Distribution features of resident CD8 + T cells in human esophageal cancer tissues and its effects on the prognosis / 中华消化杂志

Objective:To explore the distribution features of resident CD8 + T cells infiltration in human esophageal cancer tissues and its clinical significance. Methods:Data from the Cancer Genome Atlas database were retrieved, the correlation between CD103 + CD8 + T cells and infiltration degree of conventional type 1 dendritic cell (cDC1), conventional type 2 dendritic cell (cDC2), type 3 dendritic cell(DC3) was investigated. From January 2006 to December 2008, 78 esophageal cancer tissues and 75 adjacent normal tissues from 78 esophageal cancer patients were collected by Shanghai Outdo Biotechnology Co., Ltd, the clinical data of patients was followed up by telephone until July 2015. The distribution of CD8 + T cells and CD103 + CD8 + T cells in cancer tissues and adjacent normal tissues was detected by multi-color labeling techniques and multispectral tissue imaging. The differences of the number and the ratio of CD8 + T cells and CD103 + CD8 + T cells in cancer tissues and adjacent normal tissues were compared. The Kaplan-Meier survival curves of patients with tissue infiltration of CD8 + T cells and CD103 + CD8 + T cells at different levels were drawn through the R language " survminer" package, and the best cut-off value was obtained. TNM stage, pathological stage and other clinical parameters of patients with high and low infiltration of CD8 + T cells, CD103 + CD8 + T cells were compared. Wilcoxon rank sum test, chi-square test, log-rank test and Cox proportional risk regression model statistical analysis were used to evaluate the prognostic value of the above indicators. Spearman correlation analysis was used for correlation analysis. Results:In the cancer tissues of patients with esophageal cancer, the infiltration degree of CD103 + CD8 + T cells was positively correlated with the infiltration degree of cDC1 cells, cDC2 cells and DC3 cells ( r=0.67, 0.53 and 0.47, all P<0.001). The percentage of CD8 + T cells in all cells in the whole tissue core of tumor tissues (63.09% (42.14%, 76.21%)) was higher than that of adjacent normal tissues (2.56% (1.68%, 5.38%)), and the difference was statistically significant ( U=41.00, P<0.001). The proportion of CD103 + CD8 + T cells in all cells in the whole tissue core of tumor tissues (7.92% (1.60%, 20.61%)) was higher than that of adjacent normal tissues (0.04% (0.01%, 0.10%)), and the difference was statistically significant ( U=857.50, P<0.001). The percentage of high CD8 + T cells infiltration in esophageal cancer tissues of patients with pathological stage Ⅰ+ Ⅱ was lower than that of patients with stage Ⅲ+ Ⅳ (57.9%, 33/57 vs. 85.7%, 18/21); the percentage of high CD103 + CD8 + T cells in CD8 + T cells in esophageal cancer tissues of patients with TNM stage Ⅰ+ Ⅱ was lower than that of patients with stage Ⅲ+ Ⅳ (21.6%, 8/37 vs. 48.8%, 20/41), and the differences were both statistically significant ( χ2=5.25 and 6.23, P=0.022 and 0.013). The results of Kaplan-Meier survival analysis and univariate Cox proportional risk regression model showed that the overall survival (OS) of patients with high CD8 + T cell infiltration was longer than that of patients with low CD8 + T cell infiltration ( HR=0.57, 95% confidence interval (95% CI) 0.34 to 0.96, P=0.034). There was no significant difference in OS between patients with high CD103 + CD8 + T cell infiltration and patients with low CD103 + CD8 + T cell infiltration ( HR=0.66, 95% CI 0.40 to 1.08, P>0.05). Conclusion:The high infiltration of CD103 + CD8 + T cells in esophageal cancer tissues are expected to be used as a prognostic predictor for patients with esophageal cancer, which is an important component of anti-tumor immune response in tumor microenvironment of esophageal cancer.

Assessment of BMP responses in an in vitro model of acute ethanol toxicity.

The assay presented here was designed to assess the immediate effects of ethanol (EtOH) exposure on intracellular signaling activated by BMPs (Bone Morphogenetic Proteins). Previous reports of the relationship between EtOH exposure and BMP-dependent signaling have primarily assessed the expression of individual BMPs, changes in BMP target genes or effects on the phosphorylation level of key downstream mediators after days or weeks of in vivo EtOH exposure. What happens to BMP-stimulated signaling immediately following exposure to EtOH remains largely unexplored. Here, the early events of BMP-evoked intracellular signaling were examined in an in vitro model of acute EtOH toxicity. The BMP/Ethanol Stimulation Assay involved first stimulating cultured cells with recombinant BMPs. BMP-evoked intracellular signaling was then allowed to develop for 30 minutes. Next, the cells were exposed to a range of EtOH concentrations for an additional 30 minutes. Finally, the cultures were processed for Western blot analysis or immunofluorescent labeling. This short-term assay: • Permits investigation of EtOH exposure during the initial signaling events downstream of BMP receptor activation • Enables assessment of how the presence of BMPs might protect against cellular injury caused by toxic EtOH levels.

