RESUMO
BACKGROUND: Monitoring and analysing the infection rates of the vector of Trypanosoma cruzi, that causes Chagas disease, helps assess the risk of transmission. OBJECTIVES: A study was carried out on triatomine in the State of Paraná, Brazil, between 2012 and 2021 and a comparison was made with a previous study. This was done to assess the risk of disease transmission. METHODS: Ecological niche models based on climate and landscape variables were developed to predict habitat suitability for the vectors as a proxy for risk of occurrence. FINDINGS: A total of 1,750 specimens of triatomines were recorded, of which six species were identified. The overall infection rate was 22.7%. The areas with the highest risk transmission of T. cruzi are consistent with previous predictions in municipalities. New data shows that climate models are more accurate than landscape models. This is likely because climate suitability was higher in the previous period. MAIN CONCLUSION: Regardless of uneven sampling and potential biases, risk remains high due to the wide presence of infected vectors and high environmental suitability for vector species throughout the state and, therefore, improvements in public policies aimed at wide dissemination of knowledge about the disease are recommended to ensure the State remains free of Chagas disease.
Assuntos
Doença de Chagas , Insetos Vetores , Triatominae , Trypanosoma cruzi , Doença de Chagas/transmissão , Animais , Insetos Vetores/classificação , Insetos Vetores/parasitologia , Brasil/epidemiologia , Triatominae/classificação , Triatominae/parasitologia , Humanos , Fatores de Risco , Medição de Risco , EcossistemaRESUMO
Bovine cysticercosis is detected during the routine post mortem examination of carcasses by visual inspection (knife and eye method). However, the sensitivity of this procedure is several times lower than immunoassays, even when it is performed by qualified professionals. In the present study, a new generation capture antigens were screened from a phage display peptide library using antibodies from Taenia saginata-infected animals. Eight phage clones were selected, and one, Tsag 3 (VHTSIRPRCQPRAITPR), produced similar results to the T. saginata metacestode crude antigen (TsCa) when used as a capture antigen in an ELISA. The phage-displayed peptides competed with TsCa for binding sites, reducing the reactivity by approximately 30 %. Alanine scanning indicated that proline, arginine, and serine are important residues for antibody binding. Tsag 1 (HFYQITWLPNTFPAR), the most frequent affinity-selected clone, and Tsag 6 (YRWPSTPSASRQATL) shared similarity with highly conserved proteins from the Taeniidae family with known immunogenicity. Due to their epitopic or mimotopic properties, these affinity-selected phages could contribute to the rational design of an ante mortem immunodiagnosis method for bovine cysticercosis, as well as an epitope-based vaccine to interrupt the taeniosis/cysticercosis complex.
Assuntos
Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos , Doenças dos Bovinos/diagnóstico , Técnicas de Visualização da Superfície Celular/métodos , Taenia saginata/imunologia , Teníase/veterinária , Animais , Antígenos de Helmintos/genética , Bovinos , Proteínas Recombinantes/genética , Teníase/diagnósticoRESUMO
In the Americas and specially in Brazil, the Loxosceles intermedia, Loxosceles gaucho and Loxosceles laeta are the three most medically relevant brown spider species, and whose bites can lead to the condition known as loxoscelism. Here, we report the development of a tool capable of identifying a common epitope amongst Loxosceles sp. venom's toxins. A murine monoclonal antibody (LmAb12) and its recombinant fragments (scFv12P and diabody12P) have been produced and characterized. This antibody and its recombinant constructs were able to recognize proteins of Loxosceles spider venoms with specificity. The scFv12P variant was also able to detect low concentrations of Loxosceles venom in a competitive ELISA assay, displaying potential as a venom identification tool. The primary antigenic target of LmAb12 is a knottin, a venom neurotoxin, that has a shared identity of 100 % between the L. intermedia and L. gaucho species and high similarity to L. laeta. Furthermore, we observed LmAb12 was able to partially inhibit in vitro hemolysis, a cellular event typically induced by the Loxosceles sp. venoms. Such behavior might be due to LmAb12 cross-reactivity between the antigenic target of LmAb12 and the venom's dermonecrotic toxins, the PLDs, or even the existence of synergism between these two toxins.
