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Flow cytometry (FC) is widely used in microbiology, immunology, hematology, and oncology. In the veterinary field, FC enabled the study of the immune response in cattle infected with different pathogens, as well as vaccine testing. However, few fluorochrome-conjugated antibodies recognize bovine antigens, limiting the possible benefits of FC and the implementation of multiparametric analysis for more complex studies. Two cytometry panels with five colors each were designed and implemented for the study and identification of populations and subpopulations of T cells derived from the peripheral blood mononuclear cells of dairy heifers. Both panels detected differences in T cell subpopulations between heifers positively and negatively tested for tuberculin; they detected overexpression of CD25+ and CD45RO+ in tuberculin-positive heifers after stimulation with a culture filtrate protein extract (CFPE) from Mycobacterium bovis (M. bovis). We identified subpopulations of T cells from peripheral blood mononuclear cells using two multicolor panels. These panels could be used to analyze total bovine blood in immunopathogenic studies and vaccine development. The same strategy could be implemented in other species of veterinary interest.
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Drug therapy for leishmaniasis remains a major challenge as currently available drugs have limited efficacy, induce serious side-effects and are not accessible to everyone. Thus, the discovery of affordable drugs is urgently needed. Chalcones present a great potential as bioactive agents due to simple structure and functionalization capacity. The antileishmanial activity of different natural and synthetic chalcones have been reported. Here we report the synthesis of twenty-five novel prenylated chalcones that displayed antiparasitic activity in Leishmania mexicana. All the chalcones were evaluated at 5 µg/mL and eleven compounds exhibited a metabolic inhibition close to or exceeding 50%. Compounds 49, 30 and 55 were the three most active with IC50 values < 10 µM. These chalcones also showed the highest selectivity index (SI) values. Interestingly 49 and 55 possessing a substituent at a meta position in the B ring suggests that the substitution pattern influences antileishmanial activity. Additionally, a tridimensional model of fumarate reductase of L. mexicana was obtained by homology modeling. Docking studies suggest that prenylated chalcones could modulate fumarate reductase activity by binding with good affinity to two binding sites that are critical for the target. In conclusion, the novel prenylated chalcones could be considered as promising antileishmanial agents.
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Antiprotozoarios , Chalconas , Leishmaniasis , Humanos , Chalconas/química , Succinato Deshidrogenasa , Éteres , Antiprotozoarios/química , Leishmaniasis/tratamiento farmacológico , Relación Estructura-ActividadRESUMEN
Leishmaniasis is a neglected vector-borne disease; there are different manifestations of the diseases and species involved, and cutaneous leishmaniasis caused by Leishmania (L.) mexicana is the most prevalent in Mexico. Currently, the drugs available for the treatment of leishmaniasis are toxic, expensive, and often ineffective; therefore, it is imperative to carry out research and development of new therapeutic alternatives, with natural products being an attractive option. In particular, oregano is a plant with worldwide distribution; in Mexico, two species: Lippia berlandieri Schauer and Poliomintha longiflora Gray are endemic. Both essential oils (EO's) have been reported to have antimicrobial activity attributed to their main components, thymol and carvacrol. In this research, the leishmanicidal effect and mechanism of cell death induced by L. berlandieri EO, P. longiflora EO, thymol, and carvacrol in L. mexicana promastigotes were determined in vitro. Additionally, the cytotoxic activity in mammalian cells was evaluated. L. berlandieri EO presented higher leishmanicidal activity (IC50 = 41.78 µg/mL) than P. longiflora EO (IC50 = 77.90 µg/mL). Thymol and carvacrol were the major components of both Mexican oregano EO's. Thymol presented higher leishmanial inhibitory activity (IC50 = 22.39 µg/mL), above that of carvacrol (IC50 = 61.52 µg/mL). All the EO's and compounds evaluated presented lower cytotoxic activity than the reference drug; thymol was the compound with the best selectivity index (SI). In all cases, apoptosis was identified as the main mechanism of death induced in the parasites. The leishmanicidal capacity of the Mexican oregano EO is an accessible and affordable alternative that can be further explored.
