Isolation and Characterization of Intestinal Epithelial Cells from Normal and SIV-Infected Rhesus Macaques

Impairment of intestinal epithelial barriers contributes to the progression of HIV/SIV infection and leads to generalized HIV-induced immune-cell activation during chronic infection. Rhesus macaques are the major animal model for studying HIV pathogenesis. However, detailed characterization of isolated rhesus epithelial cells (ECs) from intestinal tissues is not well defined. It is also not well documented whether isolated ECs had any other cell contaminants from intestinal tissues during the time of processing that might hamper interpretation of EC preparations or cultures. In this study, we identify and characterize ECs based on flow cytometry and immunohistochemistry methods using various enzymatic and mechanical isolation techniques to enrich ECs from intestinal tissues. This study shows that normal healthy ECs differentially express HLA-DR, CD23, CD27, CD90, CD95 and IL-10R markers. Early apoptosis and upregulation of ICAM-1 and HLA-DR in intestinal ECs are thought to be the key features in SIV mediated enteropathy. The data suggest that intestinal ECs might be playing an important role in mucosal immune responses by regulating the expression of different important regulatory and adhesion molecules and their function

Synthesis, Multinuclear NMR Characterization and Dynamic Property of Organic–Inorganic Hybrid Electrolyte Membrane Based on Alkoxysilane and Poly(oxyalkylene) Diamine

Organic–inorganic hybrid electrolyte membranes based on poly(propylene glycol)-<em>block</em>-poly(ethylene glycol)-<em>block</em>-poly(propylene glycol) bis(2-aminopropyl ether) complexed with LiClO<sub>4</sub> via the co-condensation of tetraethoxysilane (TEOS) and 3-(triethoxysilyl)propyl isocyanate have been prepared and characterized. A variety of techniques such as differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) spectroscopy, alternating current (AC) impedance and solid-state nuclear magnetic resonance (NMR) spectroscopy are performed to elucidate the relationship between the structural and dynamic properties of the hybrid electrolyte and the ion mobility. A VTF (Vogel-Tamman-Fulcher)-like temperature dependence of ionic conductivity is observed for all the compositions studied, implying that the diffusion of charge carriers is assisted by the segmental motions of the polymer chains. A maximum ionic conductivity value of 5.3 × 10<sup>−5</sup> Scm<sup>−1</sup> is obtained at 30 °C. Solid-state NMR results provide a microscopic view of the effects of salt concentrations on the dynamic behavior of the polymer chains

Molecular differentiation of cryptic stage of Echinococcus granulosus and Taenia species from faecal and environmental samples

ObjectiveTo differentiate cryptic stage of Echinococcus granulosus (E. granulosus) and Taenia by PCR-RFLP and sequence information of amplicon.MethodsDNA were isolated from metacestodes stage of Taenia and E. granulosus using DNA isolation kit (Q-BIOgene kit, USA), the amplified and purified DNA product was then cloned and sent for sequencing. The generating sequence information was used for amplicons identification.ResultsOut of 112 faecal and environmental samples, 16 exhibited positive result. The product size of amplicon positive for E. granulosus was 310 bp; whereas, for Taenia spp. sizes varied from 379 to 388 bp. Restriction profile of actin II with Csp61 also differed Taenia spp. and E. granulosus.ConclusionsThe result of the study indicated that, the primers were useful to differentiate cryptic stage of the two genera which is yet to be reported earlier

SIV infection induces apoptosis and upregulation of CD54 expression on intestinal epithelial cells (ECs).

<p>(<b>A</b>) Epithelial cells apoptosis was detected in jejunum by multi-labeled immunofluorescent confocal microscopy. Note that increased apoptosis of jejunum ECs was detected during acute (AV85; 21 days post infection) and chronic (HG58; 288 days post infection) SIV infection (as indicated by the white arrows). (<b>B</b>) Mean frequencies (± standard deviation) of surface CD54, CD80, CD86 and HLA-DR expression are shown for both jejunum ECs and CD45+ leukocytes from normal (n = 6) and chronically SIV-infected (n = 4) macaques. Note that a significant increase in CD54 expression on jejunum ECs was detected in SIV-infected macaques. However, increased expression of CD54, CD80 and HLA-DR on CD45+ leukocytes was observed in SIV-infected RMs. Statistically significant differences between each group of cells are shown.</p

Schematic representation of epithelial cell and leukocyte isolation protocols from intestinal tissues after different enzymatic treatments.