Assuntos
Venenos de Aranha , Aranhas , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Antígenos/química , Antivenenos/química , Reações Cruzadas , Miniproteínas Nó de Cistina/química , Fosfolipase D/química , Venenos de Aranha/química , Aranhas/química , Epitopos/químicaRESUMO
We reconstructed a transcriptional regulatory network for adrenocortical carcinoma (ACC) using transcriptomic and clinical data from The Cancer Genome Atlas (TCGA)-ACC cohort. We investigated the association of transcriptional regulatory units (regulons) with overall survival, molecular phenotypes, and immune signatures. We annotated the ACC regulons with cancer hallmarks and assessed single sample regulon activities in the European Network for the Study of Adrenal Tumors (ENSAT) cohort. We found 369 regulons associated with overall survival and subdivided them into four clusters: RC1 and RC2, associated with good prognosis, and RC3 and RC4, associated with worse outcomes. The RC1 and RC3 regulons were highly correlated with the 'Steroid Phenotype,' while the RC2 and RC4 regulons were highly correlated with a molecular proliferation signature. We selected two regulons, NR5A1 (steroidogenic factor 1, SF-1) and CENPA (Centromeric Protein A), that were consistently associated with overall survival for further downstream analyses. The CENPA regulon was the primary regulator of MKI-67 (a marker of proliferation KI-67), while the NR5A1 regulon is a well-described transcription factor (TF) in ACC tumorigenesis. We also found that the ZBTB4 (Zinc finger and BTB domain-containing protein 4) regulon, which is negatively associated with CENPA in our transcriptional regulatory network, is also a druggable anti-tumorigenic TF. We anticipate that the ACC regulons may be used as a reference for further investigations concerning the complex molecular interactions in ACC tumors.
RESUMO
Envenoming due to Loxosceles spider bites still remains a neglected disease of particular medical concern in the Americas. To date, there is no consensus for the treatment of envenomed patients, yet horse polyclonal antivenoms are usually infused to patients with identified severe medical conditions. It is widely known that venom proteins in the 30-35 kDa range with sphingomyelinase D (SMasesD) activity, reproduce most of the toxic effects observed in loxoscelism. Hence, we believe that monoclonal antibody fragments targeting such toxins might pose an alternative safe and effective treatment. In the present study, starting from the monoclonal antibody LimAb7, previously shown to target SMasesD from the venom of L. intermedia and neutralize its dermonecrotic activity, we designed humanized antibody V-domains, then produced and purified as recombinant single-chain antibody fragments (scFvs). These molecules were characterized in terms of humanness, structural stability, antigen-binding activity, and venom-neutralizing potential. Throughout this process, we identified some blocking points that can impact the Abs antigen-binding activity and neutralizing capacity. In silico analysis of the antigen/antibody amino acid interactions also contributed to a better understanding of the antibody's neutralization mechanism and led to reformatting the humanized antibody fragment which, ultimately, recovered the functional characteristics for efficient in vitro venom neutralization.
Assuntos
Anticorpos Monoclonais , Antivenenos , Anticorpos de Cadeia Única , Venenos de Aranha/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Antivenenos/administração & dosagem , Antivenenos/imunologia , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Modelos Moleculares , Testes de Neutralização , Anticorpos de Cadeia Única/administração & dosagem , Anticorpos de Cadeia Única/imunologia , Picada de Aranha/terapia , Venenos de Aranha/efeitos adversos , Aranhas/imunologiaRESUMO
Loxoscelism pose a health issue in the South America. The treatment for these accidents is based on the administration of antivenom produced in animals immunized with Loxosceles venom. In this work, a previously produced non-toxic multiepitopic chimeric protein (rMEPlox), composed of epitopes derived from the main toxins families (sphyngomielinase-D, metalloproteases, and hyaluronidases) of Loxosceles spider venoms, was used as antigen to produce monoclonal antibodies (mAbs). A selected anti-rMEPlox mAb (Lox-mAb3) reacted with metalloprotease from L. intermedia venom and showed cross-reactivity with metalloproteses from Brazilian and Peruvian Loxosceles laeta and Loxosceles gaucho venoms in immunoassays. The sequence recognized by Lox-mAb3 (184ENNTRTIGPFDYDSIMLYGAY205) corresponds to the C-terminal region of Astacin-like metalloprotease 1 and the amino acid sequence IGPFDYDSI, conserved among the homologs metalloproteases sequences, is important for antibody recognition. Lox-mAb3 neutralizes the fibrinogenolytic activity caused by metalloprotease from L. intermedia spider venom in vitro, which may lead to a decrease in hemorrhagic disturbances caused by Loxosceles envenomation. Our results show, for the first time, the use of a non-toxic multiepitopic protein for the production of a neutralizing monoclonal antibody against a metalloprotease of medically important Loxosceles venoms. These results contribute for the production improvement of therapeutic antivenom against loxoscelism.
