Employing of the Amplified Fragment Length Polymorphism (AFLP) Methodology as an Efficient Population Genetic Tool for Symbiotic Cnidarians

Although the use of molecular markers in population genetics of marine organisms is increasingly employed, methodologic limitations still hampered the research for some taxa, such as symbiotic cnidarians, including scleractinian corals. The development of molecular tools in scleractinian corals' studies is faced with a list of obstacles, such as high cost, labor, time consuming, contamination with foreign DNA, and markers with low resolution. The AFLP (amplified fragment length polymorphism) method, overcomes most of the obstacles listed above except of the difficulty of contamination by algal symbiont DNA. We compared the implication of two pre-DNA extraction treatments to obtain coral DNA free of algal contaminations, termed as CPEM, cell population enriched method, and TTEM, total tissue extraction method. The CPEM process result in pure coral DNA for all samples, but is time consuming, whereas in the TTEM process, approximately 25% to 18% of the samples are still contaminated by algal DNA. However, algal DNA contaminations in the PCR at 2.5 x 10(-1) ng level (approximately 100 algal cells) and below, did not amplify any new AFLP band or peak for neither radioactive nor florescence analyses. Therefore, even the TTEM process may be used because it is faster, easier to handle, and easily employed on a large amount of samples, with minimal contamination artifacts. When correctly employed, both methods are applicable to wide experimental manipulations.     

Comparative assessment of passive surveillance in disease-free and endemic situation: Example of <Emphasis Type="Italic">Brucella melitensis</Emphasis> surveillance in Switzerland and in Bosnia and Herzegovina

Background

Globalization and subsequent growth in international trade in animals and animal products has increased the importance of international disease reporting. Efficient and reliable surveillance systems are needed in order to document the disease status of a population at a given time. In this context, passive surveillance plays an important role in early warning systems. However, it is not yet routinely integrated in the assessment of disease surveillance systems because different factors like the disease awareness (DA) of people reporting suspect cases influence the detection performance of passive surveillance. In this paper, we used scenario tree methodology in order to evaluate and compare the quality and benefit of abortion testing (ABT) for Brucella melitensis (Bm) between the disease free situation in Switzerland (CH) and a hypothetical disease free situation in Bosnia and Herzegovina (BH), taking into account DA levels assumed for the current endemic situation in BH.

Results

The structure and input parameters of the scenario tree were identical for CH and BH with the exception of population data in small ruminants and the DA in farmers and veterinarians. The sensitivity analysis of the stochastic scenario tree model showed that the small ruminant population structure and the DA of farmers were important influential parameters with regard to the unit sensitivity of ABT in both CH and BH. The DA of both farmers and veterinarians was assumed to be higher in BH than in CH due to the current endemic situation in BH. Although the same DA cannot necessarily be assumed for the modelled hypothetical disease free situation as for the actual endemic situation, it shows the importance of the higher vigilance of people reporting suspect cases on the probability that an average unit processed in the ABT-component would test positive.

Conclusion

The actual sensitivity of passive surveillance approaches heavily depends on the context in which they are applied. Scenario tree modelling allows for the evaluation of such passive surveillance system components under assumed disease free situation. Despite data gaps, this is a real opportunity to compare different situations and to explore consequences of changes that could be made.

     

Limited maintenance of vaccine-induced simian immunodeficiency virus-specific CD8 T-cell receptor clonotypes after virus challenge

T-cell receptors (TCRs) govern the specificity, efficacy, and cross-reactivity of CD8 T cells. Here, we studied CD8 T-cell clonotypes from Mane-A*10(+) pigtail macaques responding to the simian immunodeficiency virus (SIV) Gag KP9 epitope in a setting of vaccination and subsequent viral challenge. We observed a diverse TCR repertoire after DNA, recombinant poxvirus, and live attenuated virus vaccination, with none of 59 vaccine-induced KP9-specific TCRs being identical between macaques. The KP9-specific TCR repertoires remained diverse after SIV or simian-human immunodeficiency virus challenge but, remarkably, exhibited substantially different clonotypic compositions compared to the corresponding populations prechallenge. Within serial samples from individual pigtail macaques, only a small subset (33.9%) of TCRs induced by vaccination were maintained or expanded after challenge. Most (66.1%) of the TCRs induced by vaccination were not detectable after challenge. Our results suggest that some CD8 T cells induced by vaccination are more efficient than others at responding to a viral challenge. These findings have implications for future AIDS virus vaccine studies, which should consider the "fitness" of vaccine-induced T cells in order to generate robust responses in the face of virus exposure.     

