Recent progress in application of nanovaccines for enhancing mucosal immune responses

Mucosal vaccines that stimulate both mucosal and systemic immune responses are desirable, as they could prevent the invading pathogens at their initial infection sites in a convenient and user-friendly way. Nanovaccines are receiving increasing attention for mucosal vaccination due to their merits in overcoming mucosal immune barriers and in enhancing immunogenicity of the encapsulated antigens. Herein, we summarized several nanovaccine strategies that have been reported for enhancing mucosal immune responses, including designing nanovaccines that have superior mucoadhesion and mucus penetration capacity, designing nanovaccines with better targeting efficiency to M cells or antigen-presenting cells, and co-delivering adjuvants by using nanovaccines. The reported applications of mucosal nanovaccines were also briefly discussed, including prevention of infectious diseases, and treatment of tumors and autoimmune diseases. Future research progresses in mucosal nanovaccines may promote the clinical translation and application of mucosal vaccines.

The construction of nasal cavity-mimic M-cell model, design of M cell-targeting nanoparticles and evaluation of mucosal vaccination by nasal administration

In order to better evaluate the transport effect of nanoparticles through the nasal mucosa, an nasal cavity-mimic model was designed based on M cells. The differentiation of M cells was induced by co-culture of Calu-3 and Raji cells in invert model. The ZO-1 protein staining and the transport of fluorescein sodium and dexamethasone showed that the inverted co-culture model formed a dense monolayer and possessed the transport ability. The differentiation of M cells was observed by up-regulated expression of Sialyl Lewis A antigen (SLAA) and integrin 1, and down-regulated activity of alkaline phosphatase. After targeting M cells with iRGD peptide (cRGDKGPDC), the transport of nanoparticles increased. , the co-administration of iRGD could result in the increase of nanoparticles transported to the brain through the nasal cavity after intranasal administration. In the evaluation of immune effect , the nasal administration of OVA-PLGA/iRGD led to more release of IgG, IFN-, IL-2 and secretory IgA (sIgA) compared with OVA@PLGA group. Collectively, the study constructed M cell model, and proved the enhanced effect of targeting towards M cell with iRGD on improving nasal immunity.

Morphological and Immunohistochemical Identification of Villous M Cells in the Small Intestine of Newborn Piglets / Identificación Morfológica e Inmunohistoquímica de los Epiteliocitos Microplegados de las Vellosidades en el Intestino Delgado de Lechones Recién Nacidos

Microfold (M) cells act as antigen-sampling sites for initiating antigen specific mucosal immune responses, but they may also provide a gateway for enteropathogen entry. In this study we demonstrated villous M cells by morphological and immunohistochemical methods to be present in the three regions of the small intestine from newborn piglets. Immunohistochemical analysis, using anti- cytokeratin 18 (CK18) primary antibodies, showed a gradually decreased density of M cells from the lower crypt epithelium to the upper villous epithelium. The proportion of villous M cells was greater in the ileum than in the duodenum or the mid-jejunum. Ultrastructural observation revealed that villous M cells were mainly columnar in shape in the duodenum and the mid-jejunum, and appeared as more pocket-like structure in the ileum. These villous M cells exhibited short and irregular microvilli, rich vesicles and reduced glycocalyx, but lacked the lymphocyte-containing basolateral invagination. Our results support evidence that M cells can develop in the small intestinal villous epithelium of newborn piglets, implying that villous M cells may begin developing in the pig's small intestine during fetal stages, which depends neither on the influence of the mucosal lymphoid tissue nor the antigen from the intestinal lumen stimulation. In addition, the variable morphology and heterogeneity distribution of villous M cells in the three regions of the small intestine may be indicative of its different functional properties. This information extent our understanding of the diversity of M cells and provides important basic knowledge for further research on the actual role of villous M cells in neonate.