Assuntos
Anticorpos Monoclonais Murinos/imunologia , Anticorpos Neutralizantes/imunologia , Proteínas de Artrópodes , Epitopos , Metaloendopeptidases , Diester Fosfórico Hidrolases , Venenos de Aranha , Aranhas , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Feminino , Metaloendopeptidases/química , Metaloendopeptidases/genética , Metaloendopeptidases/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/imunologia , Engenharia de Proteínas , Venenos de Aranha/química , Venenos de Aranha/genética , Venenos de Aranha/imunologiaRESUMO
BACKGROUND Monitoring and analysing the infection rates of the vector of Trypanosoma cruzi, that causes Chagas disease, helps assess the risk of transmission. OBJECTIVES A study was carried out on triatomine in the State of Paraná, Brazil, between 2012 and 2021 and a comparison was made with a previous study. This was done to assess the risk of disease transmission. METHODS Ecological niche models based on climate and landscape variables were developed to predict habitat suitability for the vectors as a proxy for risk of occurrence. FINDINGS A total of 1,750 specimens of triatomines were recorded, of which six species were identified. The overall infection rate was 22.7%. The areas with the highest risk transmission of T. cruzi are consistent with previous predictions in municipalities. New data shows that climate models are more accurate than landscape models. This is likely because climate suitability was higher in the previous period. MAIN CONCLUSION Regardless of uneven sampling and potential biases, risk remains high due to the wide presence of infected vectors and high environmental suitability for vector species throughout the state and, therefore, improvements in public policies aimed at wide dissemination of knowledge about the disease are recommended to ensure the State remains free of Chagas disease.
RESUMO
AIM: To develop a monoclonal antibody against dehydroepiandrosterone (DHEA) and miniaturize it, generating a single-chain antibody variable fragment (scFv) against DHEA as an adrenocortical carcinoma (ACC) marker. MATERIAL & METHODS: DHEA conjugated to keyhole limpet hemocyanin was used as an immunogen to obtain anti-DHEA hybridomas. Variable fragments were cloned from hybridoma 5B7 total RNA, and used to detect DHEA in normal adrenal tissue and ACC cells. RESULTS: IgM monoclonal antibody was highly specific, and the recombinant scFv preserved parental antibody characteristics, allowing tissue localization of DHEA. CONCLUSION: Undefined small lesions are challenges for clinicians and impact clinical adrenocortical tumor management. Generating an anti-DHEA scFv facilitates development of imaging tests for early diagnosis of pediatric ACC.
