RESUMO
This is the first study aiming to determine the therapeutic effects of the Sambucus ebulus aquatic extract as an antileishmanial herbal drug and evaluate the immune responses in Leishmania major major infected BALB/c mice. The antileishmanial activity of S ebulus aquatic extract was evaluated using MTT test as well as parasite rescue and transformation assay. Footpad swelling and parasite load of infected mice were measured by several techniques. The immune responses were evaluated by measuring the levels of IFN-γ, IL-4, nitric oxide and arginase. The results indicated that S. ebulus can significantly decrease L. major promastigotes and amastigotes viability, but it was not toxic to macrophages. The lesion size, parasite burden and the level of ARG decreased in the treated infected mice, while the IFN-γ-to-IL-4 ratio and the level of NO increased significantly. Altogether, the S. ebulus extract is an effective compound for killing Leishmania parasite without excessive toxicity to the host cells and can cure the CL by switching the host immune responses towards Th1 response. Thus, it may be a perfect therapeutic option for CL treatment.
Assuntos
Leishmania major , Leishmaniose Cutânea/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Sambucus/química , Tripanossomicidas/uso terapêutico , Animais , Arginase/sangue , Linhagem Celular , Feminino , Humanos , Irã (Geográfico) , Leishmaniose Cutânea/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Camundongos Endogâmicos BALB C , Óxido Nítrico/sangue , Carga Parasitária , FitoterapiaRESUMO
Recent progress in hair follicle regeneration and alopecia treatment necessitates revisiting the concepts and approaches. In this sense, there is a need for shedding light on the clinical and surgical therapies benefitting from nanobiomedicine. From this perspective, this review attempts to recognize requirements upon which new hair therapies are grounded; to underline shortcomings and opportunities associated with recent advanced strategies for hair regeneration; and most critically to look over hair regeneration from nanomaterials and pluripotent stem cell standpoint. It is noteworthy that nanotechnology is able to illuminate a novel path for reprogramming cells and controlled differentiation to achieve the desired performance. Undoubtedly, this strategy needs further advancement and a lot of critical questions have yet to be answered. Herein, we introduce the salient features, the hurdles that must be overcome, the hopes, and practical constraints to engineer stem cell niches for hair follicle regeneration.
Assuntos
Alopecia/terapia , Cabelo/citologia , Regeneração , Células-Tronco/citologia , Engenharia Tecidual , Animais , Diferenciação Celular , Cabelo/fisiologia , HumanosRESUMO
One of the proposed approaches in cancer therapy is to induce and direct the patient's own immune system against cancer cells. In this study, we determined the epitope mapping of the rat anti-human epidermal growth factor receptor (EGFR) monoclonal antibody ICR-62 using a phage display of random peptide library and identified a 12 amino acids peptide, which was recognized as a mimotope. The peptide was synthesized and conjugated to bovine serum albumin (BSA) as carrier protein (P-BSA). We have shown that ICR-62 can react specifically with P-BSA as well as native EGFR. Two rabbits were immunized either by BSA or P-BSA and the rabbits IgGs were purified and examined for binding to the antigens, mimotope and the EGFR protein purified from the EGFR overexpressing A431 cell line. We showed that the rabbit IgG generated against the mimotope is capable of inhibiting the growth of A431 cells by 15%, but does not have any effect on the growth of EGFR-negative MDA-MB-453 cell line in vitro. Our results support the need for further investigations on the potential of vaccination with either mimotope of the EGFR or epitope displayed on the surface of phage particles for use in active immunotherapy of cancer.
