CD8 alpha alpha-mediated intraepithelial lymphocyte snatching of thymic leukemia MHC class Ib molecules in vitro and in vivo

Thymic leukemia (TL) is a MHC class Ib molecule that interacts with CD8alphaalpha homodimers. CD8alphaalpha is abundantly expressed by intraepithelial T lymphocytes (IELs) located in close proximity to TL-expressing intestinal epithelial cells. In this study, we show that CD8alphaalpha(+) IELs "snatch" TL from the plasma membrane of TL-expressing cells and express TL in its proper orientation on their own cell surface. TL snatching is enhanced by cross-linking of IEL TCRs in a phosphatidylinositol kinase-dependent manner, and results in overall alterations to the IEL cell surface detected by enhanced binding of peanut agglutinin lectin. Induction of bowel inflammation results in the presence of TL on IELs, probably via in vivo snatching, providing the initial evidence for the interaction of CD8alphaalpha IELs with intestinal cells.     

Vaccinia virus E2L null mutants exhibit a major reduction in extracellular virion formation and virus spread

The vaccinia virus E2L (VACWR058) gene is conserved in all sequenced chordopoxviruses and is predicted to encode an 86-kDa protein with no recognizable functional motifs or nonpoxvirus homologs. Although the region immediately upstream of the open reading frame lacked optimal consensus promoter motifs, expression of the E2 protein occurred after viral DNA replication. Transfection studies, however, indicated that the promoter was weak compared to well-characterized intermediate and late promoters. The E2 protein was present in mature virions purified from infected cells but was more abundant in extracellular enveloped forms. Despite the conservation of the E2L gene in chordopoxviruses, deletion mutants could be isolated from both the WR and IHD-J strains of vaccinia virus. These null mutants produced very small plaques in all cell lines tested, reduced amounts of mature infectious virions, and very low numbers of extracellular virions. Nevertheless, viral protein synthesis appeared qualitatively and quantitatively normal. The defect in extracellular virus formation was corroborated by electron microscopy, which also showed some aberration in the wrapping of virions by cisternal membranes. Extracellular virions that did form, however, were able to induce actin tail formation.     

Tensin: A potential link between the cytoskeleton and signal transduction

Cytoskeletal proteins provide the structural foundation that allows cells to exist in a highly organized manner. Recent evidence suggests that certain cytoskeletal proteins not only maintain structural integrity, but might also be associated with signal transduction and suppression of tumorigenesis. Since the time of the discovery of tensin, a fair amount of data has been gathered which supports the notion that tensin is one such protein possessing these characteristics. In this review, we discuss recent studies that: (1) elucidate a role for tensin in maintenance of cellular structure and signal transduction; (2) implicate tensin as the anchor for actin filaments at the focal adhesion; (3) describe the phosphorylation of tensin; (4) describe potential targets for its Src homology region 2 domain; (5) describe the association between tensin and the nuclear protein p130; and (6) demonstrate that increased tensin expression in a cell line appears to reduce its transformation potential.     

Chemical-based fecal source tracking methods: current status and guidelines for evaluation

Fecal source tracking is a rapidly evolving field for which there have been a number of method evaluation studies, workshops, review articles and a book that synthesize information about method efficacy. Chemicals that are specific to human wastewater offer several potential advantages over biologically based methods, but have received less scrutiny. More than 35 chemical analytes have been found to consistently occur in human waste streams and here we review these potential human-origin indicators in context of seven evaluation criteria. Some chemical methods offer advantages over microbial methods: they are generally faster to prepare and analyze, more source-specific because they are not confounded by regrowth in the environment, and some may be more geographically and temporally stable. However, they often require specialized equipment and are usually more expensive regarding sample preparation and analysis. Additionally, most chemicals that are specific to human waste-streams occur at concentrations low enough to be diluted below detection limits once the waste-stream enters the ambient environment. These two factors will likely result in chemical measures being used more often as cross-validation supplements or initial screening approaches, rather than replacements for microbial measures. Cross-validation supplements include several chemicals that are highly specific to human sources and can be important contributors when certainties about human sources are critical, such as in drinking water applications. At least one set of chemicals, fecal sterols and stanols, may have potential for identification of other sources in addition to humans. Of all the chemicals examined to date, optical brighteners (OBs) in detergents have shown considerable promise, especially for screening purposes. Optical brighteners are not as sensitive as most microbial assessments, but can be measured with a hand-held fluorometer, providing near real-time and relatively inexpensive tracking of signals in the field, if the human fecal source contains an OB concentration large enough to produce a measurable signal.     

vFLIP protects PC-12 cells from apoptosis induced by Sindbis virus: implications for the role of TNF-alpha.

Sindbis virus (SV) is an alphavirus used as a model for studying the pathogenesis of viral encephalitis. In this study we examined the effects and the mechanisms involved in the apoptosis induced by SV in PC-12 cells, and the role of a vFLIP in this process. Infection of PC-12 cells with a neurovirulent strain of SV, SVNI, induced cell apoptosis. Overexpression of vFLIP encoded by the HHV-8 or treatment with a caspase-8 inhibitor inhibited cell apoptosis. SVNI induced an increase in the expression of tumor necrosis factor alpha (TNF-alpha), and pre-treatment of the cells with an anti-TNF-alpha blocking antibody or with soluble TNF-alpha receptor abrogated the apoptotic effect of SVNI. Moreover, TNF-alpha R1 knockout mice were more resistant to the cytopathic effects of the virus as compared to control animals. Our results indicate that the apoptosis induced by SVNI is mediated by activation of caspase-8, and that TNF-alpha plays an important role in the apoptotic response.     

