RESUMO
Cell encapsulation strategies using hydrogel beads have been considered as an alternative to immunosuppression in cell-based therapies. They rely on layer-by-layer (LbL) deposition of polymers to tune beads' permeability, creating a physical barrier to the host immune system. However, the LbL approach can also create diffusion barriers, hampering the flow of essential nutrients and therapeutic cell products. In this work, the polyelectrolyte complex (PEC) methodology was used to circumvent the drawbacks of the LbL strategy by inducing hydrogel bead formation through the interaction of anionic methacrylated gellan gum (GG-MA) with cationic poly-l-lysine (PLL). The interfacial complexation between both polymers resulted in beads with a cell-friendly GG-MA hydrogel core surrounded by a PEC semipermeable membrane. The beads showed great in vitro stability over time, a semi-permeable behavior, and supported human adipose-derived stem cell encapsulation. Additionally, and regarding immune recognition, the in vitro and in vivo studies pointed out that the hydrogel beads behave as an immunocompatible system. Overall, the engineered beads showed great potential for hydrogel-mediated cell therapies, when immunoprotection is required, as when treating different metabolic disorders.
Assuntos
Polilisina , Polissacarídeos Bacterianos , Humanos , Hidrogéis , PolieletrólitosRESUMO
In our work, we aim to identify new candidate host biomarkers to discriminate between active TB patients (n = 28), latent infection (LTBI; n = 27) and uninfected (NoTBI; n = 42) individuals. For that, active TB patients and their contacts were recruited that donated serum and saliva samples. A multiplex assay was performed to study the concentration of different cytokines, chemokines and growth factors. Proteins with significant differences between groups were selected and logistic regression and the area under the ROC curve (AUC) was used to assess the diagnostic accuracy. The best marker combinations that discriminate active TB from NoTBI contacts were [IP-10 + IL-7] in serum and [Fractalkine + IP-10 + IL-1α + VEGF] in saliva. Best discrimination between active TB and LTBI was achieved using [IP-10 + BCA-1] in serum (AUC = 0.83) and IP-10 in saliva (p = 0.0007; AUC = 0.78). The levels of TNFα (p = 0.003; AUC = 0.73) in serum and the combination of [Fractalkine+IL-12p40] (AUC = 0.83) in saliva, were able to differentiate between NoTBI and LTBI contacts. In conclusion, different individual and combined protein markers could help to discriminate between active TB and both uninfected and latently-infected contacts. The most promising ones include [IP-10 + IL-7], [IP-10 + BCA-1] and TNFα in serum and [Fractalkine + IP-10 + IL-1α + VEGF], IP-10 and [Fractalkine+IL-12p40] in saliva.
Assuntos
Quimiocina CX3CL1/sangue , Quimiocina CXCL10/sangue , Interleucinas/sangue , Tuberculose Latente/sangue , Tuberculose Pulmonar/sangue , Fator A de Crescimento do Endotélio Vascular/sangue , Adulto , Idoso , Biomarcadores/sangue , Biomarcadores/metabolismo , Quimiocina CX3CL1/análise , Quimiocina CXCL10/análise , Feminino , Humanos , Interleucinas/análise , Tuberculose Latente/diagnóstico , Tuberculose Latente/metabolismo , Masculino , Pessoa de Meia-Idade , Saliva/química , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/metabolismo , Fator A de Crescimento do Endotélio Vascular/análiseRESUMO
OBJECTIVES: To identify new potential host biomarkers in blood to discriminate between active TB patients, uninfected (NoTBI) and latently infected contacts (LTBI). METHODS: A blood cell count was performed to study parent leukocyte populations. Peripheral blood mononuclear cells (PBMCs) were isolated and a multi-parameter flow cytometry assay was conducted to study the distribution of basal and Mycobacterium tuberculosis (Mtb)-stimulated lymphocytes. Differences between groups and the area under the ROC curve (AUC) were investigated to assess the diagnostic accuracy. RESULTS: Active TB patients presented higher Monocyte-to-lymphocyte and Neutrophil-to-lymphocyte ratios than LTBI and NoTBI contacts (p<0.0001; AUC>0.8). Lymphocyte subsets with differences (p >0.05; AUC >0.7) between active TB and both contact groups include the basal distribution of Th1/Th2 ratio, Th1-Th17, CD4+ Central Memory (TCM) or MAIT cells. Expression of CD154 is increased in Mtb-activated CD4+ TCM and Effector Memory T cells in active TB and LTBI compared to NoTBI. In CD4+T cells, expression of CD154 showed a higher accuracy than IFNγ to discriminate Mtb-specific activation. CONCLUSIONS: We identified different cell subsets with potential use in tuberculosis diagnosis. Among them, distribution of CD4 TCM cells and their expression of CD154 after Mtb-activation are the most promising candidates.
