93 research outputs found
Mutagenesis of the RGD Motif in the Yellow Fever Virus 17D Envelope Protein
No full textAbstractThe envelope protein of yellow fever virus 17D (YFV-17D) contains a solvent-exposed RGD motif, which has led to the suggestion that integrins may function as cellular receptors for YFV-17D. We found that mutating the RGD motif to RGE had no effect on viral titers, whereas changing RGD to TGD, TGE, TAD, TAE, or RGS led to reduced titers. Substitution of RGD by RAD or RAE yielded RNA genomes that replicated in mammalian cells but could not spread to neighboring cells at 37°C. These mutants did spread through the cell monolayer at 30°C (both in mosquito cells and in SW13 cells) and viruses grown at this temperature were capable of infecting mammalian cells at 37°C. These results strongly suggest that RGD-mediated integrin binding does not play a major role in YFV-17D entry, since the RGD to RAD mutation, as well as many or all of the other mutations studied, should disrupt all RGD-dependent integrin binding. However, the RGD to RAD or RAE mutations (as well as TAD and TAE) severely destabilized the envelope protein at 37°C, providing an explanation for the observed phenotype. Implications of these findings are discussed in light of the fact that mutations that alter tropism or virulence in different flaviviruses are often found within the loop containing the RGD motif
Characterization of T-cell immune responses in clinical trials of the candidate RTS,S malaria vaccine
No full textThe candidate malaria vaccine RTS,S has demonstrated 45.7% efficacy over 18 months against all clinical disease in a phase-III field study of African children. RTS,S targets the circumsporozoite protein (CSP), which is expressed on the Plasmodium sporozoite during the pre-erythrocyte stage of its life-cycle; the stage between mosquito bite and liver infection. Early in the development of RTS,S, it was recognized that CSP-specific cell-mediated immunity (CMI) was required to complement CSP-specific antibody-mediated immunity. In reviewing RTS,S clinical studies, associations between protection and various types of CMI (CSP-specific CD4+ T cells and INF-γ ELISPOTs) have been identified, but not consistently. It is plausible that certain CD4+ T cells support antibody responses or co-operate with other immune-cell types to potentially elicit protection. However, the identities of vaccine correlates of protection, implicating either CSP-specific antibodies or T cells remain elusive, suggesting that RTS,S clinical trials may benefit from additional immunogenicity analyses that can be informed by the results of controlled human malaria infection studies
Breeding against osteochondrosis : phenotypic and genetic analyses in horses and pigs
No full text Osteochondrosis (OC) is a disturbance in the process of endochondral ossification during skeletal growth. The objectives of this thesis were to assess the prevalence and genetic parameters of OC, and to facilitate optimization of breeding against OC in horses and pigs. In the Dutch warm blood horse, 70% of individuals showed joint abnormalities in at least one of the 28 locations examined. Joint abnormalities were divided into flattened bone contours and bone fragments. Genetic parameters were estimated for flattened bone contours, fragments and overall OC. Heritability was 0.08 for flattened bone contours, 0.22 for fragments, and 0.23 for overall OC. In fattening pigs, the prevalence of OC was 41%, and 12% of the individuals had severe OC. The prevalence was highest for individuals kept on a concrete, partially slatted floor with ad libitum feeding (58%), and lowest for individuals kept on a deep litter floor with restricted feeding (34%). These results demonstrate that the prevalence of OC can be reduced by applying deep litter floors and restricted feeding. The differences in growth patterns between pigs with and without OC were investigated also. After 28 days of age, piglets with severe OC at slaughter started to grow faster, and were significantly heavier after 70 days of age than pigs without OC at slaughter. These results suggest that OC might be related to high growth rates during a specific time period. To evaluate prospects for breeding against OC, selection responses were compared between breeding schemes using phenotypic selection based on own performance or progeny testing and genomic selection. The results show that, when genomic information has an equal accuracy as phenotypic information before selection, it will have a lower equilibrium response to selection because it has a larger Bulmer effect. Especially for low heritable traits, genomic selection was of additional value over traditional selection. The general discussion argues that OC has both economical and practical relevance for the current horse industry. Simulations showed potential for genomic selection compared to the current breeding program in the Dutch warmblood horse (KWPN), even with small reference populations. In pigs, OC is a more serious problem in sows than in fattening pigs, because sows live longer. A decrease in OC prevalence will, therefore, yield larger economic and welfare benefits then apparent from results on fattening pigs. Also for pigs, simulations indicated possibilities for genomic selection. The final section of the general discussion proposes directions for future research.</p
A protein kinase C-related enzyme activity in Dictyostelium discoideum
No full textAbstractIn crude cell lysates of the cellular slime mould Dictyostelium discoideum we identified a protein kinase C (PKC)-like enzyme activity. This activity, measured as phosphorylation of a synthetic EGF-receptor-derived peptide [(1987) J. Biol. Chem. 262, 772–777], was regulated by Ca2+, phosphatidylserine (PS), 1,2-dioleoyl-rac-glycerol (DG) and the phorbol ester PMA. PS and DG stimulated the enzyme in a synergistic manner. The stimulation by these lipids was, in contrast to what has been found for ‘classical’ mammalian PKC, not dependent on Ca2+. The D. discoideum enzyme was strongly stimulated by nanomolar concentrations of PMA, and inhibited by PKC-inhibitor staurosporine