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Lamiaceae , Leishmania mexicana , Leishmania , Lippia , Aceites Volátiles , Origanum , Animales , Apoptosis , Muerte Celular , Mamíferos , México , Aceites Volátiles/farmacología , Aceites de Plantas/farmacología , Timol/análisis , Timol/farmacologíaRESUMEN
The biological activity of essential oils and their major components is well documented. Essential oils such as oregano and cinnamon are known for their effect against bacteria, fungi, and even viruses. The mechanism of action is proposed to be related to membrane and external cell structures, including cell walls. This study aimed to evaluate the biological activity of seven essential oils and eight of their major components against Gram-negative and Gram-positive bacteria, filamentous fungi, and protozoans. The antimicrobial activity was evaluated by determination of the Minimal Inhibitory Concentration for Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Salmonella Typhimurium, Shigella sonnei, Aspergillus niger, Aspergillus ochraceus, Alternaria alternata, and Fusarium oxysporium, the half-maximal inhibitory concentration (IC50) for Trypanosoma cruzi and Leishmania mexicana, and the median lethal dose (LD50) for Giardia lamblia. Results showed that oregano essential oil showed the best antibacterial activity (66-100 µg/mL), while cinnamon essential oil had the best fungicidal activity (66-116 µg/mL), and both showed excellent antiprotozoal activity (22-108 µg/mL). Regarding the major components, thymol and carvacrol were also good antimicrobials (23-200 µg/mL), and cinnamaldehyde was an antifungal compound (41-75 µg/mL). The major components were grouped according to their chemical structure as phenylpropanoids, terpenoids, and terpinenes. The statistical analysis of the grouped data demonstrated that protozoans were more susceptible to the essential oils, followed by fungi, Gram-positive bacteria, and Gram-negative bacteria. The analysis for the major components showed that the most resistant microbial group was fungi, which was followed by bacteria, and protozoans were also more susceptible. Principal Component Analysis for the essential oils demonstrated the relationship between the biological activity and the microbial group tested, with the first three components explaining 94.3% of the data variability. The chemical structure of the major components was also related to the biological activity presented against the microbial groups tested, where the three first principal components accounted for 91.9% of the variability. The external structures and the characteristics of the cell membranes in the different microbial groups are determinant for their susceptibility to essential oils and their major components.
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Dengue is a global health problem without current specific treatment nor safe vaccines available. While severe dengue is related to pre-existing non-neutralizing dengue virus (DENV) antibodies, the role of T cells in protection or pathology is unclear. Using cutaneous DENV infection in immunocompetent mice we previously showed the generation of PNA+ germinal centers (GCs), now we assessed the activation and proliferation of B and T cells in draining lymph nodes (DLNs). We found a drastic remodelling of DLN compartments from 7 to 14 days post-infection (dpi) with greatly enlarged B cell follicles, occupying almost half of the DLN area compared to ~24% in naïve conditions. Enormous clusters of proliferating (Ki-67+) cells inside B follicles were found 14 dpi, representing ~33% of B cells in DLNs but only ~2% in non-infected mice. Inside GCs, we noticed an important recruitment of tingle body macrophages removing apoptotic cells. In contrast, the percentage of paracortex area and total T cells decreased by 14-16 dpi, compared to controls. Scattered randomly distributed Ki-67+ T cells were found, similar to non-infected mice. CD69 expression by CD4+ and CD8+ T cells was minor, while it was remarkable in B cells, representing 1764.7% of change from basal levels 3 dpi. The apparent lack of T cell responses cannot be attributed to apoptosis since no significant differences were observed compared to non-infected mice. This study shows massive B cell activation and proliferation in DLNs upon DENV infection. In contrast, we found very poor, almost absent CD4+ and CD8+ T cell responses.
