Citrobacter rodentium Induces Tissue-Resident Memory CD4+ T Cells.

Tissue-resident memory T cells (TRM cells) are a novel population of tissue-restricted antigen-specific T cells. TRM cells are induced by pathogens and promote host defense against secondary infections. Although TRM cells cannot be detected in circulation, they are the major memory CD4+ and CD8+ T-cell population in tissues in mice and humans. Murine models of CD8+ TRM cells have shown that CD8+ TRM cells maintain tissue residency via CD69 and though tumor growth factor ß-dependent induction of CD103. In contrast to CD8+ TRM cells, there are few models of CD4+ TRM cells. Thus, much less is known about the factors regulating the induction, maintenance, and host defense functions of CD4+ TRM cells. Citrobacter rodentium is known to induce IL-17+ and IL-22+ CD4+ T cells (Th17 and Th22 cells, respectively). Moreover, data from IL-22 reporter mice show that most IL-22+ cells in the colon 3 months after C. rodentium infection are CD4+ T cells. This collectively suggests that C. rodentium may induce CD4+ TRM cells. Here, we demonstrate that C. rodentium induces a population of IL-17A+ CD4+ T cells that are tissue restricted and antigen specific, thus meeting the criteria of CD4+ TRM cells. These cells expand and are a major source of IL-22 during secondary C. rodentium infection, even before the T-cell phase of the host response in primary infection. Finally, using FTY 720, which depletes circulating naive and effector T cells but not tissue-restricted T cells, we show that these CD4+ TRM cells can promote host defense.

Effect of Selenization Processes on CIGS Solar Cell Performance.

Cu(In, Ga)Se2 (CIGS) films were fabricated by a two-step process method using sputtering from Cu0.7Ga0.3 and In targets. The metallic precursor structures of In/CuGa/In were prepared, and CuGa film was adjusted to the thicknesses of 150, 200, 250 and 300 nm, in order to optimize the CIGS film. After selenization, three independent CIGS (112), CIGS (220/204) and CIGS (312/116) began to crystallize at ~280 °C and phase peaks continued growing until 560 °C. Experimental results showed that with a single stage selenization method, the excessive stoichiometry of the CIGS films was obtained. Using three sequential stages for the selenization process, with a annealing time of 20 min, the stoichiometry of the CIGS absorbers with the Cu/(In + Ga) and Ga/(In + Ga) showed atomic ratios of 0.94 and 0.34, respectively. The intensity of the (112) XRD diffraction peak became stronger, indicating an improvement in the crystallinity. Raman spectra of CIGS absorbers showed a main peak (174 cm-1) and two weak signals (212 and 231 cm-1). TEM image for electron diffraction pattern showed that the grains were randomly oriented. CIGS solar cell device prepared with a proper selenization, a maximum efficiency of 12.45% was obtained.

Large-scale mitochondrial DNA deletions in weak goslings.

1. Evidence has accumulated in mammals to support the idea that mitochondrial DNA (mtDNA) deletions and mutations might contribute to ageing and reproductive failure. White Roman geese were monitored to evaluate the effect of large-scale deletions of mtDNA in an avian species. 2. A total of 340 samples from 114 dead embryos, 111 weak goslings, and 115 normal goslings were used in this experiment. The regions of these two large-scale mtDNA deletions, ΔmtDNA6829 and ΔmtDNA6992, were between the COI and ND5 genes. A 3·6% (4 out of 111) positive sample was detected in the weak goslings. In contrast, no large-scale mitochondrial DNA deletions were detected in either the dead embryos (0 out of 114) or the normal goslings (0 out of 115). 3. Large-scale mtDNA deletions may be a factor causing weak goslings.

Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of chicken anaemia virus.

AIM: Chicken anaemia virus (CAV) causes an economically important viral disease in chickens worldwide. The main aim of this study was to establish a rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) assay for detecting CAV infection. METHODS AND RESULTS: A set of four specific LAMP primers were designed based on the nucleotide sequence of the CAV VP2 gene, which encodes a nonstructural protein. These were used for the amplification of a specific target region of the VP2 gene. LAMP amplicons were successfully amplified and detected by DNA electrophoresis and by direct naked eye SYBR Green I visualization. A sensitivity test systematically demonstrated that the LAMP assay was superior to a conventional PCR assay with a minimum concentration limit of 100 fg compared to 10 ng for the conventional PCR. The specificity of the LAMP assay for CAV detection is consistent with conventional PCR. Using this established LAMP assay, infected and uninfected clinical samples obtained from an experimental farm were fully verified. CONCLUSIONS: A novel nucleic acid-based approach of LAMP assay was successfully developed for detecting CAV infection. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, these results indicate that the developed LAMP assay herein for CAV detection is a time-effective, simple, sensitive and specific test that can be used as an alternative approach in the future for large-scaled diagnosis on the farm of CAV infection.

The origin of the white Roman goose.

In order to avoid interference from nuclear copies of mitochondrial DNA (numts), mtDNA of the white Roman goose (domestic goose) was extracted from liver mitochondria. The mtDNA control region was amplified using a long PCR strategy and then sequenced. Neighbor-joining, maximum parsimony, and maximum-likelihood approaches were implemented using the 1,177 bp mtDNA control region sequences to compute the phylogenetic relationships of the domestic goose with other geese. The resulting identity values for the white Roman geese were 99.1% (1,166/1,177) with western graylag geese and 98.8% (1,163/1,177) with eastern graylag geese. In molecular phylogenetic trees, the white Roman goose was grouped in the graylag lineage, indicating that the white Roman goose came from the graylag goose (Anser anser). Thus, the scientific name of the white Roman goose should be Anser anser 'White Roman.'

Association of adiponectin multimers with Barrett's oesophagus.

OBJECTIVE: Barrett's oesophagus is associated with abdominal obesity. Adiponectin is a peptide that is secreted from adipocytes and circulates in three multimeric forms: low molecular weight (LMW), middle molecular weight (MMW), and high molecular weight (HMW). The anti-inflammatory effects of adiponectin are specific to individual multimers, with LMW being most anti-inflammatory. We postulated that circulating levels of adiponectin and its multimers would be associated with the risk of Barrett's oesophagus. DESIGN: Cross-sectional study. SETTING: Outpatient clinic in North Carolina, USA. PATIENTS: Cases of Barrett's oesophagus and controls undergoing upper endoscopy for gastro-oesophageal reflux disease (GORD). MAIN OUTCOME MEASURES: Adjusted odds ratios of plasma adiponectin levels and its multimers for Barrett's oesophagus. RESULTS: There were 112 cases of Barrett's oesophagus and 199 GORD controls. Total adiponectin was not associated with Barrett's oesophagus (3(rd) tertile vs 1(st) tertile adjusted odds ratio (aOR) = 0.88; 95% confidence interval (CI) = 0.44 to 1.78). High levels of LMW adiponectin were associated with a decreased risk of Barrett's oesophagus (3(rd) tertile vs 1(st) tertile aOR = 0.33; 95% CI, 0.16 to 0.69), and a high LMW/total ratio appeared particularly inversely associated with Barrett's oesophagus (3(rd) tertile vs 1(st) tertile aOR = 0.27; 95% CI, 0.13 to 0.58). CONCLUSIONS: High levels of LMW adiponectin are associated with a decreased risk of Barrett's oesophagus among patients with GORD. Further human studies are required to confirm these findings, and in vitro studies are needed to understand if there is a mechanism whereby adiponectin may affect Barrett's metaplasia.

The registration of CT image to the patient head by using an automated laser surface scanning system--a phantom study.

In this paper, a practical methodology of surface-based registration supported by an automated laser surface scanning system to achieve good mapping performance is reported. The laser scanning system is used to digitize the facial feature of a phantom so as to mesh the physical space into triangular frame. The image space is established by translating the corresponding CT image into point set through using the medical image software tools. The image-to-physical registration task is carried out by a direct searching mechanism together with the objective function in an optimal fashion. The unconstrained nonlinear optimization algorithm performs the optimal searching iteration to find those parameters in the rigid-body transformation until the sum of the squared normal distances is minimized. Considering mapping the massive points in registration task would consume the computation time, there is only a suitable sample size to stand for the entire population with sufficient confidence and accuracy are extracted statistically from the CT point space to map to the laser scanning space. Registration results evaluated by gauging the position errors of the landmarks on phantom forehead show the proposed methodology has good ability to perform the image-to-physical registration.

Human gastric epithelial cells contribute to gastric immune regulation by providing retinoic acid to dendritic cells.