Type-specific impacts of silver on the protein profile of tomato (Lycopersicon esculentum L.).

Silver nanoparticles (AgNPs) are particularly among the widely used nanomaterials in medicine, industry, and agriculture. The small size and large surface area of AgNPs and other nanomaterials result in their high reactivity in biological systems. To better understand the effects of AgNPs on plants at the molecular level, tomato (Lycopersicon esculentum L.) seedlings were exposed to 30 mg/L silver in the form of nanoparticle (AgNPs), ionic (AgNO3), or bulk (Ag0) in 50% Hoagland media for 7 days. The effects of silver on the expression of plant membrane transporters H+-ATPase, vacuolar type H+-ATPase (V-ATPase), and enzymes isocitrate dehydrogenase (IDH), and catalase in roots was assessed using RT-qPCR and immunofluorescence-confocal microscopy. We observed significantly higher expression of catalase in plants exposed to AgNPs (Fold of expression 1.1) and AgNO3 (Fold of expression 1.2) than the control group. The immunofluorescence imaging of the proteins confirmed the gene expression data; the expression of the enzyme catalase was upregulated 41, 216, and 770% higher than the control group in plants exposed to AgNPs, Ag0, and AgNO3, respectively. Exposure to AgnO3 resulted in the upregulation (fold of expression 1.2) of H+-ATPase and downregulation (fold of expression 0.7) of V-ATPase. A significant reduction in the expression of the redox-sensitive tricarboxylic cycle (TCA) enzyme mitochondrial IDH was observed in plants exposed to AgNPs (38%), AgNO3 (48%), or Ag0 (77%) compared to the control. This study shows that exposure to silver affects the expression of genes and protein involved in membrane transportation and oxidative response. The ionic form of silver had the most significant effect on the expression of genes and proteins compared to other forms of silver. The results from this study improve our understanding about the molecular effects of different forms of silver on important crop species. Novelty statementSilver nanoparticles released into the environment can be oxidized and be transformed into ionic form. Both the particulate and ionic forms of silver can be taken by plants and affect plants physiological and molecular responses. Despite the extensive research in this area, there is a scarce of information about the effects of silver nanoparticles on the expression of membrane transporters especially H+-ATPase involved in regulating cells' electrochemical charge, and the activity of enzymes involved in oxidative stress responses. This is a unique study that evaluates the expression of cellular proton transporters and enzymes of redox balance and energy metabolisms such as membrane transporters, H+-ATPase, and V-ATPases, and enzymes catalase and IDH. The results provide us valuable information about the impact of silver on plants at the molecular level by evaluating the expression of genes and proteins. Key MessageThe exposure of plants to silver as an environmental stressor affects the expression of genes and proteins involved in maintaining cell's electrochemical gradient (H+-ATPase, V-ATPase) and redox potential (IDH, catalase).

Immunofluorescence Labeling of Nuclear Receptor Expression in Formalin-Fixed, Paraffin-Embedded Tissue.

Immunofluorescent staining (IF) uses antigen-antibody complexes tagged with fluorochromes to observe the expression of proteins within a tissue sample. Multiple groups have described optimized methods to visualize several proteins simultaneously within the same tissue section using immunofluorescence in both mouse and human FFPE tissues. Our group routinely uses an optimized protocol described here to examine nuclear receptor expression in experimental samples from conditional knockout in vivo studies.

Phosphorylation of different tau sites during progression of Alzheimer's disease.

Alzheimer's disease is characterized by accumulation of amyloid plaques and tau aggregates in several cortical brain regions. Tau phosphorylation causes formation of neurofibrillary tangles and neuropil threads. Phosphorylation at tau Ser202/Thr205 is well characterized since labeling of this site is used to assign Braak stage based on occurrence of neurofibrillary tangles. Only little is known about the spatial and temporal phosphorylation profile of other phosphorylated tau (ptau) sites. Here, we investigate total tau and ptau at residues Tyr18, Ser199, Ser202/Thr205, Thr231, Ser262, Ser396, Ser422 as well as amyloid-ß plaques in human brain tissue of AD patients and controls. Allo- and isocortical brain regions were evaluated applying rater-independent automated quantification based on digital image analysis. We found that the level of ptau at several residues, like Ser199, Ser202/Thr205, and Ser422 was similar in healthy controls and Braak stages I to IV but was increased in Braak stage V/VI throughout the entire isocortex and transentorhinal cortex. Quantification of ThioS-stained plaques showed a similar pattern. Only tau phosphorylation at Tyr18 and Thr231 was already significantly increased in the transentorhinal region at Braak stage III/IV and hence showed a progressive increase with increasing Braak stages. Additionally, the increase in phosphorylation relative to controls was highest at Tyr18, Thr231 and Ser199. By contrast, Ser396 tau and Ser262 tau showed only a weak phosphorylation in all analyzed brain regions and only minor progression. Our results suggest that the ptau burden in the isocortex is comparable between all analyzed ptau sites when using a quantitative approach while levels of ptau at Tyr18 or Thr231 in the transentorhinal region are different between all Braak stages. Hence these sites could be crucial in the pathogenesis of AD already at early stages and therefore represent putative novel therapeutic targets.

