RESUMO
Pollen from common ragweed is an important allergen source worldwide and especially in western and southern Romania. More than 100 million patients suffer from symptoms of respiratory allergy (e.g., rhinitis, asthma) to ragweed pollen. Among the eleven characterized allergens, Amb a 6 is a non-specific lipid transfer protein (nsLTP). nsLTPs are structurally stable proteins in pollen and food from different unrelated plants capable of inducing severe reactions. The goal of this study was to produce Amb a 6 as a recombinant and structurally folded protein (rAmb a 6) and to characterize its physicochemical and immunological features. rAmb a 6 was expressed in Spodoptera frugiperda Sf9 cells as a secreted protein and characterized by mass spectrometry and circular dichroism (CD) spectroscopy regarding molecular mass and fold, respectively. The IgE-binding frequency towards the purified protein was evaluated using sera from 150 clinically well-characterized ragweed-allergic patients. The allergenic activities of rAmb a 6 and the nsLTP from the weed Parietaria judaica (Par j 2) were evaluated in basophil activation assays. rAmb a 6-specific IgE reactivity was associated with clinical features. Pure rAmb a 6 was obtained by insect cell expression. Its deduced molecular weight corresponded to that determined by mass spectrometry (i.e., 10,963 Da). rAmb a 6 formed oligomers as determined by SDS-PAGE under non-reducing conditions. According to multiple sequence comparisons, Amb a 6 was a distinct nsLTP with less than 40% sequence identity to currently known plant nsLTP allergens, except for nsLTP from Helianthus (i.e., 52%). rAmb a 6 is an important ragweed allergen recognized by 30% of ragweed pollen allergic patients. For certain patients, rAmb a 6-specific IgE levels were higher than those specific for the major ragweed allergen Amb a 1 and analysis also showed a higher allergenic activity in the basophil activation test. rAmb a 6-positive patients suffered mainly from respiratory symptoms. The assumption that Amb a 6 is a source-specific ragweed allergen is supported by the finding that none of the patients showing rAmb a 6-induced basophil activation reacted with Par j 2 and only one rAmb a 6-sensitized patient had a history of plant food allergy. Immunization of rabbits with rAmb a 6 induced IgG antibodies which strongly inhibited IgE binding to rAmb a 6. Our results demonstrate that Amb a 6 is an important source-specific ragweed pollen allergen that should be considered for diagnosis and allergen-specific immunotherapy of ragweed pollen allergy.
Assuntos
Alérgenos , Antígenos de Plantas , Proteínas de Transporte , Imunoglobulina E , Humanos , Alérgenos/imunologia , Imunoglobulina E/imunologia , Antígenos de Plantas/imunologia , Antígenos de Plantas/química , Animais , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Proteínas de Plantas/imunologia , Proteínas de Plantas/química , Feminino , Rinite Alérgica Sazonal/imunologia , Masculino , Adulto , Ambrosia/imunologia , Spodoptera/imunologia , Proteínas Recombinantes/imunologia , Sequência de Aminoácidos , Células Sf9 , Pessoa de Meia-Idade , Extratos VegetaisRESUMO
The topic of ragweed pollen (RW) versus house dust mites (HDMs) has often been deliberated, but the increasing incidence of co-sensitization between them has been scarcely addressed. Utilizing Sprague Dawley rats, we explored the effects of co-sensitization with the combination of HDMs and RW pollen extracts in correlation with high-fructose diet (HFrD) by in vitro tracheal reactivity analysis in isolated organ bath and biological explorations. Our findings unveiled interrelated connections between allergic asthma, dyslipidemia, and HFrD-induced obesity, shedding light on their compounding role through inflammation. The increased CRP values and airway hyperresponsiveness to the methacholine challenge suggest a synergistic effect of obesity on amplifying the existing inflammation induced by asthma. One of the major outcomes is that the co-sensitization to HDMs and RW pollen led to the development of a severe allergic asthma phenotype in rats, especially in those with HFrD. Therefore, the co-sensitization to these allergens as well as the HFrD may play a crucial role in the modulation of systemic inflammation, obesity, and airway reactivity.