Los epiteliocitos microplegados (células M) actúan como receptores de antígeno para iniciar la respuesta inmune específica de las mucosas, pero también pueden proporcionar una puerta de entrada para enteropatógenos. En este estudio, se demostró por métodos morfológicos e inmunohistoquímicos que los epiteliocitos microplegados de las vellosidades están presentes en las tres regiones del intestino delgado de lechones recién nacidos. Se utilizaron anticuerpos primarios de citoqueratina 18 (CK18) para el análisis inmunohistoquímico, el cual mostró una disminución gradual de la densidad de los epiteliocitos microplegados desde el epitelio de las criptas inferiores hasta el epitelio de las vellosidades superiores. La proporción de los epiteliocitos microplegados, fue mayor en el íleon que el duodeno o yeyuno medio. La observación ultraestructural reveló que los epiteliocitos microplegados fueron principalmente de forma columnar en el duodeno y el yeyuno medio. Además, mostraron microvellosidades cortas e irregulares, muchas vesículas y glucocáliz reducidos, pero carecían de invaginaciones basolaterales contenedoras de linfocitos. Nuestros resultados apoyan la evidencia de que los epiteliocitos microplegados pueden desarrollarse en el epitelio de las vellosidades intestinales de los lechones recién nacidos, lo que implica que estas células pueden comenzar a desarrollarse en el intestino delgado del cerdo durante las etapas fetales, y no dependen ni de la influencia del tejido linfoide de las mucosas ni del antígeno para la estimulación del lumen intestinal. Además, la morfología y heterogeneidad de distribución de los epiteliocitos microplegados en las tres regiones del intestino delgado pueden ser indicativas de sus diferentes propiedades funcionales. Esta información mejora nuestra comprensión de la diversidad de los epiteliocitos microplegados y proporciona conocimientos básicos importantes para la investigación sobre el papel de los epiteliocitos microplegados en las vellosidades del neonato.

Expression of the ATP-gated P2X7 Receptor on M Cells and Its Modulating Role in the Mucosal Immune Environment

Interactions between microbes and epithelial cells in the gastrointestinal tract are closely associated with regulation of intestinal mucosal immune responses. Recent studies have highlighted the modulation of mucosal immunity by microbe-derived molecules such as ATP and short-chain fatty acids. In this study, we undertook to characterize the expression of the ATP-gated P2X7 receptor (P2X7R) on M cells and its role in gastrointestinal mucosal immune regulation because it was poorly characterized in Peyer's patches, although purinergic signaling via P2X7R and luminal ATP have been considered to play an important role in the gastrointestinal tract. Here, we present the first report on the expression of P2X7R on M cells and characterize the role of P2X7R in immune enhancement by ATP or LL-37.

T-2 toxin inhibits mitochondrial function of differentiated murine embryonic stem cells / 中国药理学与毒理学杂志

OBJECTIVE ToexplorethepossiblemechanismoractiontargetsofT-2toxinembryo toxicity by observing the effect of T-2 toxin on mitochondrial function of differentiated murine e mbryonic stemcells(mESCs).METHODS Duringdifferentiationat24,72and120h,ESCswereexposedto T-2 toxin 0.5 μg·L-1 .Meanwhile,mESCs were pre-treated with antioxidant Trolox (200 μmol·L-1 )for 30 min and exposed to T-2 toxin (0.5 μg·L-1 )for 72 h.The mitochondrial ultrasture of differentiated mESCs was observed under a transi mission electrical microscope (TEM).The differentiated ESC mito-chondrial function,including respiratory control ratio (RCR),ATP synthase activity and mitochondrial membranepotential(MMP),wasmeasuredat144hafterdifferentiation.RESULTS Significant decrease of the mitochondrial number,deformation of mitochondrial structure,and lack of complete mito-chodrial crest were observed through TEM in the groups of T-2 toxin exposed for 72 and 1 20 h,respec-tively.Compared with the normal control group,RCR declined by 49.5% and 55.1%,ATP synthase activity decreased by 84.9% and 89.3%,and MMP decreased by 23.2% and 35.2% in T-2 toxin 0.5 μg·L-1 exposure 72 and 1 20 h group,respectively.However,the inhibition of mitochondrial function by T-2 toxin in differentiated mESCs recovered significantly in the presence of the antioxidant Trolox. CONCLUSION T-2toxininducesoxidativestressandinhibitsmESCsmitochondrialfunctionindifferenti-ated mESCs,and ROS-induced mitochondrial malfunction plays an i mportant role in T-2 toxin e mbryonic toxicity mechanis m.