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Anticorpos Monoclonais/farmacologia , Anticorpos Antineoplásicos/farmacologia , Vacinas Anticâncer/farmacologia , Mapeamento de Epitopos , Receptores ErbB/imunologia , Animais , Anticorpos Antineoplásicos/biossíntese , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Biblioteca de Peptídeos , Coelhos , Soroalbumina Bovina , VacinaçãoRESUMO
BACKGROUND: Cancer is still the most challenging disease and is responsible for many deaths worldwide. Considerable research now focuses on targeted therapy in cancer using natural components to improve anti-tumor efficacy and reduce unfavorable effects. Lactoferrin is an iron-binding glycoprotein found in body fluids. Increasing evidence suggests that lactoferrin is a safe agent capable of inducing anti-cancer effects. Therefore, we conducted a study to evaluate the effects of the exosomal form of bovine milk lactoferrin on a human MDA-MB-231 breast cancer cell line. METHODS: The exosomes were isolated from cancer cells by ultracentrifugation and incorporated with bovine milk lactoferrin through the incubation method. The average size of the purified exosome was determined using SEM imaging and DLS analysis. The maximum percentage of lactoferrin-loaded exosomes (exoLF) was achieved by incubating 1 mg/ml of lactoferrin with 30 µg/ml of MDA-MB-231 cells-derived exosomes. Following treatment of MDA-MB-231 cancer cells and normal cells with 1 mg/ml exoLF MTT assay applied to evaluate the cytotoxicity, PI/ annexin V analysis was carried out to illustrate the apoptotic phenotype, and the real-time PCR was performed to assess the pro-apoptotic protein, Bid, and anti-apoptotic protein, Bcl-2. RESULTS: The average size of the purified exosome was about 100 nm. The maximum lactoferrin loading efficiency of exoLF was 29.72%. MTT assay showed that although the 1 mg/ml exoLF treatment of MDA-MB-231 cancer cells induced 50% cell growth inhibition, normal mesenchymal stem cells remained viable. PI/ annexin V analysis revealed that 34% of cancer cells had late apoptotic phenotype after treatment. The real-time PCR showed an elevated expression of pro-apoptotic protein Bid and diminished anti-apoptotic protein Bcl-2 following exoLF treatment. CONCLUSION: These results suggested that exoLF could induce selective cytotoxicity against cancer cells compared to normal cells. Incorporating lactoferrin into the exosome seems an effective agent for cancer therapy. However, further studies are required to evaluate anti-tumor efficacy and the underlying mechanism of exoLF in various cancer cell lines and animal models.
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Neoplasias da Mama , Exossomos , Lactoferrina , Animais , Feminino , Humanos , Anexina A5/metabolismo , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Lactoferrina/farmacologia , Leite , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Designing biomaterials capable of biomimicking wound healing and skin regeneration has been receiving increasing attention recently. Some biopolymers behave similarly to the extracellular matrix (ECM), supporting biointerfacial adhesion and intrinsic cellular interactions. Polydopamine (PDA) is a natural bioadhesive and bioactive polymer that endows high chemical versatility, making it an exciting candidate for a wide range of biomedical applications. Moreover, biomaterials based on PDA and its derivatives have near-infrared (NIR) absorption, excellent biocompatibility, intrinsic antioxidative activity, antibacterial activity, and cell affinity. PDA can regulate cell behavior by controlling signal transduction pathways. It governs the focal adhesion behavior of cells at the biomaterials interface. These features make melanin-like PDA a fascinating biomaterial for wound healing and skin regeneration. This paper overviews PDA-based biomaterials' synthesis, properties, and interactions with biological entities. Furthermore, the utilization of PDA nano- and microstructures as a constituent of wound-dressing formulations is highlighted.
Assuntos
Materiais Biocompatíveis , Polímeros , Materiais Biocompatíveis/farmacologia , Materiais Biocompatíveis/uso terapêutico , Indóis/química , Indóis/farmacologia , Polímeros/farmacologia , Polímeros/uso terapêutico , CicatrizaçãoRESUMO
A severe complication associated with diabetes is diabetic foot ulcer (DFU). Most patients with DFU require amputation. Although treatment of non-healing diabetic ulcers is challenging, the use of novel therapies can be effective. In this report, we present the case of a woman with type 2 diabetes with DFU-related osteomyelitis, who was treated with a combination therapy of trichloroacetic acid, calcium alginate and foam dressings, human autologous fibroblast injection, and a fibroblast cell-seeded collagen scaffold. The results showed the positive effects of combination therapy on DFU. In the initial treatment, the wound area was measured to be 14 × 7 cm2 , with a depth of 4 cm. After 6 months, the wound was measured to be 1.5 cm2 , showing a 90% reduction of the wound area. Overall, this combination therapy was highly effective in the treatment of DFU-related osteomyelitis, and could markedly prevent amputation among DFU patients.