The Envelope Protein Encoded by the A33R Gene Is Required for Formation of Actin-Containing Microvilli and Efficient Cell-to-Cell Spread of Vaccinia Virus

The vaccinia virus (VV) A33R gene encodes a highly conserved 23- to 28-kDa glycoprotein that is specifically incorporated into the viral outer envelope. The protein is expressed early and late after infection, consistent with putative early and late promoter sequences. To determine the role of the protein, two inducible A33R mutants were constructed, one with the late promoter and one with the early and late A33R promoter elements. Decreased A33R expression was associated with small plaques that formed comets in liquid medium. Using both an antibiotic resistance gene and a color marker, an A33R deletion mutant, vA33Δ, was isolated, indicating that the A33R gene is not essential for VV replication. The plaques formed by vA33Δ, however, were tiny, indicating that the A33R protein is necessary for efficient cell-to-cell spread. Rescue of the large-plaque phenotype was achieved by inserting a new copy of the A33R gene into the thymidine kinase locus, confirming the specific genetic basis of the phenotype. Although there was a reduction in intracellular virus formed in cells infected with vA33Δ, the amount of infectious virus in the medium was increased. The virus particles in the medium had the buoyant density of extracellular enveloped viruses (EEV). Additionally, amounts of vA33Δ cell-associated extracellular enveloped viruses (CEV) were found to be normal. Immunogold electron microscopy of cells infected with vA33Δ demonstrated the presence of the expected F13L and B5R proteins in wrapping membranes and EEV; however, fully wrapped vA33Δ intracellular enveloped viruses (IEV) were rare compared to partially wrapped particles. Specialized actin tails that propel IEV particles to the periphery and virus-tipped microvilli (both common in wild-type-infected cells) were absent in cells infected with vA33Δ. This is the first deletion mutant in a VV envelope gene that produces at least normal amounts of fully infectious EEV and CEV and yet has a small-plaque phenotype. These data support a new model for VV spread, emphasizing the importance of virus-tipped actin tails.     

Marginal tests with sliced average variance estimation

We present a new computationally feasible test for the dimensionof the central subspace in a regression problem based on slicedaverage variance estimation. We also provide a marginal coordinatetest. Under the null hypothesis, both the test of dimensionand the marginal coordinate test involve test statistics thatasymptotically have chi-squared distributions given normallydistributed predictors, and have a distribution that is a linearcombination of chi-squared distributions in general.     

Disintegration of Dung Pats from Cattle Treated with the Ivermectin Anthelmintic Bolus,or the Biocontrol Agent Duddingtonia flagrans

An experiment was performed during the grazing seasons of 1998, 1999 and 2000 to study the influence of the antiparasitic drug ivermectin and the nematophagous fungus Duddingtonia flagrans on cattle dung disintegration. The faeces originated from groups of animals that were part of a separate grazing experiment where different control strategies for nematode parasite infections were investigated. Each group consisted of 10 first-season grazing cattle that were either untreated, treated with the ivermectin sustained-release bolus, or fed chlamydospores of D. flagrans. Faeces were collected monthly on 4 occasions and out of pooled faeces from each group, 4 artificial 1 kg dung pats were prepared and deposited on nylon mesh on an enclosed pasture and protected from birds. The position of the new set of pats was repeated throughout the 3 years of the study. Each year, the dung pats were weighed 4, 6, 8 and 10 weeks after deposition and immediately afterwards replaced to their initial positions.

Results showed that there was no difference in faecal pat disintegration between groups. However, the time-lag between deposition and complete disintegration of the faeces varied significantly between deposition occasions. Dung pats disappeared within 2 weeks (visual observation) when subjected to heavy rainfall early after deposition, whereas an extended dry period coincided with faeces still remaining 12 months after deposition.

     

Airway cellularity, lipid laden macrophages and microbiology of gastric juice and airways in children with reflux oesophagitis

Background and methods

Human metapneumovirus (hMPV) is a recently discovered respiratory virus associated with bronchiolitis, pneumonia, croup and exacerbations of asthma. Since respiratory viruses are frequently detected in patients with acute exacerbations of COPD (AE-COPD) it was our aim to investigate the frequency of hMPV detection in a prospective cohort of hospitalized patients with AE-COPD compared to patients with stable COPD and to smokers without by means of quantitative real-time RT-PCR.

Results

We analysed nasal lavage and induced sputum of 130 patients with AE-COPD, 65 patients with stable COPD and 34 smokers without COPD. HMPV was detected in 3/130 (2.3%) AE-COPD patients with a mean of 6.5 × 10⁵ viral copies/ml in nasal lavage and 1.88 × 10⁵ viral copies/ml in induced sputum. It was not found in patients with stable COPD or smokers without COPD.

Conclusion

HMPV is only found in a very small number of patients with AE-COPD. However it should be considered as a further possible viral trigger of AE-COPD because asymptomatic carriage is unlikely.