Despite the high prevalence of chronic gastritis caused by Helicobacter pylori, the gastric mucosa has received little investigative attention as a unique immune environment. Here, we analyzed whether retinoic acid (RA), an important homeostatic factor in the small intestinal mucosa, also contributes to gastric immune regulation. We report that human gastric tissue contains high levels of the RA precursor molecule retinol (ROL), and that gastric epithelial cells express both RA biosynthesis genes and RA response genes, indicative of active RA biosynthesis. Moreover, primary gastric epithelial cells cultured in the presence of ROL synthesized RA in vitro and induced RA biosynthesis in co-cultured monocytes through an RA-dependent mechanism, suggesting that gastric epithelial cells may also confer the ability to generate RA on gastric dendritic cells (DCs). Indeed, DCs purified from gastric mucosa had similar levels of aldehyde dehydrogenase activity and RA biosynthesis gene expression as small intestinal DCs, although gastric DCs lacked CD103. In H. pylori-infected gastric mucosa, gastric RA biosynthesis gene expression was severely disrupted, which may lead to reduced RA signaling and thus contribute to disease progression. Collectively, our results support a critical role for RA in human gastric immune regulation.

Effects of artificial supplemental light on the reproductive season of geese kept in open houses.

1. New lighting programmes were designed to change the pattern of goose reproduction, based on the response of predictable avian photoperiodic stimulation. The objective of this study was to evaluate the efficiency of a long photoperiod of 20L on shifting goose reproduction to the non-breeding season in an open housing system. 2. Eighty mature White Roman geese were randomly allocated into 4 groups (male:female = 1:4). The supplemental lighting programmes with a daily photoperiod of 20 h were initiated on 22 November and withdrawn when 90% of geese were moulting in the treatment groups. Artificial light intensities of 220, 120 and 20 lux were provided to experimental groups A, B and C, respectively. In contrast, the geese in control group D were kept under natural lighting conditions throughout this study. 3. The annual reproductive curves of all the experimental groups consisted of two separate laying periods. The first period was induced by the supplemental lighting programme while the second was induced by the naturally increasing photoperiod in this subtropical region. The first laying period of the experimental groups occurred in the breeding season, and the second was relocated to the non-breeding season. 4. The supplemental lighting could shift the laying periods of geese to the non-breeding season and had no significant effect on annual reproductive performance. The supplemental light programmes described here were able to manipulate the reproductive season of geese reared in open houses, which would be of practical value.

Acute dermal toxicity: in vivo and in vitro comparisons in mice.

Mouse skin was exposed topically to solutions of dinitrochlorobenzene (DNCB), croton oil (CO), epoxyethylbenzene (EEB), ethanolamine (EtNH2), or isopropylmyristate (IPM), and the histologic response evaluated 20 hr later with light microscopy. In a parallel series of experiments, discs of mouse skin were collected, exposed in vitro to the same chemicals and examined histologically after 20 hr in culture. Additionally, cellular enzyme leakage was determined in the culture medium. DNCB was the most toxic to the skin followed by CO and EEB. IPM and EtNH2 were toxic only at the highest concentration used (10%). There were good correlations between the magnitude of the skin lesions and the levels of enzyme activity in the culture medium. The results show that morphologic responses of skin maintained in organ culture are an indicator of in vitro skin toxicity. Moreover, enzyme leakage may provide a means for detecting sublethal cell injury which might not be observed histologically.

A multisample apparatus for kinetic evaluation of skin penetration in vitro: the influence of viability and metabolic status of the skin.

An apparatus is described for convenient in vitro quantitation of the rate, extent, and character of living skin penetration by chemicals. Evidence that viability markedly affects epidermal penetration was based upon observations utilizing benzo(a)pyrene. This compound displayed saturation kinetics in the extent of penetration, marked elevation in the rate, and extent of penetration following induction of epidermal mixed function oxidase and negligible penetration of skin previously frozen. These data are interpreted to indicate that for certain compounds, such as benzo(a)pyrene where cutaneous metabolism is expected, meaningful in vitro measures of skin penetration should take into consideration the biochemical viability and metabolic status of the tissue.

Characterization of the effects of a thymine glycol residue on the structure, dynamics, and stability of duplex DNA by NMR.

A duplex DNA containing a single thymine glycol (5,6-dihydroxy-5,6-dihydrothymidine) has been studied by NMR and other methods. Oxidative stress, ionizing radiation, and other causes can induce the oxidation of thymine to thymine glycol. The presence of thymine glycol is known to have significant biological consequences, and there are repair enzymes for thymine glycol in a wide range of organisms. These studies have been carried out on the DNA duplex of d(C1G2C3A4G5Tg6C7A8G9C10C11) paired with d(G22C21G20T19C18A17G16T15C14G13G12), with Tg indicating thymine glycol. The presence of thymine glycol lowers the thermal stability of duplex DNA. The NMR results indicate that thymine glycol induces a large, localized structural change in duplex DNA with the thymine glycol base being extrahelical as well as the opposing base on the complementary strand. This structural information is consistent with the biological consequences of thymine glycol in DNA.