Comparison of plasma viremia and antibody responses in macaques inoculated with envelope variants of single-cycle simian immunodeficiency virus differing in infectivity and cellular tropism

Molecular differences in the envelope glycoproteins of human immunodeficiency virus type 1 and simian immunodeficiency virus (SIV) determine virus infectivity and cellular tropism. To examine how these properties contribute to productive infection in vivo, rhesus macaques were inoculated with strains of single-cycle SIV (scSIV) engineered to express three different envelope glycoproteins with full-length (TM_open) or truncated (TM_stop) cytoplasmic tails. The 239 envelope uses CCR5 for infection of memory CD4⁺ T cells, the 316 envelope also uses CCR5 but has enhanced infectivity for primary macrophages, and the 155T3 envelope uses CXCR4 for infection of both naive and memory CD4⁺ T cells. Separate groups of six rhesus macaques were inoculated intravenously with mixtures of TM_open and TM_stop scSIV_mac239, scSIV_mac316, and scSIV_mac155T3. A multiplex real-time PCR assay specific for unique sequence tags engineered into each virus was then used to measure viral loads for each strain independently. Viral loads in plasma peaked on day 4 for each strain and were resolved below the threshold of detection within 4 to 10 weeks. Truncation of the envelope cytoplasmic tail significantly increased the peak of viremia for all three envelope variants and the titer of SIV-specific antibody responses. Although peak viremias were similar for both R5- and X4-tropic viruses, clearance of scSIV_mac155T3 TM_stop was significantly delayed relative to the other strains, possibly reflecting the infection of a CXCR4⁺ cell population that is less susceptible to the cytopathic effects of virus infection. These studies reveal differences in the peaks and durations of a single round of productive infection that reflect envelope-specific differences in infectivity, chemokine receptor specificity, and cellular tropism.     

Effect of thymectomy on human peripheral blood T cell pools in myasthenia gravis

The human thymus is required for establishment of the T cell pool in fetal life, but postnatal thymectomy does not lead to immunodeficiency in humans. Because thymectomy in humans is performed for treatment of myasthenia gravis (MG), we have studied patients with MG for effects of thymectomy on peripheral blood (PB) naive (CD45RA(+), CD62L(+)) and memory (CD45RO(+)) T cells. We have also determined the effect of thymectomy on levels of PB cells containing signal joint TCR delta excision circles (TRECs), a molecular marker of thymus emigrants that have divided few times after leaving the thymus. In 17 nonthymectomized and 26 thymectomized MG patients studied at varying times after thymectomy (1 day to 41 years), we found no significant mean difference in PB T cell TREC levels between ages 40 and 80 years. However, both thymectomized and nonthymectomized MG patients had lower PB T cell TREC levels than did age-matched normal subjects (p < 0.0001 for both). These data demonstrated that MG itself or treatment for MG decreased thymopoiesis independent of thymectomy. Next, to control for disease activity and treatment, we prospectively studied 10 MG patients before and from 27 to 517 days after thymectomy. We found that thymectomy decreased CD4 or CD8 T cell TREC concentrations most when thymopoiesis was active before thymectomy (six of six patients), but had little effect in patients when thymopoiesis was minimal (four of four patients). In contrast, there was no significant effect of thymectomy on absolute numbers of naive PB T cells. Thus, in MG, removal of a thymus with active thymopoiesis resulted in a significant fall in PB TREC(+) T cells postthymectomy.     

Differential impact of simultaneous migration on coevolving hosts and parasites

Background  

The dynamics of antagonistic host-parasite coevolution are believed to be crucially dependent on the rate of migration between populations. We addressed how the rate of simultaneous migration of host and parasite affected resistance and infectivity evolution of coevolving meta-populations of the bacterium Pseudomonas fluorescens and a viral parasite (bacteriophage). The increase in genetic variation resulting from small amounts of migration is expected to increase rates of adaptation of both host and parasite. However, previous studies suggest phages should benefit more from migration than bacteria; because in the absence of migration, phages are more genetically limited and have a lower evolutionary potential compared to the bacteria.     

A versatile approach to multiple gene RNA interference using microRNA-based short hairpin RNAs

Background  

Effective and stable knockdown of multiple gene targets by RNA interference is often necessary to overcome isoform redundancy, but it remains a technical challenge when working with intractable cell systems.     

Comparison of different delivery systems of DNA vaccination for the induction of protection against tuberculosis in mice and guinea pigs

The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs.