iL3) and parthenogenetic females (pF). IgY antibodies were then evaluated by their recognition of antigenic proteins, evolutive helminth forms, and serological diagnosis of human strongyloidiasis by the detection of immune complexes in serum samples. Egg yolks were fractionated to obtain IgY antibodies by thiophilic interaction chromatography. Immune complex detection in serum samples showed diagnostic values for anti-iL3 IgY and anti-pF IgY antibodies at 95.56% and 88.89% sensitivity and 95.56% and 91.11% specificity, respectively. Therefore, IgY technology is a promising tool for the detection of blood circulating Strongyloides antigens, with possible application as a serological diagnostic method.


Subject(s)
Immunoglobulins/immunology , Immunologic Tests/methods , Strongyloides/immunology , Strongyloidiasis/diagnosis , Animals , Antibodies, Helminth/blood , Antigens, Helminth , Chickens , Egg Yolk , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunoglobulin G/blood , Larva/immunology , Sensitivity and Specificity , Serologic Tests , Strongyloidiasis/immunology
15.
Toxins (Basel) ; 11(1)2019 01 17.
Article in English | MEDLINE | ID: mdl-30658385

ABSTRACT

Fumonisins are a group of toxic secondary metabolites that are produced by Fusarium verticillioides which are associated with poultry health hazard and great economic losses. The objective of the present study was to develop an immunological method to detect F. verticillioides in poultry feed samples. An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on a polyclonal antibody against 67 kDa protein of the F. verticillioides 97K exoantigen was developed to detect this fungus. Antibody anti-67 kDa protein showed cross-reactivity against F. graminearum (2⁻7%) and F. sporotrichioides (10%), but no or low cross-reactivity against Aspergillus sp. and Penicillium sp. exoantigens. The detection limit for the 67 kDa protein of F. verticillioides was 29 ng/mL. Eighty-one poultry feed samples were analyzed for Fusarium sp. count, 67 kDa protein of F. verticillioides and fumonisin concentrations. Eighty of the 81 feed samples (98.6%) showed Fusarium sp. contamination (mean 6.2 x 104 CFU/g). Mean 67 kDa protein and fumonisin concentration in the poultry feed samples was 21.0 µg/g and 1.02 µg/g, respectively. The concentration of 67 kDa protein, as determined by ic-ELISA correlated positively (p < 0.05) with fumonisin levels (r = 0.76). These results suggest that this ic-ELISA has potential to detect F. verticillioides and predict fumonisin contamination in poultry feed samples.


Subject(s)
Animal Feed/analysis , Enzyme-Linked Immunosorbent Assay/methods , Food Contamination/analysis , Fumonisins/analysis , Fungal Proteins/analysis , Fusarium/isolation & purification , Animals , Immunoglobulins/immunology , Poultry
16.
Fish Shellfish Immunol ; 87: 51-61, 2019 Apr.
Article in English | MEDLINE | ID: mdl-30599256

ABSTRACT

The biochemical mechanisms involved in phagocytosis and the intracellular survival of Aeromonas hydrophila (Ah) in host macrophages (MΦs) are complex processes that affect infection success or failure. Thus, in the present study, we described the in vitro infection of Nile tilapia MΦs by a homologous bacterium and tested the effects of anti-A. hydrophila immunoglobulin Y (IgY) on the phagolysosomal activity and intracellular survival of the pathogen. The anti-Ah IgY modulated lysosomal acid phosphatase (LAP) activity as well as the production of reactive oxygen intermediates (ROIs) and nitric oxide (NO), thereby potentiating phagocytosis and the elimination of Ah. Thus, we assume that the specific IgY had a beneficial effect on infection control and postulated the use of the Nile tilapia MΦs as an important in vitro experimental model for the functional and therapeutic study of Ah infection.


Subject(s)
Aeromonas hydrophila/physiology , Cichlids/immunology , Cichlids/microbiology , Fish Diseases/microbiology , Gram-Negative Bacterial Infections/veterinary , Macrophages/microbiology , Acid Phosphatase/metabolism , Animals , Fish Diseases/immunology , Gram-Negative Bacterial Infections/immunology , Gram-Negative Bacterial Infections/microbiology , Immunoglobulins/immunology , In Vitro Techniques , Macrophages/immunology , Nitric Oxide/metabolism , Phagocytosis , Reactive Oxygen Species/metabolism
17.
Cell Immunol ; 334: 20-30, 2018 12.
Article in English | MEDLINE | ID: mdl-30170688

ABSTRACT

This work aims to study the immunomodulation of B lymphocytes during L. amazonensis infection. We demonstrated in this study that follicular B cells from draining lymph nodes of infected wild type BALB/c mice are the major source of IL-10 during infection. We infected BALB/Xid mice that developed smaller lesions in comparison with the control, but the parasite load obtained from the infected tissues was similar in both groups. We observed a reduction in the number of follicular B cells from BALB/Xid mice in relation to WT mice and, consequently, lower levels of IgM, IgG, IgG1, IgG2a and IgG2b in the serum of BALB/Xid when compared with wild type mice. BALB/Xid mice also presented lower levels of IL-10 in the infected footpad, draining lymph nodes and in the spleen when compared with WT infected tissues. We did not detect differences in the number of IL-10 producing CD4+ and CD8+ T cells between WT and BALB/Xid mice; however, a strong reduction of IL-10 producing follicular B cells was noted in BALB/Xid mice. When analyzed together, our data indicate that B cells are related with lesion pathogenesis through the production of antibodies and IL-10.