Assuntos
Neoplasias do Córtex Suprarrenal/diagnóstico , Carcinoma Adrenocortical/diagnóstico , Biomarcadores Tumorais/análise , Desidroepiandrosterona/análise , Anticorpos de Cadeia Única/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Desidroepiandrosterona/metabolismo , Expressão Gênica , Humanos , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Conformação Proteica , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticorpos de Cadeia Única/genética , Zona Reticular/metabolismoRESUMO
The biotechnological evolution towards the development of antigens to detect leprosy has been progressing. However, the identification of leprosy in paucibacillary patients, based solely on the antigen-antibody interaction still remains a challenge. The complexity of clinical manifestations requires innovative approaches to improve the sensitivity of assays to detect leprosy before the onset of symptoms, thus avoiding disabilities and contributing, indirectly, to reduce transmission. In this study, the strategies employed for early leprosy diagnosis were: i. using a phage-displayed mimotope (APDDPAWQNIFNLRR) which mimics an immunodominant sequence (PPNDPAWQRNDPILQ) of an antigen of Mycobacterium leprae known as Ag85B; ii. engineering the mimotope by adding a C-terminal flexible spacer (SGSG-C); iii. conjugating the mimotope to a carrier protein to provide better exposure to antibodies; iv. amplifying the signal using biotin-streptavidin detection system in an ELISA; and v. coating the optimized mimotope on a quartz crystal microbalance (QCM) sensor for label-free biosensing. The ELISA sensitivity increased up to 91.7% irrespective of the immunological profile of the 132 patients assayed. By using comparative modeling, the M. tuberculosis Ag85B was employed as a template to ascertain which features make the mimotope a good antigen in terms of its specificity. For the first time, a sensitive QCM-based immunosensor to detect anti M. leprae antibodies in human serum was used. M. leprae antibodies could also be detected in the sera of paucibacillary patients; thus, the use of a mimotope-derived synthetic peptide as bait for antibodies in a novel analytical label-free immunoassay for leprosy diagnosis exhibits great potential.
Assuntos
Técnicas Biossensoriais , Imunoensaio , Hanseníase/diagnóstico , Mycobacterium leprae/isolamento & purificação , Técnicas de Microbalança de Cristal de Quartzo , Adulto , Animais , Biomarcadores/análise , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Breast cancer is a heterogeneous disease with differences in its clinical, molecular and biological features. Traditionally, immunohistochemical markers together with clinicopathologic parameters are used to classify breast cancer and to predict disease outcome. Triple-negative breast cancer (TNBC) is a particular type of breast cancer that is defined by a lack of expression of hormonal receptors and the HER2 gene. Most cases of TNBC also have a basal-like phenotype (BLBC) with expression of cytokeratin 5/6 and/or EGFR. A basal marker alone is insufficient for a better understanding of the tumor biology of TNBC. In that regard, the ADAM33 gene is silenced by DNA hypermethylation in breast cancer, which suggests that ADAM33 might be useful as a molecular marker. In the present study, we have produced monoclonal antibodies against the ADAM33 protein and have investigated the role of ADAM33 protein in breast cancer. We used 212 breast tumor samples and lower levels of ADAM33 were correlated with TNBC and basal-like markers. A lower level of ADAM33 was also correlated with shorter overall survival and metastasis-free survival and was considered an independent prognostic factor suggesting that ADAM33 is a novel molecular biomarker of TNBC and BLBC that might be useful as a prognostic factor.
Assuntos
Proteínas ADAM/genética , Biomarcadores Tumorais/genética , Prognóstico , Neoplasias de Mama Triplo Negativas/genética , Adulto , Idoso , Metilação de DNA/genética , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Células MCF-7 , Pessoa de Meia-Idade , Receptor ErbB-2/genética , Receptores de Estrogênio/genética , Neoplasias de Mama Triplo Negativas/epidemiologia , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Many processes have been used for recycling of carton packaging wastes. The pyrolysis highlights as a promising technology to be used for recovering the aluminum from polyethylene and generating products with high heating value. In this paper, a study on pyrolysis reactions of carton packaging wastes and its pure components was performed in order to estimate the kinetic parameters of these reactions. For this, dynamic thermogravimetric analyses were carried out and two different kinds of kinetic models were used: the isoconversional and Independent Parallel Reactions. Isoconversional models allowed to calculate the overall activation energy of the pyrolysis reaction, in according to their conversions. The IPR model, in turn, allowed the calculation of kinetic parameters of each one of the carton packaging and paperboard subcomponents. The carton packaging pyrolysis follows three separated stages of devolatilization. The first step is moisture loss. The second stage is perfectly correlated to devolatilization of cardboard. The third step is correlated to devolatilization of polyethylene.