Assuntos
Biomarcadores , Frutose , Pyroglyphidae , Ratos Sprague-Dawley , Animais , Ratos , Biomarcadores/sangue , Pyroglyphidae/imunologia , Asma/imunologia , Asma/sangue , Asma/etiologia , Masculino , Alérgenos/imunologia , Obesidade/imunologia , Obesidade/sangue , Antígenos de Plantas/imunologia , Pólen/imunologia , Extratos VegetaisRESUMO
Common ragweed pollen allergy has become a health burden worldwide. One of the major allergens in ragweed allergy is Amb a 1, which is responsible for over 90% of the IgE response in ragweed-allergic patients. The major allergen isoform Amb a 1.01 is the most allergenic isoform in ragweed pollen. So far, no recombinant Amb a 1.01 with similar allergenic properties to its natural counterpart (nAmb a 1.01) has been produced. Hence, this study aimed to produce a recombinant Amb a 1.01 with similar properties to the natural isoform for improved ragweed allergy management. Amb a 1.01 was expressed in insect cells using a codon-optimized DNA construct with a removable N-terminal His-Tag (rAmb a 1.01). The recombinant protein was purified by affinity chromatography and physicochemically characterized. The rAmb a 1.01 was compared to nAmb a 1.01 in terms of the IgE binding (enzyme-linked immunosorbent assay (ELISA), immunoblot) and allergenic activity (mediator release assay) in well-characterized ragweed-allergic patients. The rAmb a 1.01 exhibited similar IgE reactivity to nAmb a 1.01 in different IgE-binding assays (i.e., IgE immunoblot, ELISA, quantitative ImmunoCAP inhibition measurements). Furthermore, the rAmb a 1.01 showed comparable dose-dependent allergenic activity to nAmb a 1.01 regarding basophil activation. Overall, the results showed the successful expression of an rAmb a 1.01 with comparable characteristics to the corresponding natural isoform. Our findings provide the basis for an improvement in ragweed allergy research, diagnosis, and immunotherapy.
Assuntos
Alérgenos , Ambrosia , Antígenos de Plantas , Imunoglobulina E , Proteínas Recombinantes , Humanos , Antígenos de Plantas/imunologia , Antígenos de Plantas/genética , Antígenos de Plantas/química , Imunoglobulina E/imunologia , Animais , Alérgenos/imunologia , Alérgenos/genética , Ambrosia/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genética , Feminino , Adulto , Proteínas de Plantas/imunologia , Proteínas de Plantas/genética , Proteínas de Plantas/química , Rinite Alérgica Sazonal/imunologia , Masculino , Pessoa de Meia-Idade , Extratos Vegetais/químicaRESUMO
CONTEXT: Oleanolic and ursolic acids are antitumor and antibacterial agents which are extensively studied. Their major disadvantage is the poor water solubility which limits their applications. OBJECTIVES: Oleanolic and ursolic acid were encapsulated into polyurethane nanostructures that act as drug carriers. In order to evaluate the effectiveness of the particles, anti-microbial and anti-proliferative activity compared to un-encapsulated active compounds was tested. MATERIALS AND METHODS: Using an interfacial polycondensation technique, combined with spontaneous emulsification, structures with nanoscale dimensions were obtained. Scanning electron microscopy, differential scanning calorimetry and X-ray assays confirmed the encapsulation process. Concentrations of 10 and 30 µM particles and un-encapsulated compounds were tested by MTT viability assay for several breast cancer lines, with an exposure time of 72 h. For the antibacterial studies, the dilution method with MIC determination was used. RESULTS: Ursolic acid had an excellent inhibitory effect with IC50 value of 2.47, 1.20, 1.26 and 1.34 µM on MCF7, T47D, MDA-MB-231 and MDA-MB-361, respectively. Oleanolic acid did not show anti-proliferative activity. The pure compounds showed their antibacterial activity only against Bacillus species and Candida albicans, but MIC values were too high to be considered efficient antimicrobial agents (2280 and 4570 µg mL - 1, respectively). Polyurethane nanoparticles which incorporated the agents did not show any biological activity. DISCUSSION AND CONCLUSION: Although the active compounds did not fully exert their anti-proliferative activity following encapsulation inside polymeric nanoparticles, in vivo evaluation is needed in order to obtain an exhaustive conclusion, as the active compounds could be released as a result of metabolic activity.
