RESUMEN
P-glycoprotein (encoded by the ABCB1 gene) has a dual role in regulating inflammation and reducing chemotherapy efficacy in various diseases, but there are few studies focused on pulmonary TB patients. In this study, our objective was to identify a list of genes that correlate with high and low levels of ABCB1 gene expression in the lungs of pulmonary TB patients with different activity of chronic granulomatous inflammation. We compared gene expression in two groups of samples (with moderate and high activity of tuberculomas) to identify their characteristic gene signatures. Gene expression levels were determined using quantitative PCR in samples of perifocal area of granulomas, which were obtained from 65 patients after surgical intervention. Subsequently, two distinct gene signatures associated with high inflammation activity were identified. The first signature demonstrated increased expression of HIF1a, TGM2, IL6, SOCS3, and STAT3, which correlated with high ABCB1 expression. The second signature was characterized by high expression of TNFa and CD163 and low expression of ABCB1. These results provide insight into various inflammatory mechanisms and association with P-gp gene expression in lung tissue of pulmonary TB patients and will be useful in the development of a host-directed therapy approach to improving the effectiveness of anti-TB treatment.
Asunto(s)
Tuberculosis Pulmonar , Humanos , Tuberculosis Pulmonar/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Pulmón/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Inflamación/genéticaRESUMEN
Metal-organic framework nanoparticles (nanoMOFs) are promising nanomaterials for biomedical applications. Some of them, including biodegradable porous iron carboxylates are proposed for encapsulation and delivery of antibiotics. Due to the high drug loading capacity and fast internalization kinetics, nanoMOFs are more beneficial for the treatment of intracellular bacterial infections compared to free antibacterial drugs, which poorly accumulate inside the cells because of the inability to cross membrane barriers or have low intracellular retention. However, nanoparticle internalization does not ensure their accumulation in the cell compartment that shelters a pathogen. This study shows the availability of MIL-100(Fe)-based MOF nanoparticles to co-localize with Chlamydia trachomatis, an obligate intracellular bacterium, in the infected RAW264.7 macrophages. Furthermore, nanoMOFs loaded with photosensitizer methylene blue (MB) exhibit complete photodynamic inactivation of C. trachomatis growth. Simultaneous infection and treatment of RAW264.7 cells with empty nanoMOFs resulted in a bacterial load reduction from 100 to 36% that indicates an intrinsic anti-chlamydial effect of this iron-containing nanomaterial. Thus, our findings suggest the use of iron-based nanoMOFs as a promising drug delivery platform, which contributes to antibacterial effect, for the treatment of chlamydial infections.
Asunto(s)
Chlamydia trachomatis , Azul de Metileno , Chlamydia trachomatis/fisiología , Azul de Metileno/farmacología , Antibacterianos/farmacología , Sistemas de Liberación de Medicamentos , HierroRESUMEN
The existing methods of quantitative analysis of phagocytosis are characterized by a number of limitations. The usual method of manually counting phagocytosed objects on photographs obtained by confocal microscopy is very labor-intensive and time-consuming. As well, the resolution of conventional flow cytometry does not allow the fluorescence detection of a large number of phagocytosis objects. Thus, there is a need to combine the rapid analysis by flow cytometry and the visualization capability by confocal microscopy. This is possible due to imaging flow cytometry. However, until now, no protocols have allowed one to quantify phagocytosis at its high intensity. The present paper presents the developed and tested algorithm for assessing the level of phagocytic activity using flow cytometry with visualization and IDEAS software.
Asunto(s)
Macrófagos , Fagocitosis , Humanos , Citometría de Flujo/métodos , Microesferas , FagocitosRESUMEN
The magnetic compass is an important element of the avian navigation system, which allows migratory birds to solve complex tasks of moving between distant breeding and wintering locations. The photochemical magnetoreception in the eye is believed to be the primary biophysical mechanism behind the magnetic sense of birds. It was shown previously that birds were disoriented in presence of weak oscillating magnetic fields (OMF) with frequencies in the megahertz range. The OMF effect was considered to be a fingerprint of the photochemical magnetoreception in the eye. In this work, we used miniaturized portable magnetic coils attached to the bird's head to specifically target the compass receptor. We performed behavioural experiments on orientation of long-distance migrants, garden warblers (Sylvia borin), in round arenas. The OMF with the amplitude of about 5 nT was applied locally to the birds' eyes. Surprisingly, the birds were not disoriented and showed the seasonally appropriate migratory direction. On the contrary, the same birds placed in a homogeneous 5 nT OMF generated by large stationary coils showed clear disorientation. On the basis of these findings, we suggest that the disruption of magnetic orientation of birds by oscillating magnetic fields is not related to photochemical magnetoreceptors in their eyes.