Assuntos
Diabetes Mellitus Tipo 2/complicações , Pé Diabético/terapia , Fibroblastos/transplante , Osteomielite/terapia , Alicerces Teciduais , Ácido Tricloroacético/uso terapêutico , Adulto , Colágeno , Terapia Combinada , Pé Diabético/etiologia , Feminino , Humanos , Osteomielite/etiologiaRESUMO
Tissue engineering intends to create functionalized tissues/organs for regenerating the injured parts of the body using cells and scaffolds. A scaffold as a supporting substrate affects the cells' fate and behavior, including growth, proliferation, migration, and differentiation. Hydrogel as a biomimetic scaffold plays an important role in cellular behaviors and tissue repair, providing a microenvironment close to the extracellular matrix with adjustable mechanical and chemical features that can provide sufficient nutrients and oxygen. To enhance the hydrogel performance and compatibility with native niche, the cell-laden hydrogel is an attractive choice to mimic the function of the targeted tissue. Injectable hydrogels, due to the injectability, are ideal options for in vivo minimally invasive treatment. Cell-laden injectable hydrogels can be utilized for tissue regeneration in a noninvasive way. This article reviews the recent advances and future opportunities of cell-laden injectable hydrogels and their functions in tissue engineering. It is expected that this strategy allows medical scientists to develop a minimally invasive method for tissue regeneration in clinical settings. Impact statement Cell-laden hydrogels have been vastly utilized in biomedical application, especially tissue engineering. It is expected that this upcoming review article will be a motivation for the community. Although this strategy is still in its early stages, this concept is so alluring that it has attracted all scientists in the community and specialists at academic health centers. Certainly, this approach requires more development, and a bunch of crucial challenges have yet to be solved. In this review, we discuss this various aspects of this approach, the questions that must be answered, the expectations associated with it, and rational restrictions to develop injectable cell-laden hydrogels.
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Hidrogéis , Engenharia Tecidual , Diferenciação Celular , Matriz ExtracelularRESUMO
The ability of mesenchymal stem cells (MSCs) to enhance cutaneous wound healing has been well established. Extensive expansion of cells to reach sufficient cell numbers for regenerating tissues has always limited cell-based therapies. An ingenious solution to address this challenge is to develop a strategy to increase the immunomodulatory effects of MSCs without expanding them. In this study, we employed a simple characteristic of cells. It was observed that an optimized three-dimensional (3D) MSC culture in spheroid forms significantly improved their paracrine effects. An electrospray (ES) encapsulation apparatus was used to encapsulate individual or 3D spheroid MSCs into microscale alginate beads (microbeads). Furthermore, alginate microbeads were embedded in an injectable thermosensitive hydrogel matrix, which gels at skin temperature. The hydrogel fills and seals the wounds cavities, maintains high humidity at the wound area, absorbs exudate, and fixes microbeads, protecting them from direct contact with the harsh wound environment. In vitro investigations revealed that secretion of interleukin 10 (IL-0) and transforming growth factor ß1 (TGF-ß1) gene was gradually enhanced, providing a delivery platform for prolonged release of bioactive molecules. In vivo study on full-thickness wounds showed granulation and re-epithelialization, only after 7 days. Moreover, increased expression of α-smooth muscle actin (α-SMA) in the first 14 days after treatment ensured wound contraction. Besides, a gradual decrease in α-SMA secretion resulted in reduced scar formation. Well-organized collagen fibrils and high expression of the angiogenesis biomarker CD31 confirmed the promoting effect of the hydrogel on the wound-healing process. The proposed wound-dressing system would potentially be used in scalable and effective cell-based wound therapies.
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Células-Tronco Mesenquimais , Hidrogéis , Regeneração , Pele , CicatrizaçãoRESUMO
Infection with Toxoplasma gondii causes serious health problems in congenitally-infected and immunocompromised individuals. Numerous studies have shown usefulness of dense granule antigens of T. gondii in serodiagnosis of the infection and induction of protective immunity. This study describes cloning, expression, purification and antigenicity evaluation of recombinant GRA7 protein (rGRA7). DNA encoding GRA7, amino acids 18 to 236, was obtained from Toxoplasma gondii RH strain by polymerase chain reaction amplification and cloned in prokaryotic expression plasmid pET-28b(+). Sequence analysis showed 97% similarity between GRA7 gene fragment and published sequence of gra7. Recombinant protein was expressed in Escherichia coli and purified in a single step by immobilized metal ion affinity chromatography. Antigenicity of the protein was evaluated in Western blot analysis showing human sera from acute T. gondii infection strongly reacted with rGRA7 while sera from chronic infection weakly recognized the protein. Negative sera failed to react with rGRA7. The antigenic rGRA7 might be used, in combination with other T. gondii antigen, to develop more efficacious diagnostic tests and/or in vaccine formulations.
