Evaluation of apoptosis markers in different cell lines infected with equine arteritis virus

Equine arteritis virus (EAV) induces apoptosis in infected cells. Cell death caused by EAV has been studied mainly using three cell lines, BHK-21, RK-13 and Vero cells. The mechanism of apoptosis varies among cell lines and results cannot be correlated owing to differences in EAV strains used. We evaluated different markers for apoptosis in BHK-21, RK-13 and Vero cell lines using the Bucyrus EAV reference strain. Acridine orange/ethidium bromide staining revealed morphological changes in infected cells, while flow cytometry indicated the extent of apoptosis. We also observed DNA fragmentation, but the DNA ladder was detected at different times post-infection depending on the cell line, i.e., 48, 72 and 96 h post-infection in RK-13, Vero and BHK-21 cells, respectively. Measurement of viral titers obtained with each cell line indicated that apoptosis causes interference with viral replication and therefore decreased viral titers. As an unequivocal marker of apoptosis, we measured the expression of caspase-3 and caspases-8 and -9 as extrinsic and intrinsic markers of apoptosis pathways, respectively. Caspase-8 in BHK-21 cells was the only protease that was not detected at any of the times assayed. We found that Bucyrus EAV strain exhibited a distinctive apoptosis pathway depending on the cell line.     

Phylogenetic relationships among prokaryotic and eukaryotic catalases

Seventy-four catalase protein sequences, including 29 bacterial, 8 fungal, 7 animal, and 30 plant sequences, were compiled, and 70 were used for phylogenetic reconstruction. The core of the resulting tree revealed unique, separate groups of plant and animal catalases, two groups of fungal catalases, and three groups of bacterial catalases. The only overlap of kingdoms occurred within one branch and involved fungal and bacterial large-subunit enzymes. The other fungal branch was closely linked to the group of animal enzymes. Group I bacterial catalases were more closely related to the plant enzymes and contained such diverse taxa as the Gram-positive Listeria seeligeri, Deinocococcus radiodurans, and gamma-proteobacteria. Group III bacterial sequences were more closely related to fungal and animal sequences and included enzymes from a broad range of bacteria including high- and low-GC Gram positives, proteobacteria, and a bacteroides species. Group II was composed of large-subunit catalases from diverse sources including Gram positives (low-GC Bacilli and high-GC Mycobacteria), proteobacteria, and species of the filamentous fungus Aspergillus. These data can be interpreted in terms of two gene duplication events that produced a minimum of three catalase gene family members that subsequently evolved in response to environmental demands. Horizontal gene transfer may have been responsible for the group II mixture of bacterial and fungal large-subunit catalases.      

Sequence variation in the guillemot (Alcidae: Cepphus) mitochondrial control region and its nuclear homolog

We describe sequence variation in the mitochondrial control region and its nuclear homolog in three species and seven subspecies of guillemots (Cepphus spp.). Nuclear homologs of the 5' end of the control region were found in all individuals. Nuclear sequences were approximately 50% divergent from their mitochondrial counterparts and formed a distinct phylogenetic clade; the mitochondrial-nuclear introgression event must have predated the radiation of Cepphus. As in other vertebrates, the guillemot control region has a relatively conserved central block flanked by hypervariable 5' and 3' ends. Mean pairwise interspecific divergence values among control regions were lower than those in other birds. All individuals were heteroplasmic for the number of simple tandem nucleotide repeats (A(n)C) at the 3' end of the control region. Phylogenetic analyses suggest that black guillemots are basal to pigeon and spectacled guillemots, but evolutionary relationships among subspecies remain unresolved, possibly due to incomplete lineage sorting. Describing molecular variation in nuclear homologs of mitochondrial genes is of general interest in phylogenetics because, if undetected, the homologs may confound interpretations of mitochondrial phylogenies.      