Subject(s)
B-Lymphocytes/immunology , Immunomodulation/immunology , Interleukin-10/immunology , Leishmania mexicana/immunology , Leishmaniasis, Cutaneous/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/parasitology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/parasitology , Immunoglobulins/immunology , Leishmaniasis, Cutaneous/parasitology , Lymph Nodes/immunology , Mice , Mice, Inbred BALB C , Skin/immunology , Skin/parasitology , Spleen/immunology , Spleen/parasitology
18.
Biomedica ; 38(2): 232-243, 2018 06 15.
Article in English | MEDLINE | ID: mdl-30184353

ABSTRACT

Introduction: The use of specific antibodies capable of detecting allergens of the group 1 of house dust mites represents a potential strategy to reduce exposure and clinical symptomatology associated with asthma and allergic rhinitis. Objective: To produce and purify chicken antibodies specific for the dust mites Dermatophagoides sp. and B. tropicalis using the IgY technology. Materials and methods: We designed and synthesized oligopeptides showing immunogenic epitopes of Der p1, Der f1, and Blo t1. These were used to produce IgY antibodies in Hy Line Brown chickens. IgY were extracted from egg yolk using thiophilic chromatography. The immunogenicity and specificity were assayed by indirect ELISA and Dot Blot. Results: We obtained high reactivity of IgY antibodies against epitopes of allergens present in whole body mites extracts of D. farinae, D. pteronyssinus, and B. tropicalis. The highest IgY levels were registered between days 32 and 40 after immunization. The antibodies showed high immunoreactivity and specificity towards D. farinae proteins with detection limits above 0.03 µg of mite proteins under the experimental conditions used. Purified IgY did not show significant reactivity when binding to Periplaneta americana extract. Conclusion: The IgY technology allowed the production of specific antibodies against house dust mites group 1 allergens using non-glycosylated synthetic peptides. To our knowledge, this is the first time that this immunochemicals are used in the detection of mites of medical relevance.


Subject(s)
Antibodies/immunology , Antigens, Dermatophagoides/immunology , Immunoglobulins/immunology , Oligopeptides/immunology , Pyroglyphidae/immunology , Animals , Chickens
19.
J Immunol Methods ; 461: 100-105, 2018 10.
Article in English | MEDLINE | ID: mdl-30158073

ABSTRACT

Influenza A virus (IAV) causes an important respiratory disease in mammals and birds leading to concerns in animal production industry and public health. Usually, antibodies produced in mammals are employed in diagnostic tests. However, due to animal welfare concerns, technical advantages and the high cost of production, alternatives to the production of antibodies in mammals have been investigated. The aim of this study was to produce egg yolk immunoglobulin (IgY) in laying hens against a highly conserved protein (nucleoprotein- NP) of IAV and to evaluate the application of anti-NP IgY antibodies in virus detection by immunocytochemistry (ICC) and immunohistochemistry (IHC). Three laying hens of the White Leghorn line were inoculated seven times with a recombinant NP protein and their eggs collected seven days after the 3rd, 5th and 7th inoculations. Immunoglobulin Y antibodies were purified from egg yolk through precipitation with ammonium sulfate. The titers and specificity of the purified antibodies were determined by ELISA, western blotting, ICC and IHC. High levels of specific anti-NP antibodies were detected by ELISA after the 5th inoculation, reaching a peak after the 7th inoculation. The mean yield of total protein in yolk after the 7th inoculation was 13.5 mg/mL. The use of western blotting and ICC demonstrated that anti-NP IgY binds specifically to NP protein. Moreover, the use of anti-NP IgY antibody in ICC test revealed positive staining of MDCK cells infected with IAV of the three subtypes circulating in swine (H1N1, H1N2, and H3N2). However, no staining was observed in lung tissues through the IHC test. The data obtained showed that anti-NP IgY antibodies bound specifically to influenza virus NP protein, detecting the main virus subtypes circulating in swine, reinforcing their usefulness in the influenza diagnosis.


Subject(s)
Antibodies, Viral , Immunoglobulins , Influenza A virus , Orthomyxoviridae Infections , Swine Diseases , Viral Core Proteins , Animals , Antibodies, Viral/chemistry , Antibodies, Viral/immunology , Chickens/immunology , Dogs , Enzyme-Linked Immunosorbent Assay/methods , Humans , Immunoglobulins/chemistry , Immunoglobulins/immunology , Influenza A virus/immunology , Influenza A virus/metabolism , Madin Darby Canine Kidney Cells , Orthomyxoviridae Infections/blood , Orthomyxoviridae Infections/immunology , Orthomyxoviridae Infections/veterinary , Swine , Swine Diseases/blood , Swine Diseases/immunology , Swine Diseases/virology , Viral Core Proteins/blood , Viral Core Proteins/immunology
20.
J Immunoassay Immunochem ; 39(3): 235-248, 2018.
Article in English | MEDLINE | ID: mdl-30044696

ABSTRACT

Immunoglobulin Y (IgY), an antibody present in birds, reptiles, and amphibians, is actively transported from the serum to egg yolks, where it is stored in large quantities. The use of chicken polyclonal IgY instead of mammalian IgG antibodies for biomedical applications has ethical and economic advantages, such as the lack of a need for animal bleeding because the antibodies are extracted from eggs after hen immunization and the low cost of the production and purification methods. This article reviews the latest IgY applications in diagnostic virology and the therapeutic use of IgY in viral gastroenteritis.


Subject(s)
Chickens/immunology , Gastroenteritis/drug therapy , Gastroenteritis/virology , Immunoglobulins/immunology , Virology/methods , Animals , Gastroenteritis/immunology , Immunoglobulins/economics , Immunoglobulins/isolation & purification , Immunoglobulins/therapeutic use
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