Larger sperm size may contribute to reproductive isolation between Etheostoma species.

Etheostoma is a genus of North American darter fish whose species have similar habitats and breeding seasons, yet hybridization is rare. Behavioral barriers have been demonstrated to play a key role in maintaining species boundaries. Further, conspecific (same species) sperm precedence has also been observed when the gametes of two different species come into contact. In this study, we investigated if physical characteristics of sperm could be a mechanism for the lower fertilization success of heterospecific (different species) males when eggs are simultaneously exposed to conspecific and heterospecific sperm. We chose to examine the sperm of two closely related species, E. zonale and E. barrenense. Using toluidine blue and immunofluorescent labeling methods, we compared head diameter and tail length of sperm cells between the two species. We found that head diameter was significantly larger for E. barrenense sperm compared to E. zonale. This difference in cell morphology may point to a physical mechanism underlying conspecific sperm precedence in Etheostoma. Our results are the first to describe a morphological difference in sperm between species in this genus and provide initial evidence for the role of sperm morphology in prezygotic reproductive isolation.

A combined acetylcholinesterase and immunohistochemical method for precise anatomical analysis of intrinsic cardiac neural structures.

A significant challenge when investigating autonomic neuroanatomy is being able to reliably obtain tissue that contains neuronal structures of interest. Currently, histochemical staining for acetylcholinesterase (AChE) remains the most feasible and reliable method to visualize intrinsic nerves and ganglia in whole organs. In order to precisely visualize and sample intrinsic cardiac nerves and ganglia for subsequent immunofluorescent labeling, we developed a modified histochemical AChE method using material from pig and sheep hearts. The method involves: (1) chemical prefixation of the whole heart, (2) short-term and weak histochemical staining for AChE in situ, (3) visual examination and extirpation of the stained neural structures from the whole heart, (4) freezing, embedding and cryostat sectioning of the tissue of interest, and (5) immunofluorescent labeling and microscopic analysis of neural structures. Firstly, our data demonstrate that this modified AChE protocol labeled intrinsic cardiac nerves as convincingly as our previously published data. Secondly, there was the added advantage that adrenergic, cholinergic and peptidergic neuropeptides, namely protein gene product 9.5 (PGP 9.5), neurofilament (NF), tyrosine hydroxylase (TH), vesicular monoamine transporter (VMAT2), neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), calcitonin gene related peptide (CGRP), and substance P may be identified. Our method allows the precise sampling of neural structures including autonomic ganglia, intrinsic nerves and bundles of nerve fibers and even single neurons from the whole heart. This method saves time, effort and a substantial amount of antisera. Nonetheless, the proof of specific staining for many other autonomic neuronal markers has to be provided in subsequent studies.

Proteoglycans affect monomer infiltration in the etch-and-rinse bonding technique.

OBJECTIVES: To identify proteoglycans (PGs) and collagen fibrils (CF) within human dentin by means of a dual immunofluorescent labeling technique and to investigate the monomer infiltration of two etch-and-rinse adhesives to tosyl-phenylalanine chloromethyl-ketone (TPCK)-treated trypsin (TRY)-pretreated dentin. METHODS: Thirty-micrometer sections of middle coronal dentin were obtained and etched with 37% phosphoric acid gel for 15 s. After preconditioning with or without TRY digestion, the sections were subjected to dual immunofluorescent labeling and observed with a confocal laser scanning microscope (CLSM). Demineralized dentin matrixes treated with or without TRY were observed with field emission scanning electron microscope (FE-SEM). Two etch-and-rinse adhesives, Adper Single Bond 2 (SB) and Prime & Bond NT (PBNT), were applied to the dentin surfaces that were pretreated with or without TRY. The thickness of the hybrid layers was evaluated under confocal micro-Raman spectroscopy and analyzed with a two-way ANOVA. RESULTS: Green and red fluorescence was used to represent the PGs and the CF that were colocalized in the same section with different distributions. PGs were localized in the lumens of the dentin tubules and in peritubular dentin, while the type-I collagen fibrils were localized in intertubular dentin and peritubular dentin. After preconditioning with TRY digestion, the red fluorescence decreased or disappeared, the organic filaments in the lumens of the dentin tubules disappeared, the tubules were enlarged, and the hybrid layer thickness for adhesives bonded to the TRY-pretreated dentin surfaces were significantly increased (p<0.001 for both SB and PBNT). SIGNIFICANCE: The dual immunofluorescence labeling methodology can be used to study the human dentin matrix without decalcifying the entire dentin fragment. Proteoglycans were localized in the lumens of the dentin tubules and in peritubular dentin, which could depress the infiltration of the adhesive resin monomers. The use of TRY digestion increased the thickness of the hybrid layer created by the tested two-step etch-and-rinse adhesive.