Assuntos
Antígenos de Protozoários/biossíntese , Proteínas de Protozoários/biossíntese , Proteínas Recombinantes/biossíntese , Toxoplasma/imunologia , Animais , Especificidade de Anticorpos , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/isolamento & purificação , Escherichia coli , Humanos , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/isolamento & purificação , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
BACKGROUND: Dense granule antigens (GRA antigens) of Toxoplasma gondii induce strong antibody response in humans and are considered as useful diagnostic antigens. Previous studies reported expression of amino terminal GRA8 protein in fusion with large tags such as glutathione-S-transferase. The present study aimed to produce soluble full length immunogenic GRA8 in bacteria. METHODS: GRA8 complementary DNA (cDNA), encoding amino acids 24 to 258, was amplified from tachyzoites of RH strain and cloned in prokaryotic expression vector pET-28b(+). Expression of recombinant GRA8 (rGRA8) was analyzed by SDS-PAGE. Antigenicity and immunogenicity of the protein were evaluated by Western-blotting. RESULTS: The cloned gene fragment exhibited complete similarity with the published sequence of gra8 gene by sequence analysis. rGRA8 was expressed in Escherichia coli in fusion with a very small tag and the soluble protein was purified by immobilized metal ion affinity chromatography. In immunoblot, serum sample from a rabbit immunized with rGRA8 recognized a single antigen of T. gondii tachyzoite at the expected molecular weight of native GRA8. Sera from acutely-infected pregnant women strongly reacted with rGRA8 in Western-blotting, while sera from chronically-infected or T. gondii-negative women failed to recognize the protein. CONCLUSION: The full length soluble rGRA8 was successfully produced in E. coli and shown to be a highly immunogenic protein. As a result it could be used in immunological as well as molecular biology experiments.
Assuntos
Antígenos de Protozoários/biossíntese , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/isolamento & purificação , Transformação Bacteriana , Animais , Formação de Anticorpos , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/isolamento & purificação , Clonagem Molecular , Técnicas e Procedimentos Diagnósticos , Ensaio de Imunoadsorção Enzimática , Epitopos , Feminino , Humanos , Camundongos , Gravidez , Engenharia de Proteínas/métodos , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Coelhos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Toxoplasma/genética , Toxoplasma/imunologia , Toxoplasmose/sangue , Toxoplasmose/diagnóstico , Toxoplasmose/imunologiaRESUMO
Induced pluripotent stem (iPS) cells are generated from differentiated adult somatic cells by reprogramming them. Unlimited self-renewal, and the potential to differentiate into any cell type, make iPS cells very promising candidates for basic and clinical research. Furthermore, iPS cells can be genetically manipulated for use as therapeutic tools. DNA can be introduced into iPS cells, using lentiviral vectors, which represent a helpful choice for efficient transduction and stable integration of transgenes. In this study, we compare two methods of lentiviral transduction of iPS cells, namely, the suspension method and the hanging drop method. In contrast to the conventional suspension method, in the hanging drop method, embryoid body (EB) formation and transduction occur concurrently. The iPS cells were cultured to form EBs, and then transduced with lentiviruses, using the conventional suspension method and the hanging drop method, to express miR-128 and green fluorescent protein (GFP). The number of transduced cells were assessed by fluorescent microscopy and flow cytometry. MTT assay and real-time PCR were performed to determine the cell viability and transgene expression, respectively. Morphologically, GFP+ cells were more detectable in the hanging drop method, and this finding was quantified by flow cytometric analysis. According to the results of the MTT assay, cell viability was considerably higher in the hanging drop method, and real-time PCR represented a higher relative expression of miR-128 in the iPS cells introduced with lentiviruses in drops. Altogether, it seems that lentiviral transduction of challenging iPS cells using the hanging drop method offers a suitable and sufficient strategy in their gene transfer, with less toxicity than the conventional suspension method.