<p>Note that this protocol explains cell isolation procedures with initial EDTA treatment. No prior DTT treatment has been used in this protocol.</p

Phenotyping epithelial cells of normal rhesus macaques.

<p>(<b>A</b>) Representative expression of cytokeratin (CK; epithelial cell marker) and CD45 (leukocyte marker) in jejunum intestinal cells isolated from lower layer (“lymphocyte enriched”) of percoll gradient after treating with DTT and EDTA solution (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>; Step IX). Note that 2.8% of cells were double positive for CK and CD45 receptors. Fractions of double negative CK−CD45− cells were also evident from isolated cells. Live cells were gated first from all acquired cells and plotted based on CK and CD45 markers. Visualization of epithelial cells both in (<b>B</b>) colon at 20× and (<b>C</b>) jejunum at 5× resolution were detected by immunohistochemistry staining using anti-CK monoclonal antibody with hematoxylin counterstain.</p

Phenotyping colon and jejunum epithelial cells (ECs) by confocal microscopy.

<p>Essentially all colon (<b>A–E</b>) ECs (cytokeratin positive) were negative for (<b>A</b>) Ham56 (macrophage marker), and (<b>B</b>) CD11c (dendritic cell marker). Very few colon ECs were double positive for (<b>C</b>) both CD45 (leukocyte marker) and cytokeratin expression. A major population of colon ECs was positive for (<b>D</b>) HLA-DR expression. However, very few colon ECs were positive for (<b>E</b>) IL-10R expression which was evident in apical regions. Jejunum ECs were also negative for (<b>F</b>) CD54 (ICAM-1, cell adhesion marker). White arrow denotes the presence of double positive cells (cytokeratin and CD45/HLA-DR/IL-10R) for the specified sample and fluorochrome.</p

Percentages of epithelial cells retrieved from different isolation protocols.

<p>Mean percentages (± standard deviation) of isolated cytokeratin (CK)+ CD45− cells from jejunum either after DTT, EDTA or collagenase treatment with or without prior DTT wash (as discussed in details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Figs. 1</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g002" target="_blank">2</a>) are shown from uninfected normal healthy rhesus macaques (n = 5). Note that there was increased yield of ECs either by DTT or EDTA only treatment compared to other protocols followed in this experiment. Statistical significant differences between each group of cells are shown. * Indicates significant differences in ECs isolated from either DTT or EDTA only treatment compared to other group of cells.</p

Schematic representation of epithelial cell and leukocyte isolation protocols from intestinal tissues after different enzymatic treatments.

<p>Note that this protocol explains cell isolation procedures with initial DTT treatment.</p

Isolation of epithelial cells and leukocytes from intestinal tissues with DTT, EDTA and collagenase treatments.

<p>Representative dot plots of total cells from (<b>A</b>) DTT wash (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>, step IV); (<b>B</b>) lower layer of percoll density gradient, isolated after EDTA treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>, step IX); (<b>C</b>) lower layers of percoll density gradient, isolated after collagenase treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>, step XIII) from jejunum tissue showing distribution of epithelial cells (ECs; cytokeratin as an epithelial cell marker) and CD45 leukocytes in a normal uninfected healthy rhesus macaque. Each quadrant shows percentages of specified cell populations. Note increased percentage of ECs were isolated from DTT wash compared to other methods examined. Mean frequencies of cytokeratin (CK) positive, CD45 positive, double positive and double negative for CK and CD45 cell subsets are shown as box and whisker vertical bars for different isolation protocols as specified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>. In summary all the specified bars represent (<b>D</b>) cells isolated from DTT wash (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>, step IV); (<b>E</b>) upper layer cells from percoll density gradient isolated after EDTA treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>, step IX); (<b>F</b>) lower layer cells from percoll density gradient isolated after EDTA treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>, step IX); (<b>G</b>) upper layer cells from percoll density gradient isolated after collagenase treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>, step XIII); and (<b>H</b>) lower layer cells from percoll density gradient isolated after collagenase treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030247#pone-0030247-g001" target="_blank">Fig. 1</a>, step XIII) from jejunum in healthy, normal, uninfected rhesus macaques (n = 5). Note that in all isolation protocols, there was a variable amount of CD45+ and double negative CK−CD45− cells contamination observed. * Indicates significant differences between CK+CD45− and other different subsets of total cells within the specified isolation protocol.</p