Effect of phosphocreatine on transient outward potassium current in ischemic ventricular mid-myocardial cells of rats / 中国病理生理杂志

AIM: To determine the effect of exogenous phosphocreatine (PCr) at different concentrations on transient outward potassium (Ito) current in rat ischemic ventricular mid-myocardial (M) cells and to explore the antiarrhythmia mechanism in the treatment of ischemic heart disease. METHODS: M cells were isolated enzymatically from left ventricular mid-myocardium of rats. Peak Ito current was recorded by patch-clamp technique in the whole-cell configuration when M cells were superfused with normal Tyrode solution,simple ischemic solution,and simulated ischemic solution containing PCr at concentrations of 5,10,20 and 30 mmol/L for 10 min. RESULTS: Peak Ito current density of M cells superfused with simple simulated ischemic solution was significantly reduced by (76.1±6.3)% (P0.05). CONCLUSION: PCr reverses the inhibition of Ito current under ischemic condition in M cells,which may be the mechanism responsible for arrhythmia prevention in ischemic heart disease. PCr at concentrations of 0～10 mmol/L exerts significant dose-effect relationship.

Apoptosis induction by E2F decoy DNA of the prostate cancer cell line

To observe effect of E2F decoy DNA on proliferation and apoptosis of androgen-independent prostate cancer cell line PC-3M the binding specificity of the E2F decoy DNA to the PC-3M nuclear protein was detected by electrophoretic mobility shift assay (EMSA). E2F decoy DNA, ARE decoy DNA and Control decoy DNA were respectively transfected into PC-3M cells with lipofectamine.Their influence on cell proliferative activity was detected by MTT assay.The cell apoptotic rate was determined by flow cytometric(FCM) analysis and chromosome DNA ladder was detected by DNA gel electrophoresis.The change of mRNA expression of C-myc and CyclinD1 were detected by RT-PCR.The change of mRNA expression of C-myc and CyclinD1 were detected by Western-blot. EMSA demonstrated specific binding of the E2F decoy to E2F transcription factor.The PC-3M cell growth was inhibited after transfection. The apoptotic rate was 26.35 percent and DNA ladder could be observed after transfection.The expression of C-myc and CyclinD1 were inhibited. All these results indicated that E2F decoy DNA induced apoptosis of androgen-independent prostate cancer cell lines PC-3M and inhibited cell proliferation via inhibiting expression of C-myc and CyclinD1.

Effect of vitamin K_3 on apoptosis induced by androgen-independent prostate cancer cell PC-3M / 中国病理生理杂志

AIM:To study the effect of vitamin K3(VK3) on the induction of apoptosis in androgen-independent prostate cancer cell PC-3M in vitro.METHODS:Cell viability was estimated by MTT assay.AO/EB staining was performed to detect apoptotic cells.Apoptosis and the changes of cell cycle were detected by flow cytometry.NAC was used to observe the effect of growth inhibition by VK3.RT-PCR was used to confirm the changes in gene expression.Levels of intracellular peroxides were estimated by using an oxidation-sensitive fluorescent probe DCFH-DA.RESULTS:PC-3M cells growth was significantly inhibited by VK3(≥60 ?mol/L,P

Apoptosis of the prostate cancer cell line PC-3M induced by E2F decoy DNA / 中国病理生理杂志

AIM: To observe the effect of E2F decoy DNA on proliferation and apoptosis of androgen-independent prostate cancer cell line PC-3M.METHODS: E2F decoy DNA,ARE decoy DNA and control decoy DNA were transfected into PC-3M cells with lipofectamine,respectively.Their effects on cell proliferation were detected by MTT assay.The changes of cell morphology were observed by inverted phase contrast microscope.The cell apoptotic rate was determined by flow cytometry(FCM) analysis and chromosome DNA ladder was detected by DNA gel electrophoresis.The expression of c-Myc mRNA and cyclin D1 mRNA was detected by RT-PCR.The protein levels of c-Myc and cyclin D1 were detected by Western blotting.RESULTS: The growth of PC-3M cells was inhibited after transfection.The transfected PC-3M cells displayed typical apoptotic morphological changes.The apoptotic rate was 26.35% and DNA ladder was observed after transfection.The expression of c-Myc and cyclin D1 were inhibited.CONCLUSION: These results indicate that E2F decoy DNA induces apoptosis of androgen-independent prostate cancer cell lines PC-3M and inhibits cell proliferation via inhibiting expression of c-Myc and cyclin D1.