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Virus del Dengue , Dengue , Animales , Linfocitos B , Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T , RatonesRESUMEN
BACKGROUND: Tuberculosis is the leading cause of death by an infectious microorganism worldwide. Conventional treatment lasts at least six months and has adverse effects; therefore, it is important to find therapeutic alternatives that reduce the bacterial load and may reduce the treatment duration. The immune response against tuberculosis can be modulated by several mechanisms, including extracellular vesicles (EVs), which are nano-sized membrane-bound structures that constitute an efficient communication mechanism among immune cells. METHODS: The EVs released by the J774A.1 mouse macrophage cell line, both spontaneously (S-EV) and after infection with Mycobacterium tuberculosis H37Rv (Mtb-EV), were purified by ultra-centrifugation and size-exclusion chromatography. The size distribution and chemical composition of these EVs were evaluated, and their effect on the bacterial load and the production of cytokines was determined in both in vitro and in vivo models of M. tuberculosis infection. RESULTS: Mtb-EV are larger than S-EV, they contain M. tuberculosis-specific antigens (not detected in EVs released from M. fortuitum-infected J774A.1 cells) and are rich in phosphatidylserine, present in their outer membrane layer. S-EV, but not Mtb-EV, reduced the bacterial load and the production of MCP-1 and TNF-α in M. tuberculosis-infected macrophages, and these effects were reversed when phosphatidylserine was blocked with annexin V. Both S-EV and Mtb-EV significantly reduced the lung bacterial load in mice infected with M. tuberculosis after 60 days of treatment, but they had no effect on survival or on the lung pneumonic area of these mice. CONCLUSION: J774A.1 macrophages infected with M. tuberculosis H37Rv released EVs that differed in size and phosphatidylserine content from spontaneously released EVs, and these EVs also had different biological effects: S-EV reduced the mycobacterial load and the cytokine production in vitro (through a phosphatidylserine-dependent mechanism), while both EVs reduced the lung bacterial load in vivo. These results are the basis for further experiments to evaluate whether EVs improve the efficiency of the conventional treatment for tuberculosis.
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Vesículas Extracelulares/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiología , Tuberculosis/terapia , Animales , Carga Bacteriana , Línea Celular , Citocinas/metabolismo , Modelos Animales de Enfermedad , Vesículas Extracelulares/química , Vesículas Extracelulares/trasplante , Masculino , Ratones Endogámicos BALB C , Mycobacterium tuberculosis/patogenicidad , Tuberculosis/microbiologíaRESUMEN
Ursolic and oleanolic acids are natural isomeric triterpenes known for their anticancer activity. Here, we investigated the effect of triterpenes on the viability of A549 human lung cancer cells and the role of autophagy in their activity. The induction of autophagy, the mitochondrial changes and signaling pathway stimulated by triterpenes were systematically explored by confocal microscopy and western blotting. Ursolic and oleanolic acids induce autophagy in A549 cells. Ursolic acid activates AKT/mTOR pathways and oleanolic acid triggers a pathway independent on AKT. Both acids promote many mitochondrial changes, suggesting that mitochondria are targets of autophagy in a process known as mitophagy. The PINK1/Parkin axis is a pathway usually associated with mitophagy, however, the mitophagy induced by ursolic or oleanolic acid is just dependent on PINK1. Moreover, both acids induce an ROS production. The blockage of autophagy with wortmannin is responsible for a decrease of mitochondrial membrane potential (Δψ) and cell death. The wortmannin treatment causes an over-increase of p62 and Nrf2 proteins promote a detoxifying effect to rescue cells from the death conducted by ROS. In conclusion, the mitophagy and p62 protein play an important function as a survival mechanism in A549 cells and could be target to therapeutic control.