Assuntos
Embalagem de Alimentos , Incineração/métodos , Termogravimetria , ResíduosRESUMO
Bites by spiders from Loxosceles genus often lead to a wide variance in envenomation profile of patients and diagnosis is difficult due to the number of diseases that mimic loxoscelism. In such a context, it is of interest to consider the design of standardized recombinant colorimetric antibodies for diagnosis and specific detection of individual circulating toxins in biological fluids of envenomed patients. We have previously prepared a monoclonal murine IgG (LiMab7) that reacts with Loxosceles intermedia venom components of 32-35kDa and neutralizes the dermonecrotic activity of the venom. Here, we re-engineered LiMab7 into a colorimetric bifunctional protein consisting in the corresponding single-chain antibody fragment (scFv) fused to alkaline phosphatase (AP) of Escherichia coli. The immune tracer was tested in two different types of immunoassays and it proved to be efficient in both. Thus, this recombinant fusion protein (scFv-LiMab7/AP) can be used for rapid and specific immunotitration of L. intermedia venom with a linear range of 39-20000ng/mL and a detection limit of 39ng/mL without any cross-reaction.
Assuntos
Aranha Marrom Reclusa/fisiologia , Imunoensaio/métodos , Neurotoxinas/análise , Diester Fosfórico Hidrolases/análise , Pele/metabolismo , Picada de Aranha/diagnóstico , Venenos de Aranha/análise , Fosfatase Alcalina/genética , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Diagnóstico Diferencial , Humanos , Camundongos , Neurotoxinas/imunologia , Diester Fosfórico Hidrolases/imunologia , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/metabolismo , Padrões de Referência , Sensibilidade e Especificidade , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Pele/patologia , Picada de Aranha/imunologia , Venenos de Aranha/imunologiaRESUMO
Loxosceles spider bites often lead to serious envenomings and no definite therapy has yet been established. In such a context, it is of interest to consider an antibody-based targeted therapy. We have previously prepared a murine monoclonal IgG (LiMab7) that binds to 32-35kDa components of Loxosceles intermedia venom and neutralizes the dermonecrotic activity of the venom. Here, we re-engineered LiMab7 into a recombinant diabody. The protein was produced in bacteria and then it was functionally characterized. It proved to be efficient at neutralizing sphingomyelinase and hemolytic activities of the crude venom despite the slightly altered binding kinetic constants and the limited stability of the dimeric configuration. This is the first report of a specific recombinant antibody for a next-generation of Loxosceles antivenoms.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antivenenos/uso terapêutico , Imunoterapia/métodos , Diester Fosfórico Hidrolases/imunologia , Proteínas Recombinantes/uso terapêutico , Anticorpos de Cadeia Única/uso terapêutico , Picada de Aranha/terapia , Venenos de Aranha/imunologia , Animais , Células Cultivadas , Expressão Gênica , Hemólise , Camundongos , Engenharia de Proteínas , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Picada de Aranha/imunologia , Aranhas/imunologiaRESUMO
Advanced Glycation End Products (AGEs) are toxins that are involved in structural and functional alterations of several organs and tissues, resulting in various pathologies. Several types of AGEs have been described but carboxymethyllysine (CML) is the major antigenic AGE compound. In this study, three different immunogenic carrier proteins (KLH, keyhole limpet hemocyanin; BSA, bovine serum albumin; and HSA, human serum albumin) were modified by glycation. The glycated molecules were used to produce epitope-specific monoclonal antibodies able to recognize the CML domain and to detect uremic toxins in the serum of patients with chronic kidney disease (CKD). A competitive ELISA was standardized in order to quantify CML in the sera of CKD patients. An increase in uremic toxins can compromise the clinical condition of these patients, thus, the detection and quantification of these toxins should contribute to a better management and understanding of this disease.
RESUMO
Advanced glycation end products (AGEs) are compounds classified as uremic toxins in patients with chronic kidney disease that have several pro-inflammatory effects and are implicated in the development of cardiovascular diseases. To explore the mechanisms of AGEs-endothelium interactions through the receptor for AGEs (RAGE) in the PKC-ß pathway, we evaluated the production of MCP-1 and VCAM-1 in human endothelial cells (HUVECs), monocytes, and a coculture of both. AGEs were prepared by albumin glycation and characterized by absorbance and electrophoresis. The effect of AGEs on cell viability was assessed with an MTT assay. The cells were also treated with AGEs with and without a PKC-ß inhibitor. MCP-1 and VCAM-1 in the cell supernatants were estimated by ELISA, and RAGE was evaluated by immunocytochemistry. AGEs exposure did not affect cell viability, but AGEs induced RAGE, MCP-1, and VCAM-1 expression in HUVECs. When HUVECs or monocytes were incubated with AGEs and a PKC-ß inhibitor, MCP-1 and VCAM-1 expression significantly decreased. However, in the coculture, exposure to AGEs and a PKC-ß inhibitor produced no significant effect. This study demonstrates, in vitro, the regulatory mechanisms involved in MCP-1 production in three cellular models and VCAM-1 production in HUVECs, and thus mimics the endothelial dysfunction caused by AGEs in early atherosclerosis. Such mechanisms could serve as therapeutic targets to reduce the harmful effects of AGEs in patients with chronic kidney disease.