Defining the phosphodiesterase superfamily members in rat brain microvessels.

Eleven phosphodiesterase (PDE) families are known, each having several different isoforms and splice variants. Recent evidence indicates that expression of individual PDE family members is tissue-specific. Little is known concerning detailed PDE component expression in brain microvessels where the blood-brain-barrier and the local cerebral blood flow are thought to be regulated by PDEs. The present study attempted to identify PDE family members that are expressed in brain microvessels. Adult male F344 rats were sacrificed and blocks of the cerebral cortex and infratentorial areas were dissected. Microvessels were isolated using a filtration method, and total RNA was extracted. RNA quality and quantity were determined using an Agilent bioanalyzer. The isolated cortical and infratentorial microvessel total RNA amounts were 2720 ± 750 ng (n = 2) and 250 ± 40 ng (n = 2), respectively. Microarrays with 22 000 transcripts demonstrated that there were 16 PDE transcripts in the PDE superfamily, exhibiting quantifiable density in the microvessels. An additional immunofluorescent study verified that PDE4D (cAMP-specific) and PDE5A (cGMP-specific) were colocalized with RECA-1 (an endothelial marker) in the cerebral cortex using both F344 rats and Sprague-Dawley rats (n = 3-6/strain). In addition, PDE4D and PDE5A were found to be colocalized with alpha-smooth muscle actin which delineates cerebral arteries and arterioles as well as pericytes. In conclusion, a filtration method followed by microarray analyses allows PDE components to be identified in brain microvessels, and confirmed that PDE4D and PDE5A are the primary forms expressed in rat brain microvessels.

Direct nitrogen and phosphorus limitation of arbuscular mycorrhizal fungi: a model and field test.

• Since mycorrhizal fungi constitute an important component of the soil-plant interface, their responses to changes in nutrient availability may mediate shifts in ecosystem function. We tested the hypothesis that initial soil nutrient availability may determine effects of nitrogen (N) and phosphorus (P) additions on the growth and community of arbuscular mycorrhizal (AM) fungi. • Extraradical hyphal lengths and degree of root colonization of AM fungi were measured in control and fertilized plots along a soil fertility gradient in Hawaii. Responses of individual AM genera were assessed through immunofluorescent labeling. • The AM biomass was increased by N and P additions in the N- and P-limited sites, respectively, and reduced by P fertilization in the fertile site only. The abundance of Scutellospora was lower under N than under P fertilization, whereas the incidence of Glomus was higher in the fertile site than the N-limited site. Gigaspora and Acaulospora did not vary among sites or treatments. • Our results indicate that a decrease in AM abundance following nutrient additions cannot be assumed to occur and the effects may differ among AM genera and ecosystems with varying soil nutrients. Limitation of N and P may be one possible explanation.

A MORPHOLOGICAL STUDY OF CONNECTIONS BETWEEN ENKEPHALIN IMMUNOREACTIVE TERMINALS AND ?-AMINOBUTYRIC ACID IMMUNOREACTIVE NEURONS IN THE ROSTRAL PORTION OF THE NUCLEUS TRACTUS SOLITARIOUS OF THE RAT / 解剖学报

Objective To observe the connections between enkephalin immunoreactive(ENK-ir) terminals and ?-aminobutyric acid immunoreactive(GABA-ir) neurons in the rostral portion of the nucleus tractus solitarious(rNTS) of the rat. Methods Double-immunofluorescent labeling method and the pre-embedding immunohistochemical staining combined with immuno-gold particles labeling technique for electron microscopic detection were used in the present study. Results Under the laser scanning confocal microscope,dense ENK-ir fibers and terminals and some GABA-ir neurons were observed in the rNTS.Close contacts between ENK-ir terminals and GABA-ir cell bodies were also detected.By using the electron microscopic technique,ENK-ir reaction products were found to be mainly localized on the surface of round clear vesicles and dense-cored vesicles in the axon terminals.ENK-ir terminals formed symmetric,which was a dominant pattern,and asymmetric synaptic connections with GABA-ir and immunonegative cell bodies and dendrites.Conclusion The ENK-ir terminals might take part in the transmission and regulation of the taste information in the rNTS through inhibiting,exciting the GABAergic neurons or inhibiting directly the activity of neurons within the rNTS.