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Mitofagia/efectos de los fármacos , Ácido Oleanólico/farmacología , Triterpenos/farmacología , Células A549 , Humanos , Proteínas Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ácido UrsólicoRESUMEN
The available drugs for treating Leishmaniasis and American trypanosomiasis have high toxicity and multiple side effects, among other problems. More effective and less toxic treatments are urgently needed. A series of chalcones that contained a prenyloxy or geranyloxy substituent was synthesized and characterized. Each substituent was attached to the A ring in some compounds and to the B ring in others, with additional substituents placed on the chalcone moiety. The present aim was to evaluate the effect of the substitution pattern on leishmanicidal and trypanocidal activity. When tested at a single concentration, the compounds exerting a metabolic inhibition close to or exceeding 50% for Leishmania mexicana were 11, 17 and 12, and for Trypanosoma cruzi were 11, 17, 15 and 26. Upon determining the selectivity index (SI =IC50/CC50), the values were 80.9, 1.24 and 55.12 for 11, 17 and 12 (respectively) versus L. mexicana, and 75.1, 1.43, 27.36 and 33.52 for 11, 17, 15 and 26 (respectively) versus T. cruzi. Structural isomers 11 and 17 showed activity for both the L. mexicana and T. cruzi strains, though the greater cytotoxic activity of 17 led to a lower SI. Compounds 12, 15 and 26 were species specific. For T. cruzi, the SI was higher for 11, 15 and 26 than for the reference drugs nifurtimox and benznidazole. The examination of promastigote morphology after exposing L. mexicana and T. cruzi to 11 revealed a decrease in cell density. The current findings suggest that 11 could be a useful lead compound for further SAR studies.
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Antiprotozoarios/síntesis química , Chalconas/síntesis química , Chalconas/farmacología , Diseño de Fármacos , Tripanocidas/síntesis química , Animales , Antiprotozoarios/farmacología , Enfermedad de Chagas/tratamiento farmacológico , Humanos , Leishmania mexicana/efectos de los fármacos , Leishmaniasis/tratamiento farmacológico , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacosRESUMEN
Leishmaniasis is a neglected tropical disease caused by the parasite of the genus Leishmania. About 13 million people are infected worldwide, and it is estimated that 350 million are at risk of infection. Clinical manifestations depend on the parasite species and factors related to the host such as the immune system, nutrition, housing, and financial resources. Available treatments have severe side effects; therefore, research currently focuses on finding more active and less toxic compounds. Quinoxalines have been described as promising alternatives. In this context, 17 isopropyl quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives were evaluated as potential leishmanicidal agents. Their effect on the cell metabolism of Leishmania mexicana promastigotes and their cytotoxic effects on the J774.A1 cell line and on erythrocytes were evaluated, and their selectivity index was calculated. Compounds T-069 (IC50 = 1.49 µg/mL), T-070 (IC50 = 1.71 µg/mL), T-072 (IC50 = 6.62 µg/mL), T-073 (IC50 = 1.25 µg/mL), T-085 (IC50 = 0.74 µg/mL), and T-116 (IC50 = 0.88 µg/mL) were the most active against L. mexicana promastigotes and their mechanism of action was characterized by flow cytometry and microscopy. Compound T-073, the most selective quinoxaline derivative, induced cell membrane damage, phosphatidylserine exposition, reactive oxygen species production, disruption of the mitochondrion membrane potential, and DNA fragmentation, all in a dose-dependent manner, indicating the induction of regulated necrosis. Light and transmission electron microscopy showed the drastic morphological changes induced and the mitochondrion as the most sensitive organelle in response to T-073. This study describes the mechanism by which active isopropyl quinoxaline-7-carboxylate 1,4-di-N-oxide quinoxalines affect the parasite.