Assuntos
Quimiocina CCL2/metabolismo , Produtos Finais de Glicação Avançada/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Proteína Quinase C beta/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células U937RESUMO
We have produced 13 mAbs for Loxosceles intermedia crude venom. Twelve were reactive against proteins of 32-35 kDa and one of these Li mAb(7) showed high neutralizing potency for the dermonecrotic activity of L. intermedia venom. This Li mAb(7) showed no cross-reactivity, with Loxosceles laeta (Brazil), L. laeta (Perú) and Loxosceles gaucho venoms. The mAbs were produced by immunization with the crude venom and screened by enzyme-linked immunosorbent assay (ELISA) using L. intermedia whole venom or dermonecrotic fraction (DNF) as antigens coated onto microtitre plates. A sensitive two-site immunometric assay was designed and shown to be useful for identifying and quantifying DNF from L. intermedia in biological samples. The Li mAb(7) coated onto microtitre plates and hyperimmune horse anti-L. intermedia IgGs prepared by immunoaffinity chromatography and conjugated to horseradish peroxidase were used to set up a sandwich-type ELISA. Measurable absorbance signals were obtained with 0.2 ng of L. intermedia crude venom per assay.
Assuntos
Anticorpos Monoclonais/farmacologia , Antivenenos/farmacologia , Venenos de Aranha/antagonistas & inibidores , Venenos de Aranha/imunologia , Aranhas , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Especificidade de Anticorpos , Antivenenos/química , Western Blotting , Ensaio de Imunoadsorção Enzimática , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Coelhos , Picada de Aranha/sangue , Picada de Aranha/imunologia , Venenos de Aranha/químicaRESUMO
Microcystins (MCs) are heptapeptide toxins produced by cyanobacteria. Their global occurrence in aquatic ecosystems has prompted the development of several detection methods, including antibody-based methods. Here, we propose to apply recombinant antibody technologies to the production of a bivalent colorimetric immunoprobe (scFv-AP) made of the so-called scFv fused to the alkaline phosphatase (AP) of Escherichia coli. Recombinant antibody technologies allow the development of specific probes with improved properties and suitable for the detection of MCs. The fusion protein was produced in the periplasm of recombinant bacteria and was used to develop a direct competitive enzyme immunoassay for specific detection of MCs without requiring further purification. The epitope recognized by the recombinant molecule was circumscribed to a motif common to all MCs. Such a genetic approach offers many advantages over chemical cross-linking of antibodies to colorimetric enzymes and may be adaptable to the analysis of water samples and in situ detection.