Assuntos
Biotecnologia/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Lentivirus/genética , Transdução Genética/métodos , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/citologiaRESUMO
Controlling cellular orientation, proliferation, and differentiation is valuable in designing organ replacements and directing tissue regeneration. In the present study, we developed a hybrid microfluidic system to produce a dynamic microenvironment by placing aligned PDMS microgrooves on surface of biodegradable polymers as physical guidance cues for controlling the neural differentiation of human induced pluripotent stem cells (hiPSCs). The neuronal differentiation capacity of cultured hiPSCs in the microfluidic system and other control groups was investigated using quantitative real time PCR (qPCR) and immunocytochemistry. The functionally of differentiated hiPSCs inside hybrid system's scaffolds was also evaluated on the rat hemisected spinal cord in acute phase. Implanted cell's fate was examined using tissue freeze section and the functional recovery was evaluated according to the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale. Our results confirmed the differentiation of hiPSCs to neuronal cells on the microfluidic device where the expression of neuronal-specific genes was significantly higher compared to those cultured on the other systems such as plain tissue culture dishes and scaffolds without fluidic channels. Although survival and integration of implanted hiPSCs did not lead to a significant functional recovery, we believe that combination of fluidic channels with nanofiber scaffolds provides a great microenvironment for neural tissue engineering, and can be used as a powerful tool for in situ monitoring of differentiation potential of various kinds of stem cells. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1534-1543, 2016.
Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Microfluídica/métodos , Neurônios/citologia , Animais , Comportamento Animal , Sobrevivência Celular , Feminino , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Imuno-Histoquímica , Ácido Láctico/química , Nanofibras/química , Nanofibras/ultraestrutura , Neurônios/transplante , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/terapia , Coloração e Rotulagem , Alicerces Teciduais/químicaRESUMO
Neurodegenerative diseases are one of the most challenging subjects in medicine. Investigation of their underlying genetic or epigenetic factors is hampered by lack of suitable models. Patient-specific induced pluripotent stem cells (iPS cells) represent a valuable approach to provide a proper model for poorly understood mechanisms of neuronal diseases and the related drug screenings. miR-124 and miR-128 are the two brain-enriched miRNAs with different time-points of expression during neuronal development. Herein, we transduced human iPS cells with miR-124 and miR-128 harboring lentiviruses sequentially. The transduced plasmids contained GFP and puromycin antibiotic-resistant genes for easier selection and identification. Morphological assessment and immunocytochemistry (overexpressions of beta-tubulin and neuron-specific enolase) confirmed that induced hiPS cells by miR-124 and miR-128 represent similar characteristics to those of mature neurons. In addition, the upregulation of neuron-specific enolase, beta-tubulin, Map2, GFAP, and BDNF was detected by quantitative real-time PCR. In conclusion, it seems that our novel protocol remarks the combinatorial effect of miR-124 and miR-128 on neural differentiation in the absence of any extrinsic factor. Moreover, such cellular models could be used in personalized drug screening and applied for more effective therapies.
Assuntos
Células-Tronco Pluripotentes Induzidas/fisiologia , MicroRNAs/administração & dosagem , Células-Tronco Neurais/fisiologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Neurônios/fisiologia , Diferenciação Celular/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , MicroRNAs/biossíntese , MicroRNAs/genética , Células-Tronco Neurais/citologia , Doenças Neurodegenerativas/tratamento farmacológico , Neurônios/citologia , Plasmídeos/administração & dosagem , Plasmídeos/genética , Transdução Genética/métodosRESUMO
Recombinant antigens are increasingly applied to replace native antigens in serological tests. Surface antigen 1 (SAG1) is a highly immunogenic antigen and probably represents the most explored and used antigen of Toxoplasma gondii for development of serological test kits. The presence of six disulfide bridges in its structure makes SAG1 a highly conformational protein. In fact, antigenicity of SAG1 is greatly dependent on proper disulfide bonding and folding. In-vitro refolding of SAG1 inclusion bodies, produced in Escherichia coli, was reported to result in soluble and antigenic protein. We produced SAG1 in E. coli and highly purified it by a single denaturing immobilized metal affinity chromatography. Refolding of denatured SAG1 was performed by (a) dialysis in the presence of reduced/oxidized glutathione, (b) drop-wise dilution and (c) drop-wise dilution in the presence of CuSo4. Refolding in the presence of oxido-shuffling reagent was much more efficient in producing presumably correctly-folded and highly antigenic SAG1 as demonstrated by non-reducing SDS-gel electrophoresis, ELISA, Western blotting and reversed-phase HPLC. An IgG ELISA developed using SAG1 refolded in the presence of oxido-shuffling reagent displayed high sensitivity and specificity for detection of Toxoplasma IgG antibodies in pregnant women.