Assuntos
Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Sistemas de Liberação de Medicamentos , Ácido Oleanólico/farmacologia , Poliuretanos/administração & dosagem , Triterpenos/farmacologia , Antibacterianos/administração & dosagem , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Humanos , Microscopia Eletrônica de Varredura , Nanoestruturas , Ácido Oleanólico/administração & dosagem , Tamanho da Partícula , Triterpenos/administração & dosagem , Ácido UrsólicoRESUMO
Currently, allergen-specific immunotherapy (AIT) for ragweed allergy is still based on natural allergen extracts. This study aimed to analyse the ability of four commercially available AIT vaccines (CLUSTOID, TYRO-SIT, POLLINEX Quattro Plus and Diater Depot) regarding their ability to induce IgG antibodies against ragweed pollen allergens in rabbits. Accordingly, the IgG reactivity of AIT-induced rabbit sera was tested for ten different ragweed pollen allergens (Amb a 1, 3, 4, 5, 6, 8, 9, 10, 11 and 12) by an ELISA. Furthermore, the ability of rabbit AIT-specific sera to block allergic patients' IgE binding to relevant ragweed allergens (Amb a 1, 4, 6, 8 and 11) and to inhibit allergen-induced basophil activation was evaluated by an IgE inhibition ELISA and a mediator release assay. Only two AIT vaccines (Diater Depot > CLUSTOID) induced relevant IgG antibody levels to the major ragweed allergen Amb a 1. The IgG responses induced by the AIT vaccines against the other ragweed allergens were low and highly heterogeneous. Interestingly, the kinetics of IgG responses were different among the AIT vaccines and even within one AIT vaccine (Diater Depot) for Amb a 1 (long-lasting) versus Amb a 8 and Amb a 11 (short-lived). This could be due to variations in allergen contents, the immunogenicity of the allergens, and different immunization protocols. The IgE inhibition experiments showed that rabbit AIT-specific sera containing high allergen-specific IgG levels were able to inhibit patients' IgE binding and prevent the mediator release with Diater Depot. The high levels of allergen-specific IgG levels were associated with their ability to prevent the recognition of allergens by patients' IgE and allergen-induced basophil activation, indicating that the measurement of allergen-induced IgG could be a useful surrogate marker for the immunological efficacy of vaccines. Accordingly, the results of our study may be helpful for the selection of personalized AIT vaccination strategies for ragweed-allergic patients.
RESUMO
Human skeletal muscle tissue displays specific cellular architecture easily damaged during individual existence, requiring multiple resources for regeneration. Congruent with local prerequisites, heterogeneous muscle stem cells (MuSCs) are present in the muscle interstitium. In this study, we aimed to characterize the properties of human muscle interstitial cells that had the characteristic morphology of telocytes (TCs). Immunocytochemistry and immunofluorescence showed that cells with TC morphology stained positive for c-kit/CD117 and VEGF. C-kit positive TCs were separated with magnetic-activated cell sorting, cultured in vitro and expanded for study. These cells exhibited high proliferation capacity (60% expressed endoglin/CD105 and 80% expressed nuclear Ki67). They also exhibited pluripotent capacity limited to Oct4 nuclear staining. In addition, 90% of c-kit positive TCs expressed VEGF. C-kit negative cells in the MuSCs population exhibited fibroblast-like morphology, low trilineage differential potential and negative VEGF staining. These results suggested that c-kit/CD117 positive TCs represented a unique cell type within the MuSC niche.
Assuntos
Músculo Esquelético/citologia , Nicho de Células-Tronco , Separação Celular , Células Cultivadas , Humanos , Proteínas Proto-Oncogênicas c-kit/análise , Regeneração , Fator A de Crescimento do Endotélio Vascular/análiseRESUMO
Tumour-associated fibroblasts (TAFs) are part of the tumour stroma, providing functional and structural support for tumour progression and development. The origin and biology of TAFs are poorly understood, but within the tumour environment, TAFs become activated and secrete different paracrine and autocrine factors involved in tumorigenesis. It has been shown that bone marrow mesenchymal stem cells (MSCs) can be recruited into the tumours, where they proliferate and acquire a TAF-like phenotype. We attempted to determine to what extent TAFs characteristics in vitro juxtapose to MSCs' definition, and we showed that TAFs and MSCs share immunophenotypic similarities, including the presence of certain cell surface molecules [human leukocyte antigen-DR subregion (HLA-DR), CD29, CD44, CD73, CD90, CD106 and CD117]; the expression of cytoskeleton and extracellular matrix proteins, such as vimentin, α-smooth muscle actin, nestin and trilineage differentiation potential (to adipocytes, chondrocytes and osteoblasts). When compared to MSCs, production of cytokines, chemokines and growth factors showed a significant increase in TAFs for vascular endothelial growth factor, transforming growth factor-ß1, interleukins (IL-4, IL-10) and tumour necrosis factor α. Proliferation rate was highly increased in TAFs and fibroblast cell lines used in our study, compared to MSCs, whereas ultrastructural details differentiated the two cell types by the presence of cytoplasmic elongations, lamellar content lysosomes and intermediate filaments. Our results provide supportive evidence to the fact that TAFs derive from MSCs and could be a subset of 'specialized' MSCs.
Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular , Fibroblastos/patologia , Células-Tronco Mesenquimais/citologia , Actinas/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Células da Medula Óssea/metabolismo , Células da Medula Óssea/ultraestrutura , Neoplasias da Mama/patologia , Linhagem Celular , Proliferação de Células , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Antígenos HLA-DR/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Integrina beta1/metabolismo , Interleucina-1/metabolismo , Interleucina-10/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/ultraestrutura , Microscopia Eletrônica de Transmissão , Músculo Liso/química , Osteoblastos/citologia , Osteoblastos/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Vimentina/metabolismoRESUMO
In recent years, identification of new sources of adult stem cells developed rapidly, pursuing to find easily available tissues, which will give rise to homogenous stem cells populations. Up to present, bone marrow-derived mesenchymal stem cells (BM-MSCs) are unanimously considered to fulfill the criteria for being used in clinical settings, but adipose stem cells, placental and umbilical cord stem cells, and other tissue-derived stem cells are making their way to being used at least in autologous transplantation. We isolated cellular populations from placental tissue and umbilical cord using the explants method. The placental (PL) and umbilical cord (UC)-derived cells were cultured and expanded in appropriate conditions for generation of stem cells. We assessed the stemness characteristics of the tissue-isolated cells and compared them to an established MSCs line. For this purpose, we determined the immunophenotype, morphological and ultrastructural characteristics, as well as functional abilities of PL- and UC-derived cells. Flow cytometric evaluation of cells revealed presence of CD90, CD73, and CD105 stem cells markers, while the cells were negative for CD34, CD45 and HLA-DR. Immunocytochemical staining showed that 100% of PL- and UC-derived cells are positive for vimentin and CD105 expression, while cytokeratin was revealed in less than 10% in both tissue-isolated cells. Morphological and ultrastructural characteristics of cells exposed analogous cellular size and intracellular organization, similar to MSCs, but detailed view of UC-derived cells by transmission electron microscopy (TEM) demonstrated presence of intercellular junctions-desmosomes, similar to epithelial cells. Both PL- and UC-derived cells confirmed their trilineage potential, being able to differentiate into adipocytes, osteoblasts, and chondrocytes in different proportions. Flow chamber in vitro assay was used to determine to what extent PL- and UC-derived cells are able to adhere to substrates (VCAM and ICAM) and we showed progressively decreased adhesion of both cellular types, inversely proportional to the generated shear stress. We may conclude that explants-isolated placental and umbilical cord cells are endowed with characteristics of both epithelial and mesenchymal stem cells, and purification procedures are additionally required for safe use of these cells in diverse clinical applications.
Assuntos
Separação Celular/métodos , Células Epiteliais/citologia , Células-Tronco Mesenquimais/citologia , Placenta/citologia , Cordão Umbilical/citologia , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Biomarcadores/metabolismo , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Membrana Celular/metabolismo , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Imunofenotipagem , Molécula 1 de Adesão Intercelular/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Gravidez , Cordão Umbilical/ultraestrutura , Molécula 1 de Adesão de Célula Vascular/farmacologia , Vimentina/metabolismoRESUMO
INTRODUCTION: The antimicrobial role of probiotic Lactobacillus casei subspecies casei DG (L. casei DG) and of the mix culture of probiotic Lactobacillus acidophilus LA-5 and Bifidobacterium BB-12 was tested on species of Staphylococcus, Streptococcus, Pasteurella, and Neisseria genera from supragingival sites from dogs with dental disease of different breed, age, sex, weight, and diet. The research was conducted on these four genera because of their importance in zoonotic infections after dog bites. METHODOLOGY: Species from Staphylococcus, Streptococcus, Pasteurella, and Neisseria genera were isolated and identified. To test the antimicrobial efficacy of L. casei DG and the mixed culture of probiotic L. acidophilus LA-5 and Bifidobacterium bifidum BB-12 on the pathogenic species, the agar overlay method was used. RESULTS: L. casei DG had a bactericidal effect on all analyzed species isolated from Staphylococcus, Streptococcus, Pasteurella, and Neisseria genera after 24 hours of incubation. The mixed probiotic culture made up of L. acidophilus LA-5 and Bifidobacterium BB-12 species had no bactericidal effect on the species of Staphylococcus and Streptococcus genera, which were resistant. However, it had a bacteriostatic effect on several species of Pasteurella and Neisseria genera. CONCLUSIONS: This work highlights the antimicrobial potential of probiotics in vitro, demonstrating that the probiotic L. casei DG has a bactericidal effect on all analyzed species isolated from dental plaque and that the mix culture of probiotic L. acidophilus LA-5 and Bifidobacterium BB-12 has only a bacteriostatic effect.