Assuntos
Fosfatase Alcalina/imunologia , Colorimetria/métodos , Cianobactérias/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas de Escherichia coli/imunologia , Microcistinas/análise , Proteínas Recombinantes/imunologia , Anticorpos de Cadeia Única/imunologia , Microbiologia da Água , Fosfatase Alcalina/genética , Corantes , Epitopos/imunologia , Proteínas Recombinantes/genética , Anticorpos de Cadeia Única/genéticaRESUMO
Dematiaceous fungi constitute a large and heterogeneous group, characterized by having a dark pigment, the dihydroxynaftalen melanin-DHN, inside their cell walls. In nature they are found mainly as soil microbiota or decomposing organic matter, and are spread in tropical and subtropical regions. The fungus Fonsecaea monophora causes chromoblastomycosis in humans, and possesses essential mechanisms that may enhance pathogenicity, proliferation and dissemination inside the host. Glycoconjugates confer important properties to these pathogenic microorganisms. In this work, structural characterization of glycan structures present in two different strains of F. monophora MMHC82 and FE5p4, from clinical and environmental origins, respectively, was performed. Each one were grown on Minimal Medium (MM) and Czapeck-Dox (CD) medium, and the water soluble cell wall glycoconjugates and exopolysaccharides (EPS) were evaluated by NMR, methylation and principal component analysis (PCA). By combining the methylation and 2D NMR analyses, it was possible to visualize the glycosidic profiles of the complex carbohydrate mixtures. Significant differences were observed in ß-D-Galf-(1â5) and (1â6) linkages, α- and ß-D-Glcp-(1â3), (1â4), and (1â6) units, as well as in α-D-Manp. PCA from (1)H-NMR data showed that MMHC82 from CD medium showed a higher variation in the cell wall carbohydrates, mainly related to O-2 substituted ß-D-Galf (δ 106.0/5.23 and δ 105.3/5.23) units. In order to investigate the antigenic response of the glycoconjugates, these were screened against serum from chromoblastomycosis patients. The antigen which contained the cell wall of MMHC82 grown in MM had ß-D-Manp units that promoted higher antigenic response. The distribution of these fungal species in nature and the knowledge of how cell wall polysaccharides and glycoconjugates structure vary, may contribute to the better understanding and the elucidation of the pathology caused by this fungus.
Assuntos
Antígenos de Fungos/química , Antígenos de Fungos/imunologia , Ascomicetos/imunologia , Polissacarídeos Fúngicos/química , Polissacarídeos Fúngicos/imunologia , Micoses/imunologia , Micoses/microbiologia , Anticorpos Antifúngicos/sangue , Anticorpos Antifúngicos/imunologia , Ascomicetos/crescimento & desenvolvimento , Ascomicetos/isolamento & purificação , Ascomicetos/metabolismo , Parede Celular/metabolismo , Cromoblastomicose/imunologia , Cromoblastomicose/microbiologia , Microbiologia Ambiental , Glicoconjugados/metabolismo , Humanos , Estrutura Molecular , Ressonância Magnética Nuclear BiomolecularRESUMO
Serum therapy remains the only specific treatment against envenoming, but anti-venoms are still prepared by fragmentation of polyclonal antibodies isolated from hyper-immunized horse serum. Most of these anti-venoms are considered to be efficient, but their production is tedious, and their use may be associated with adverse effects. Recombinant antibodies and smaller functional units are now emerging as credible alternatives and constitute a source of still unexploited biomolecules capable of neutralizing venoms. This review will be a walk through the technologies that have recently been applied leading to novel antibody formats with better properties in terms of homogeneity, specific activity and possible safety.
Assuntos
Anticorpos Neutralizantes , Antivenenos , Peçonhas/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Antivenenos/imunologia , Antivenenos/uso terapêutico , Mordeduras e Picadas/tratamento farmacológico , Humanos , Fragmentos de Imunoglobulinas/imunologia , Fragmentos de Imunoglobulinas/uso terapêutico , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/uso terapêuticoRESUMO
A chimeric protein (rCpLi) was constructed expressing three epitopes of rLiD1, a dermonecrotic toxin from the venom of Loxosceles intermedia spider. We have analyzed the neutralization potential of sera obtained by immunization of horses with rCpLi and rCpLi combined with initial doses of venoms and compared these with antivenom traditionally produced in horses using crude Loxosceles gaucho, Loxosceles laeta and L. intermedia venoms as antigens. We have demonstrated by ELISA that horses immunized with three initial doses of crude venom containing mixtures of L. intermedia, L. gaucho and L. laeta followed by nine doses of rCpLi generate antibodies with the same reactivity as those produced following immunization with traditional antivenom, towards the venoms of the three Loxosceles sp. species. Results from in vivo and in vitro neutralization assays showed that the new horse sera are able to neutralize the dermonecrotic activity of Loxosceles venoms, which are of medical importance in Brazil and some of these sera are capable of meeting the necessary potency requirements that could allow for their therapeutic use in humans. This immunization strategy combining both antigens used approximately 67% less crude Loxosceles venoms compared to traditional immunization protocol and can mean the development of Loxosceles antivenoms with the consequent reduction of devastation of arachnid fauna.