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Antiprotozoarios/farmacología , Leishmania mexicana/efectos de los fármacos , Quinoxalinas/farmacología , Animales , Muerte Celular/efectos de los fármacos , Línea Celular , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Quinoxalinas/química , Especies Reactivas de OxígenoRESUMEN
BACKGROUND: Quinoxalines have shown a wide variety of biological activities including as antitumor agents. The aims of this study were to evaluate the activity of quinoxaline 1,4-di-N-oxide derivatives on K562 cells, the establishment of the mechanism of induced cell death, and the construction of predictive QSAR models. MATERIAL AND METHODS: Sixteen esters of quinoxaline-7-carboxylate 1,4-di-N-oxide were evaluated for antitumor activity on K562 chronic myelogenous leukemia cells and their IC50 values were determined. The mechanism of induced cell death by the most active molecule was assessed by flow cytometry and an in silico study was conducted to optimize and calculate theoretical descriptors of all quinoxaline 1,4-di-N-oxide derivatives. QSAR and QPAR models were created using genetic algorithms. RESULTS & CONCLUSIONS: Our results show that compounds C5, C7, C10, C12 and C15 had the lowest IC50 of the series. C15 was the most active compound (IC50= 3.02 µg/mL), inducing caspase-dependent apoptotic cell death via the intrinsic pathway. QSAR and QPAR studies are discussed.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Óxidos N-Cíclicos/farmacología , Relación Estructura-Actividad Cuantitativa , Teoría Cuántica , Quinoxalinas/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Óxidos N-Cíclicos/síntesis química , Óxidos N-Cíclicos/química , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Células K562 , Estructura Molecular , Quinoxalinas/síntesis química , Quinoxalinas/química , Células Tumorales CultivadasRESUMEN
Amphipterygium adstringens is an endemic species in Mexico commonly known as "cuachalalate." Healers to treat gastritis, gastric ulcers, and gastrointestinal cancer have traditionally used the bark. We investigated the effects of alcoholic extract of A. adstringens (AaEE) in DSS-induced colitis in mice. The protective effect of AaEE was determined at 200 mg/kg by oral gavage for 10 days. We determine the effect of AaEE on clinical features (disease activity index), antioxidants, anti-inflammatory, and immunomodulatory activities in relation to the activity of SOD, CAT, and GPx, levels of proinflammatory cytokines, and changes both macroscopic and microscopic of the colonic mucosa. AaEE significantly reduced the inflammation of colon and significantly increased SOD and GPx activities. AaEE also significantly decreased TNF-α, IFN-γ, and IL-1ß cytokine levels compared to DSS-treated mice and reduced both infiltration of inflammatory cells and the mucosal damage in colon. The results suggested the protective potential of AaEE in DSS-induced colitis and this might be attributed to its phytochemicals compounds that have been found to induce a wide spectrum of activities such as reduction in oxidative stress, suppression of inflammation, modulating numerous signal transduction pathways, and induction of apoptosis. The findings of this study suggest that AaEE has substantial potential for the treatment of inflammatory colitis.
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Colitis Ulcerosa/tratamiento farmacológico , Extractos Vegetales/farmacología , Sapindaceae/química , Animales , Antioxidantes/metabolismo , Apoptosis , Catalasa/metabolismo , Colitis Ulcerosa/inducido químicamente , Colon/efectos de los fármacos , Citocinas/metabolismo , Sulfato de Dextran , Femenino , Glutatión Peroxidasa/metabolismo , Inflamación/tratamiento farmacológico , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos BALB C , Estrés Oxidativo , Superóxido Dismutasa/metabolismoRESUMEN
Bovine lactoferrin (bLf) up-modulates intestinal IgA that is essential for homeostasis and which might confer protection to the distal small intestine that is vulnerable to inflammation. This study analyzed the effects of bLf administered orally on the IgA response at inductive (Peyer's patches) and effector (lamina propria) sites of the distal small intestine in mice. Groups of five healthy male BALB/c mice were orally treated with 5 mg of bLf for 7, 14, 21, or 28 days. Then, mice were killed and the distal small intestine was dissected. Intestinal fluid samples were analyzed to determine IgA and IgM levels by enzyme-immuno assay. Peyer's patches and lamina propria were analyzed for IgA(+) or IgM(+) plasma cells, B, CD4(+) T and CD8(+) T cells as well as CD4(+) T cells positive for either pro-inflammatory cytokines [tumor necrosis factor (TNF)-α, interferon-γ and interleukin (IL)-12] or for IgA-producing ILs (IL-4, -5, -10 and -6) by cytofluorometry. Antibodies, antibody-secreting cells, and B and T responses in both Peyer's patches and lamina propria were higher in bLf-treated than bLf-untreated mice. The generation of IL-10 and IL-6 CD4(+) T cells in Peyer's patches or TNF-α and IL-12 CD4(+) T cells in lamina propria showed similar response patterns. On days 14 and 28, cytokine/IL CD4(+) T cell responses were increased in Peyer's patches or decreased in lamina propria. The effect of bLf on the elicitation of IgA indicates a potential application of bLf as a nutraceutical to control inflammation in the distal small intestine.