Assuntos
Tuberculose Latente , Mycobacterium tuberculosis , Tuberculose , Linfócitos T CD4-Positivos , Citometria de Fluxo , Humanos , Imunofenotipagem , Tuberculose Latente/diagnóstico , Leucócitos Mononucleares , Tuberculose/diagnósticoRESUMO
A better understanding of the response against Tuberculosis (TB) infection is required to accurately identify the individuals with an active or a latent TB infection (LTBI) and also those LTBI patients at higher risk of developing active TB. In this work, we have used the information obtained from studying the gene expression profile of active TB patients and their infected -LTBI- or uninfected -NoTBI- contacts, recruited in Spain and Mozambique, to build a class-prediction model that identifies individuals with a TB infection profile. Following this approach, we have identified several genes and metabolic pathways that provide important information of the immune mechanisms triggered against TB infection. As a novelty of our work, a combination of this class-prediction model and the direct measurement of different immunological parameters, was used to identify a subset of LTBI contacts (called TB-like) whose transcriptional and immunological profiles are suggestive of infection with a higher probability of developing active TB. Validation of this novel approach to identifying LTBI individuals with the highest risk of active TB disease merits further longitudinal studies on larger cohorts in TB endemic areas.
Assuntos
Tuberculose Latente/diagnóstico , Modelos Imunológicos , Análise de Sequência de RNA/métodos , Linfócitos T/imunologia , Tuberculose/diagnóstico , Doença Aguda , Adulto , Idoso , Células Cultivadas , Progressão da Doença , Feminino , Humanos , Interferon gama/metabolismo , Tuberculose Latente/genética , Tuberculose Latente/imunologia , Ativação Linfocitária , Aprendizado de Máquina , Masculino , Pessoa de Meia-Idade , Tuberculose/genética , Tuberculose/imunologiaRESUMO
In this study, methacrylated gellan-gum (GG-MA) heteropolysaccharide is proposed as a hydrogel for drug delivery and bone tissue engineering applications. Calcium-enriched beads obtained from the crosslinking of 1% (w/v) GG-MA solutions with 0.1â¯MCaCl2 were investigated, considering their intrinsic capacity to promote self-mineralization by ion binding and deposition. Indeed, when immersed in a physiological environment, the Ca-enriched beads promoted the development of a bone-like apatite layer, as confirmed by EDS and XRD chemical analysis. Additionally, the mild production process is compatible with drugs incorporation and release. After encapsulation, Dextran with different molecular weights as well as Dexamethasone 21-phosphate were efficiently released to the surrounding environment. The engineered system was also evaluated considering its biocompatibility, by means of qualitative determination of total complement activation, macrophage proliferation, cytokine release and in vitro cell culture. These experiments showed that the developed hydrogels may not stimulate a disproportionate pro-inflammatory reaction once transplanted. At last, when implanted subcutaneously in CD1 male mice up to 8â¯weeks, the beads were completely calcified, and no inflammatory reaction was observed. Summing up, these results show that calcium-enriched GG-MA hydrogel beads hold great potential as news tools for bone tissue regeneration and local drug delivery applications. STATEMENT OF SIGNIFICANCE: This work describes a low-cost and straightforward strategy to prepare bioactive methacrylated gellan gum (GG-MA) hydrogels, which can be used as drug delivery systems. GG-MA is a highly anionic polymer, that can be crosslinked with divalent ions, as calcium. Taking advantage of this feature, it was possible to prepare Ca-enriched GG-MA hydrogel beads. These beads display a bioactive behavior, since they promote apatite deposition when placed in physiological conditions. Studies on the immune response suggest that the developed beads do not trigger severe immune responses. Importantly, the mild processing method render these beads compliant with drug delivery strategies, paving the way for the application of dual-functional materials on bone tissue engineering.
Assuntos
Calcificação Fisiológica/efeitos dos fármacos , Cálcio , Hidrogéis , Teste de Materiais , Polissacarídeos Bacterianos , Engenharia Tecidual , Adolescente , Adulto , Animais , Osso e Ossos/metabolismo , Cálcio/química , Cálcio/farmacologia , Feminino , Humanos , Hidrogéis/química , Hidrogéis/farmacologia , Masculino , Metacrilatos/química , Metacrilatos/farmacologia , Camundongos , Pessoa de Meia-Idade , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/farmacologiaRESUMO
We studied the immunoglobulin genes from either the genomes or RNAs of amphibians. In particular, we obtained data from one frog genome (Nanorana parkeri) and three transcriptomes of the Caudata order (Andrias davidianus, Notophthalmus viridescens and Cynops pyrrhogaster). Apart from the immunoglobulins IgM and IgY previously described, we identified several IgD related immunoglobulins. The species N. parkeri, N. viridescens and C. pyrrhogaster have two IgD genes, while Andrias davidianus has three such genes. The three Caudata species have long IgD immunoglobulins similar to IgD of reptiles, and could be an ancient relic from the common ancestor of IgD of all mammals and reptiles. We also found two IgA isotypes. The results suggest that one of the IgA may be the ancestor of IgA in crocodiles and birds, while the other could be the ancestor IgA found in mammals. These results provide information that could help understand the evolution of immunoglobulins in terrestrial vertebrates.
Assuntos
Proteínas de Anfíbios/genética , Anfíbios/genética , Evolução Biológica , Genes de Imunoglobulinas , Imunoglobulina A/genética , Imunoglobulina D/genética , Sequência de Aminoácidos , Animais , Isotipos de Imunoglobulinas/genética , Dados de Sequência MolecularRESUMO
The Squamata order represents a major evolutionary reptile lineage, yet the structure and expression of immunoglobulins in this order has been scarcely studied in detail. From the genome sequences of four Squamata species (Gekko japonicus, Ophisaurus gracilis, Pogona vitticeps and Ophiophagus hannah) and RNA-seq datasets from 18 other Squamata species, we identified the immunoglobulins present in these animals as well as the tissues in which they are found. All Squamata have at least three immunoglobulin classes; namely, the immunoglobulins M, D, and Y. Unlike mammals, however, we provide evidence that some Squamata lineages possess more than one Cµ gene which is located downstream from the Cδ gene. The existence of two evolutionary lineages of immunoglobulin Y is shown. Additionally, it is demonstrated that while all Squamata species possess the λ light chain, only Iguanidae species possess the κ light chain.
Assuntos
Imunoglobulinas/biossíntese , Répteis/genética , Répteis/imunologia , Animais , Feminino , Genoma , Imunoglobulinas/genética , Imunoglobulinas/imunologia , MasculinoRESUMO
Magnetic nanoparticles (NPs) hold great promise for biomedical applications. The core composition and small size of these particles produce superparamagnetic behavior, thus facilitating their use in magnetic resonance imaging and magnetically induced therapeutic hyperthermia. However, the development and control of safe in vivo applications for NPs call for the study of cell-NP interactions and cell viability. Furthermore, as for most biotechnological applications, it is desirable to prevent unspecific cell internalization of these particles. It is also crucial to understand how the surface composition of the NPs affects their internalization capacity. Here, through accurate control over unspecific protein adsorption, size distribution, grafting density, and an extensive physicochemical characterization, we correlated the cytotoxicity and cellular uptake mechanism of 6 nm magnetic NPs coated with several types and various densities of biomolecules, such as glucose, galactose, and poly(ethylene glycol). We found that the density of the grafted molecule was crucial to prevent unspecific uptake of NPs by Vero cells. Surprisingly, the glucose-coated NPs described here showed cellular uptake as a result of lipid raft instead of clathrin-mediated cellular internalization. Moreover, these glucose-functionalized NPs could be one of the first examples of NPs being endocytosed by caveolae that finally end up in the lysosomes. These results reinforce the use of simple carbohydrates as an alternative to PEG molecules for NPs functionalization when cellular uptake is required.
Assuntos
Monossacarídeos/química , Nanopartículas/química , Polietilenoglicóis/química , Adsorção , Animais , Transporte Biológico , Células HeLa , Humanos , Camundongos , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
A comparative study was conducted to determine the feasibility of enzyme-linked immunosorbent assays (ELISAs) for the detection of amnesic shellfish poisoning (ASP) and paralytic shellfish poisoning (PSP) toxins in nine naturally contaminated species in fresh, frozen, boiled and canned fish and shellfish. PSP and ASP were analyzed in 138 shellfish samples (mussels, clams, barnacles, razor shells, scallops and cockles) and anchovies by mouse bioassay (MBA) and high performance liquid chromatography with ultraviolet detection (HPLC-UV), respectively. Results were compared with toxin concentrations obtained using two commercial competitive ELISAs, saxitoxin and ASP kits. Immunoassays were able to quantify toxins in different matrices showing excellent Pearson's correlation coefficients (r = 0.974 for saxitoxin ELISA and r = 0.973 for ASP ELISA) and to detect PSP and ASP with a lower limit of detection (LOD), namely, 50 microg saxitoxin equivalent/kg shellfish meat for PSP and 60 microg/kg domoic acid in shellfish flesh for ASP, than the reference methods (350 microg saxitoxin equivalent/kg shellfish meat and 1.6 mg/kg domoic acid in shellfish flesh, respectively). These results suggest that the ELISA method could be used as screening systems in a variety of species without matrix interference.