Spontaneous CD4+ T cell responses against TRAG-3 in patients with melanoma and breast cancers

The taxol resistance gene TRAG-3 was initially isolated from cancer cell lines that became resistant to taxol in vitro. TRAG-3 is a cancer germline Ag expressed by tumors of different histological types including the majority of melanoma, breast, and lung cancers. In the present study, we report that patients with stage IV melanoma and breast cancers developed spontaneous IFN-gamma-producing CD4+ T cell responses against a single immunodominant and promiscuous peptide epitope from TRAG-3 presented in the context of multiple HLA-DR molecules. The TRAG-3-specific CD4+ T cells and clones were expanded in vitro and recognized not only peptide pulsed APCs but also autologous dendritic cells (DCs) loaded with the TRAG-3 protein. All stage IV melanoma patients with TRAG-3-expressing tumors developed spontaneous CD4+ T cell responses against TRAG-3, demonstrating its strong immunogenicity. None of these patients had detectable IgG Ab responses against TRAG-3. TCRbeta gene usage studies of TRAG-3-specific CD4+ T cell clones from a melanoma patient and a normal donor suggested a restricted TCR repertoire in patients with TRAG-3-expressing tumors. Altogether, our data define a novel profile of spontaneous immune responses to cancer germline Ag-expressing tumors, showing that spontaneous TRAG-3-specific CD4+ T cells are directed against a single immunodominant epitope and exist independently of Ab responses. Because of its immunodominance, peptide TRAG-3(34-48) is of particular interest for the monitoring of spontaneous immune responses in patients with TRAG-3-expressing tumors and for the development of cancer vaccines.     

Systematic review and meta-analysis: rapid diagnostic tests versus placental histology, microscopy and PCR for malaria in pregnant women

Background

To prepare field sites for malaria vaccine trials, it is important to determine baseline antibody and T cell responses to candidate malaria vaccine antigens. Assessing T cell responses is especially challenging, given genetic restriction, low responses observed in endemic areas, their variability over time, potential suppression by parasitaemia and the intrinsic variability of the assays.

Methods

In Part A of this study, antibody titres were measured in adults from urban and rural communities in Ghana to recombinant Plasmodium falciparum CSP, SSP2/TRAP, LSA1, EXP1, MSP1, MSP3 and EBA175 by ELISA, and to sporozoites and infected erythrocytes by IFA. Positive ELISA responses were determined using two methods. T cell responses to defined CD8 or CD4 T cell epitopes from CSP, SSP2/TRAP, LSA1 and EXP1 were measured by ex vivo IFN-γ ELISpot assays using HLA-matched Class I- and DR-restricted synthetic peptides. In Part B, the reproducibility of the ELISpot assay to CSP and AMA1 was measured by repeating assays of individual samples using peptide pools and low, medium or high stringency criteria for defining positive responses, and by comparing samples collected two weeks apart.

Results

In Part A, positive antibody responses varied widely from 17%-100%, according to the antigen and statistical method, with blood stage antigens showing more frequent and higher magnitude responses. ELISA titres were higher in rural subjects, while IFA titres and the frequencies and magnitudes of ex vivo ELISpot activities were similar in both communities. DR-restricted peptides showed stronger responses than Class I-restricted peptides. In Part B, the most stringent statistical criteria gave the fewest, and the least stringent the most positive responses, with reproducibility slightly higher using the least stringent method when assays were repeated. Results varied significantly between the two-week time-points for many participants.

Conclusions

All participants were positive for at least one malaria protein by ELISA, with results dependent on the criteria for positivity. Likewise, ELISpot responses varied among participants, but were relatively reproducible by the three methods tested, especially the least stringent, when assays were repeated. However, results often differed between samples taken two weeks apart, indicating significant biological variability over short intervals.     

Resting CD4+ effector memory T cells are precursors of bystander-activated effectors: a surrogate model of rheumatoid arthritis synovial T-cell function

Background

Previously we described a system whereby human peripheral blood T cells stimulated for 8 days in a cytokine cocktail acquired effector function for contact-dependent induction of proinflammatory cytokines from monocytes. We termed these cells cytokine-activated (Tck) cells and found that the signalling pathways elicited in the responding monocytes were identical whether they were placed in contact with Tck cells or with T cells isolated from rheumatoid arthritis (RA) synovial tissue.