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Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Inmunomodulación , Inflamación/inmunología , Intestino Delgado/efectos de los fármacos , Lactoferrina/administración & dosificación , Administración Oral , Animales , Bovinos , Citocinas/metabolismo , Suplementos Dietéticos , Humanos , Inmunidad Humoral/efectos de los fármacos , Inmunoglobulina A/metabolismo , Inflamación/tratamiento farmacológico , Mediadores de Inflamación/metabolismo , Intestino Delgado/inmunología , Lactoferrina/efectos adversos , Masculino , Ratones Endogámicos BALB CRESUMEN
PD-L1 expression plays a critical role in the impairment of T cell responses during chronic infections; however, the expression of PD-L1 on T cells during acute viral infections, particularly during the pandemic influenza virus (A(H1N1)pdm09), and its effects on the T cell response have not been widely explored. We found that A(H1N1)pdm09 virus induced PD-L1 expression on human dendritic cells (DCs) and T cells, as well as PD-1 expression on T cells. PD-L1 expression impaired the T cell response against A(H1N1)pdm09 by promoting CD8⺠T cell death and reducing cytokine production. Furthermore, we found increased PD-L1 expression on DCs and T cells from influenza-infected patients from the first and second 2009 pandemic waves in Mexico City. PD-L1 expression on CD8⺠T cells correlated inversely with T cell proportions in patients infected with A(H1N1)pdm09. Therefore, PD-L1 expression on DCs and T cells could be associated with an impaired T cell response during acute infection with A(H1N1)pdm09 virus.
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Antígeno B7-H1/genética , Regulación de la Expresión Génica , Subtipo H1N1 del Virus de la Influenza A/inmunología , Gripe Humana/genética , Gripe Humana/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Adolescente , Adulto , Anciano , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Estudios de Casos y Controles , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Transducción de Señal , Adulto JovenRESUMEN
Recently it was discovered that neutrophils can generate extracellular fibers called NET (neutrophil extracellular traps), which are composed of a skeleton of DNA "decorated" with many cytoplasmic including enzymes and nuclear components.The NET are a physical barrier that prevent the spread of microorganisms and facilitate the cell death by promoting a high local concentration of antimicrobial molecules. On the other hand, the fibrous structure limits the damage to the tissue where they are generated by restricting the range of molecules that are released by the neutrophil. This paper describes this new form of cell death and the implications this may have on different diseases.
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Autofagia/fisiología , Neutrófilos/fisiología , Humanos , Neutrófilos/ultraestructuraRESUMEN
The effects of stress on the mucosal immune responses in inflammatory disorders of the gut, as well as on salivary and intestinal IgA levels are well known. However, its effects on the structure and function of the NALT have not yet been reported, and are examined in the present study. Balb/c mice were submitted to restraint stress for 3h per day during 4 or 8d. The immunohistochemistry and flow cytometric analysis revealed that repeated restraint stress (4 and 8d) decreased the percentage, compared to the control group, of CD3(+) and CD4(+) T cells, without affecting the percentage of CD8(+) T cells or B220(+) cells (B cells). The numbers of IELs (CD4(+) and CD8(+) T cells) were lower at 4d of stress and higher at 8d. IgA(+) cells in NALT and nasal IgA levels showed a similar pattern, being significantly lower at 4d of stress and significantly higher at 8d. In summary, repeated restraint stress altered the distribution and number of lymphocytes and IgA(+) cells in nasal mucosa, probably due to changes in norepinephrine and corticosterone levels.