Assuntos
Anti-Infecciosos/administração & dosagem , Bifidobacterium/crescimento & desenvolvimento , Biota , Placa Dentária/veterinária , Doenças do Cão/microbiologia , Lactobacillus/crescimento & desenvolvimento , Probióticos/administração & dosagem , Animais , Placa Dentária/microbiologia , Cães , Feminino , Masculino , Neisseria/isolamento & purificação , Pasteurella/isolamento & purificação , Staphylococcus/isolamento & purificação , Streptococcus/isolamento & purificaçãoRESUMO
The metal alloys used in dental practice are the subject of numerous discussions referring to the effect they have on oral cavity tissues. Numerous scientists established that the organic molecules did not exert a significant influence on the degradation of metallic implants. The aim of this study was to test the cytotoxic potential of the most used dental alloys, the Ni-Cr alloy and the Co-Cr alloy. The tests were made on cell culture of pure cell line dermal fibroblasts and of those obtained from skin biopsies, for both, dental alloys and their eluates. The results were compared with control samples. At seven days after inoculation, we observed the relative similarity between the Ni-Cr alloy and the Co-Cr alloy, where the cells did not detach from the plate and they grow to the edge of the material. In case of the eluates, there were no fragments detached, the cells having a relatively high confluence. Therefore, the cytotoxic effects of the two alloys are similar, even if there are speculations in the literature according to which Ni-Cr alloys would have a more pronounced effect. In conclusion, our study revealed non-cytotoxicity of these two dental alloys, and we believe so they can be used successfully in dental practice.
Assuntos
Ligas Dentárias/toxicidade , Fibroblastos/patologia , Adulto , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Pele/citologiaRESUMO
Our study examined whether human bone marrow-derived MSCs are able to differentiate, in vitro, into functional epithelial-like cells. MSCs were isolated from the sternum of 8 patients with different hematological disorders. The surface phenotype of these cells was characterized.To induce epithelial differentiation, MSCs were cultured using Epidermal Growth Factor, Keratinocyte Growth Factor, Hepatocyte Growth Factor and Insulin-like growth Factor-II. Differentiated cells were further characterized both morphologically and functionally by their capacity to express markers with specificity for epithelial lineage. The expression of cytokeratin 19 was assessed by immunocytochemistry, and cytokeratin 18 was evaluated by quantitative RT-PCR (Taq-man). The data demonstrate that human MSCs isolated from human bone marrow can differentiate into epithelial-like cells and may thus serve as a cell source for tissue engineering and cell therapy of epithelial tissue.
Assuntos
Diferenciação Celular , Linhagem da Célula , Células Epiteliais/citologia , Células-Tronco Mesenquimais/citologia , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Células Epiteliais/metabolismo , Citometria de Fluxo , Regulação da Expressão Gênica , Humanos , Queratina-18/genética , Queratina-18/metabolismo , Células-Tronco Mesenquimais/metabolismoRESUMO
Hematopoietic stem cells have a remarkable plastic capacity, which allows them to differentiate into various cells, such as immune cells, nervous cells, muscle cells, bone and cartilaginous cells. The aim of this study was to show the capacity of stem cells to differentiate into endothelial cells, in culture, after addition of endothelial cells growth supplement (ECGS). We also compared the behavior of these cells with that of endothelial cells obtained from human umbilical vein (HUVEC). CD34+ cells obtained by immunomagnetic separation from human umbilical cord and placental blood were used. After 12-15 days of culture in a medium containing ECGS, the cells showed morphological changes characteristic to endothelial cells and immunocytochemical analysis revealed the presence of CD31 surface antigen and von Willebrand factor. The flow-cytometric analysis of endothelial cells adhesion molecules (ECAM) showed that endothelial cells derived from CD34+ cells expressed CD54/ICAM-1 9.65+/-0.2% and CD106/VCAM 7.73+/-0.3%, values similar to those expressed by HUVECs. After TNF incubation, ECAM expression increased only in HUVECs. These data demonstrate that a fraction of circulating CD34+ cells may develop some endothelial cell characteristics when cultured with ECGS, but they are functionally different from HUVECs.