Methods

Here, using magnetic beads and fluorescence-activated cell sorting, we extensively phenotype the Tck effector cells and conclude that effector function resides within the CD4⁺CD45RO⁺, CCR7^-, CD49d^high population, and that these cells are derived from the effector memory CD4⁺ T cells in resting blood.

Results

After stimulation in culture, these cells produce a wide range of T-cell cytokines, undergo proliferation and differentiate to acquire an extensively activated phenotype resembling RA synovial T cells. Blocking antibodies against CD69, CD18, or CD49d resulted in a reduction of tumour necrosis factor-α production from monocytes stimulated with CD4⁺CD45RO⁺ Tck cells in the co-culture assay. Moreover, blockade of these ligands also resulted in inhibition of spontaneous tumour necrosis factor-α production in RA synovial mononuclear cell cultures.

Conclusion

Taken together, these data strengthen our understanding of T-cell effector function, highlight the multiple involvement of different cell surface ligands in cell-cell contact and, provide novel insights into the pathogenesis of inflammatory RA disease.     

Expression of ATP6V1C1 during oral carcinogenesis

We investigated the gene and protein expressions of V-type ATPase protein subunit C1 (ATP6V1C1) in cases of oral squamous cell carcinoma (OSCC) and contralateral normal mucosa in smokers, nonsmokers and former smokers. Subjects were separated into five groups of 15: group 1, smokers with OSCC; group 2, normal contralateral mucosa of OSCC patients; group 3, chronic smokers; group 4, former smokers who had stopped smoking 1 year earlier; group 5, individuals who had never smoked. Exfoliative cytology specimens from oral mucosa of smokers, former smokers and nonsmokers showed normal gene and protein expression. We found significantly greater gene expression in the OSCC group than in the nonsmoker groups. No difference in gene expression was observed between normal contralateral mucosa and nonsmoker groups, smoker and nonsmoker groups or former smoker and nonsmoker groups. We observed intense immunostaining for ATP6V1C1 protein in all cases of OSCC and weak or no staining in smoker, former smoker and nonsmoker groups. Significantly greater expression of ATP6V1C1 protein was observed in the OSCC group compared to the other groups, which supports the role of ATP6V1C1 in effecting changes associated with oral cancer. Analysis of the mucosae of chronic smokers, former smokers and the normal contralateral mucosa of patients with OSCC showed unaltered ATP6V1C1 gene and protein expression. Early stages of carcinogenesis, represented by altered epithelium of chronic smokers, had neither gene nor protein alterations as seen in OSCC. Therefore, we infer that the changes in ATP6V1C1 occur during later stages of carcinogenesis. Our preliminary study provides a basis for future studies of using ATP6V1C1 levels for detecting early stage OSCC.     

Physical model for membrane protrusions during spreading

During cell spreading onto a substrate, the kinetics of the contact area is an observable quantity. This paper is concerned with a physical approach to modeling this process in the case of ameboid motility where the membrane detaches itself from the underlying cytoskeleton at the leading edge. The physical model we propose is based on previous reports which highlight that membrane tension regulates cell spreading. Using a phenomenological feedback loop to mimic stress-dependent biochemistry, we show that the actin polymerization rate can be coupled to the stress which builds up at the margin of the contact area between the cell and the substrate. In the limit of small variation of membrane tension, we show that the actin polymerization rate can be written in a closed form. Our analysis defines characteristic lengths which depend on elastic properties of the membrane-cytoskeleton complex, such as the membrane-cytoskeleton interaction, and on molecular parameters, the rate of actin polymerization. We discuss our model in the case of axi-symmetric and non-axi-symmetric spreading and we compute the characteristic time scales as a function of fundamental elastic constants such as the strength of membrane-cytoskeleton adherence.