Effects of the presence of an aldehydic abasic site on the thermal stability and rates of helix opening and closing of duplex DNA.

The presence of an abasic site in duplex DNA lowers the thermodynamic stability, as monitored by the optical melting temperature, and decreases the rate of imino proton exchange with water, by about an order of magnitude, as monitored by direct measurement of both the exchange lifetimes and the imino proton T1S. The exchange lifetimes of the imino protons with water as a function of base catalyst concentration were analyzed to determine the origin of the effect of the abasic site on imino exchange lifetimes. Analysis of the results showed that the helix opening rate is not significantly changed by the presence of an abasic site. The differences in exchange lifetimes are attributed to a faster helix closing rate in the presence of an abasic site. The faster rate of helix closing may be an important contribution to the stability of abasic sites in duplex DNA to base-catalyzed elimination reaction. It is noted that duplex DNAs containing analogues of the aldehydic abasic site apparently do not exhibit these exchange lifetime effects.

An improved method for conjugating monoclonal antibodies with N-hydroxysulfosuccinimidyl DOTA.

A simple, water-soluble procedure for conjugation of monoclonal antibodies to 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) has been improved by optimizing pH, buffer, and temperature conditions for the preparation of N-hydroxysulfosuccinimidyl DOTA and its conjugation to the human/murine chimeric anti-carcinoembryonic antigen antibody cT84.66. This improved method results in a 6-fold increase in conjugation efficiency, a 3-7-fold decrease in antibody cross-linking, a more homogeneous population of conjugate species, and a 5-fold decrease in the quantities of reagents needed for conjugation. The cT84.66-DOTA conjugate was labeled to high specific activity with 111In, 90Y, 88Y, 64Cu, and 67Cu, affording near-quantitative incorporation of the majority of these radiometals. This improved conjugation procedure facilitates large-scale production and radiometal labeling of cT84.66-DOTA for clinical radioimmunotherapy trials.

The low frequency reflection characteristics of the fundamental antisymmetric Lamb wave a0 from a rectangular notch in a plate.

An analysis of the reflection of the fundamental Lamb mode a0 from surface-breaking rectangular notches in isotropic plates is presented. The results are obtained from finite element time domain simulations together with experimental measurements. Good agreement is found between the simulations and the measurements. Results are shown for a range of notch widths and depths, including the special case of a crack, defined as a zero-width notch. The reflection coefficient, when plotted as a function of the notch width, exhibits a cosinusoidal periodic shape, and this is explained by interference between the separate reflections from the start and the end of the notch. The reflection coefficient, when plotted as a function of notch depth, shows that in general the reflection increases with both frequency and notch depth, but the shapes of the functions are complex and there are some surprising features. An analysis of the reflection from cracks using the S-parameter scattering approach and some simplified descriptions of the crack-opening behavior yields physical explanations of the nature of these reflection functions. It is found that opening of the crack can be described adequately by a quasistatic assumption only when the crack is small, and in other cases a ray theory approach is more representative. The reflection function is shown to be a result of contributions from both the axial stress and the shear stress in the wave, and the relative importance of these varies with the crack depth and the frequency.

Use of laser-excited fluorescence to measure mixed-function oxidase activity.

The microsomal mixed-function oxidase (MFO) system is involved in the metabolism of various chemical compounds. Polycyclic aromatic hydrocarbons are metabolized by the cytochrome P-448 enzyme system, which contains MFOs. Induction of this MFO activity may be useful as an indicator of the toxicity of the inducer material. We have successfully used a portable centrifugal analyzer equipped with an argonion laser light source to quantify cytochrome P-448 activity induced in mouse-liver microsomes by exposing the animal's skin to different doses of liquids derived from a coal-liquefaction process. The MFO activity was determined kinetically by measuring the rate at which the highly fluorescent compound, resorufin, produced by the oxidation of 7-ethoxyresorufin substrate, was formed. The 514.5-nm laser excitation beam was directed with a fiber-optic bundle from the laser to the cuvets of a specially designed rotor; emitted fluorescence was monitored at 90 degrees to the incident beam through a 560-nm cut-on secondary filter. Use of the laser excitation source allowed very low MFO activities to be measured: picomole quantities of resorufin could be determined. We anticipate that the increased sensitivity of the method described here will allow MFO activities to be determined in body fluids and skin.