Asunto(s)
Ojo/efectos de la radiación , Campos Magnéticos , Pájaros Cantores/fisiología , Migración Animal , Animales , Proteínas Aviares/metabolismo , Estaciones del Año , Taxia/efectos de la radiaciónRESUMEN
One of the key requirements for the diagnosis of pulmonary tuberculosis is the identification of M. tuberculosis in tissue. In this paper, we present the advantages of specific fluorescent antibody labelling, combined with laser scanning confocal microscopy (LSCM), for the detection of M. tuberculosis in histological specimens of lung tissues. We demonstrate that the application of LSCM allows: (i) The automatic acquisition of images of the whole slice and, hence, the determination of regions for subsequent analysis; (ii) the acquisition of images of thick (20-40 µm) slices at high resolution; (iii) single bacteria identification; and (iv) 3D reconstruction, in order to obtain additional information about the distribution, size, and morphology of solitary M. tuberculosis; as well as their aggregates and colonies, in various regions of tuberculosis inflammation. LSCM allows for the discrimination of the non-specific fluorescence of bacteria-like particles and their aggregates presented in histological lung samples, from the specific fluorescence of labelled M. tuberculosis, using spectrum emission analysis. The applied method was effective in the identification of M. tuberculosis in lung histological samples with weak Ziehl-Neelsen staining. Altogether, combining immunofluorescent labelling with the application of LSCM visualization significantly increases the effectiveness of M. tuberculosis detection.
RESUMEN
The propensity of multi-walled carbon nanotubes (MWCNTs) for biodegradation is important for their safe use in medical and technological applications. Here, we compared the oxidative degradation of two samples of industrial-grade MWCNTs-we called them MWCNT-d and MWCNT-t-upon their treatment with sodium hypochlorite (NaOCl). The MWCNTs had a similar inner diameter but they differed about 2-fold in the outer diameter. Electron microscopy combined with morphometric analysis revealed the different degradation of the two types of MWCNTs after their incubation with NaOCl-the thicker MWCNT-d were damaged more significantly than the thinner MWCNT-t. The both types of MWCNTs degraded at the inner side, but only MWCNT-d lost a significant number of the outer graphitic layers. Raman spectroscopy demonstrated that both MWCNTs had a similar high defectiveness. Using energy-dispersive X-ray spectroscopy, we have shown that the more degradable MWCNT-d contained the same level of oxygen as MWCNT-t, but more metal impurities. The obtained results suggest that the biodegradability of MWCNTs depends not only on the wall thickness but also on the defects and impurities. Thus, the biodegradability can be regulated by the synthesis conditions or the post-synthesis modifications. Such degradation flexibility may be important for both medical and industrial applications.
RESUMEN
Entosis, or cell death by invading another cell, is typical for tumor epithelial cells. The formation of cell-in-cell structures is extensively studied in suspension cultures, but remains poorly understood in substrate-dependent cells. Here, we used electron, confocal and time-lapse microscopy in combination with pharmacological inhibition of intracellular components to study the kinetics of entosis using two human substrate-dependent tumor cultures, A431 and MCF7. In total, we identified and characterized five consecutive stages of entosis, which were common for both examined cell lines. We further demonstrated that actin filaments in the entotic as well as invading cells were crucial for entosis. Microtubules and the Golgi apparatus of entotic cells provided membrane expansion required for internalization of the invading cell. Depolymerization of microfilaments and microtubules, and disintegration of the Golgi complex inhibited entosis. We confirmed the presence of adhesive junctions and discovered the formation of desmosomes between the invading and entotic cells. The internalized cell was shown to be degraded due to the lysosomal activation in both cells whereas the disintegration of the Golgi apparatus did not affect the process. Thus, in the substrate-dependent cultures, entosis requires microfilaments, microtubules and the Golgi complex for cell invasion, but not for internalized cell degradation.