Assuntos
Antígenos de Protozoários/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Imunoglobulina G/imunologia , Proteínas de Protozoários/imunologia , Toxoplasma/imunologia , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/genética , Antígenos de Protozoários/metabolismo , Antígenos de Superfície/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Humanos , Gravidez , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Sensibilidade e Especificidade , Testes Sorológicos/métodos , Toxoplasma/genética , Toxoplasma/metabolismoRESUMO
Diagnosis of Toxoplasma gondii (T.gondii) infection is of great medical importance especially for pregnant women and immunosuppressed patients. Numerous studies have shown that the recombinant production of several toxoplasma antigens, including dense granule antigens (GRAs) has a great potential as diagnostic reagents. Previous studies reported expression of amino terminal GRA8 protein in fusion with large tags. In the present study, we produced truncated GRA8 (GRA8), excluded from the signal peptide and C-terminal transmembrane domain, with a short fusion tag in Escherichia coli (E.coli). GRA8 was purified using an optimized single-step Immobilized Metal ion Affinity Chromatography (IMAC). The purity and yield of GRA8 was highest at pH = 9.25. At this pH, 13.6 mg of GRA8 was obtained with the purity of 97.97%. Immunogenicity of the protein was evaluated in Western blot analysis showing the serum sample from a rabbit immunized with GRA8 recognized a single antigen of T.gondii tachyzoite at the expected molecular weight of native GRA8. To diagnosis acute toxoplasma infection in pregnant women, an indirect immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) was developed using GRA8 resulting in a test specificity and sensitivity of 97.1% and 60.6%, respectively. These results demonstrated that immunogenic GRA8 can be produced in fusion with a short tag and purified near to homogeneity using an optimized IMAC. GRA8-IgM-ELISA was useful for detection of acute toxoplasma infection.
RESUMO
The usefulness of a specific immunoglobulin G (IgG) avidity enzyme-linked immunosorbent assay (ELISA) based on recombinant GRA6 antigen for distinguishing between acute and chronic Toxoplasma infection was investigated. Two sets of serum samples obtained from pregnant women with acute, chronic, or no Toxoplasma infection collected in France and Iran were used. Among the French subjects, 19 of 20 (95%) women who experienced seroconversion during the past 4 months before sampling displayed low-avidity IgG antibodies against GRA6, while all 17 (100%) women with chronic infection had high-avidity antibodies. When the Euroimmun IgG avidity ELISA was used, 15 of 19 (78.9%) recently infected women had low-avidity antibodies, and 20 of 22 (90.9%) women with chronic infection displayed high-avidity antibodies. The results suggested better performance of the GRA6 avidity ELISA than the Euroimmun avidity ELISA for exclusion of a recent infection occurring less than 4 months previously. Similarly, all 35 Iranian women with acute Toxoplasma infection had low-avidity antibodies against GRA6, whereas all 34 women with chronic infection displayed IgG antibodies of high avidity, indicating the value of GRA6 avidity testing for ruling out a recent infection. Avidity tests based on lysed whole-cell Toxoplasma gondii antigen are currently used to exclude recently acquired infections; however, the use of recombinant antigen(s) might improve the diagnostic performance of avidity tests and facilitate the development of more standardized assays.
Assuntos
Anticorpos Antiprotozoários/imunologia , Afinidade de Anticorpos , Antígenos de Protozoários , Imunoglobulina G/imunologia , Parasitologia/métodos , Complicações Parasitárias na Gravidez/diagnóstico , Proteínas de Protozoários , Toxoplasmose/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , França , Humanos , Irã (Geográfico) , GravidezRESUMO
PR81 is an anti-MUC1 monoclonal antibody (MAb) which was generated against human MUC1 mucin that reacted with breast cancerous tissue, MUC1 positive cell line (MCF-7, BT-20, and T-4 7 D), and synthetic peptide, including the tandem repeat sequence of MUC1. Here we characterized the binding properties of PR81 against the tandem repeat of MUC1 by two different epitope mapping techniques, namely, PEPSCAN and phage display. Epitope mapping of PR81 MAb by PEPSCAN revealed a minimal consensus binding sequence, PDTRP, which is found on MUC1 peptide as the most important epitope. Using the phage display peptide library, we identified the motif PD(T/S/G)RP as an epitope and the motif AVGLSPDGSRGV as a mimotope recognized by PR81. Results of these two methods showed that the two residues, arginine and aspartic acid, have important roles in antibody binding and threonine can be substituted by either glycine or serine. These results may be of importance in tailor making antigens used in immunoassay.