Immunogenic anti-cancer chemotherapy as an emerging concept
No full textTumors can acquire mutations or hijack regulatory pathways of the host immune system to render them resistant to immune attack. Standard first line therapies such as chemotherapy and radiation were not thought to provoke natural immunity to cancer, but recent findings demonstrating that dying tumor cells present and release key signals to stimulate or evade neighboring leukocytes are challenging that view. Killing tumor cells in a manner that provides danger signals and tumor antigens in the right context promotes the engagement of innate and adaptive immunity; however, this response alone will not be effective against established cancer. Coincidently driving the immune response with specific monoclonal antibodies and other immunomodulators that activate and mature dendritic cells and co-stimulate T cells and other lymphocytes is one approach. Additionally releasing immune checkpoints and inhibiting tumor-derived molecules that prevent effective tumor immunity is another. Combined these approaches have enormous potential to improve the current outcomes from conventional cancer therapy
HLA-DP4 presents an immunodominant peptide from the RSV G protein to CD4 T cells
No full textAbstractCD4 T cells play a crucial role during virus infections by producing antiviral cytokines and by regulating humoral and cellular immune responses. Unfortunately however, exaggerated CD4 T cell responses can cause significant immune-mediated disease as was observed during RSV infections in children previously vaccinated with a formalin-inactivated virus in the 1960s. It has been observed that vaccination with the G protein of RSV tends to prime mice for a similar Th2-mediated enhanced disease. Whether the G protein may play a role in enhanced disease in man is unclear. In the present study, we identified an immunodominant epitope in the conserved region of the G protein encompassing amino acid residues 162–175. This epitope is presented in the context of HLA-DPB1*0401 and DPB1*0402, the most prevalent HLA class II alleles. Importantly, in some patients, a mixed Th1/Th2 response against this epitope was found in bronchoalveolar lavage samples during primary RSV infections
Free-living protozoa in drinking water supplies: community composition and role as hosts for Legionella pneumophila
No full text Free-living protozoa in drinking water supplies: community composition and role as hosts for Legionella pneumophila Free-living protozoa, which feed on bacteria, play an important role in the communities of microorganisms and invertebrates in drinking water supplies and in (warm) tap water installations. Several bacteria, including opportunistic human pathogens such as Legionella pneumophila, are able to survive and replicate within protozoan hosts, and certain free-living protozoa are opportunistic human pathogens as well. However, not much is known about the occurrence of these organisms in drinking water supplies in relation to water quality and other environmental conditions. This lack of knowledge may in part be attributed to the limitations of microscopic techniques and cultivation methods for detection and identification of protozoa. In the investigations described in this thesis, molecular methods were applied to identify communities of free-living protozoa in drinking water supplies and to elucidate the effects of environmental conditions on the growth of these organisms. Molecular methods were also used to detect and identify protozoan hosts, both known and yet-undescribed, for L. pneumophila. The eukaryotic communities were studied using terminal restriction fragment length polymorphism (T-RFLP), clone library analyses of partial 18S rRNA gene fragments, and qPCR assays for Acanthamoeba spp. and Hartmannella vermiformis. In two groundwater supplies in the Netherlands (T In one drinking water supply in the Caribbean, the concentration of H. vermiformis correlated with the concentration of Legionella spp., and clones related to Amoebozoa predominated the protozoan community. These observations, in combination with the low turbidity and the varying ATP concentrations, indicated that biofilms in this system promoted growth of amoebae and also Legionella spp. Ciliophora represented 25\\% of the protozoan OTUs in another Caribbean supply with elevated ATP concentrations and turbidity, related to corroding cast-iron pipes. Thus, sediments seem to favor growth of ciliates. Cercozoan types predominated in one of the Caribbean supplies with low ATP concentrations ( A biofilm batch test (BBT) was applied to amplify and subsequently identify protozoan hosts for L. pneumophila. In this test 21 freshwater samples, with added polyethylene cylinders to promote biofilm formation, were inoculated with L. pneumophila and subsequently incubated in duplicate at 37°C. Growth of L. pneumophila was observed in 16 of 18 water types when the host protozoan H. vermiformis had been added. Indigenous H. vermiformis was indicated as host in 12 of the 19 BBT flasks with growth of L. pneumophila. In none of the water samples incubated at 37°C in the BBT system, growth of Acanthamoeba spp. was observed. In several flasks with growth of L. pneumophila, the protozoa Diphylleia rotans, Echinamoeba thermarum and Neoparamoeba sp. were identified as candidate hosts. In vitro studies are needed to confirm their role as hosts for L. pneumophila. To assess the effects of water temperature on indigenous free-living protozoan communities, four water types were incubated in a BBT system at 20, 30, 37 and 42°C. Only two of the 53 OTUs related to free-living protozoa obtained from these freshwater types at day 0 were also detected after incubation. Multiplication of indigenous Acanthamoeba spp. was observed at 20 and 30°C, with only limited growth in one flask at 37°C. Indigenous H. vermiformis multiplied at 20, 30 and 37°C, but at 42°C proliferation was only observed in water collected from the river Rhine. Growth of organisms related to Amoebozoa and Stramenopiles was observed at all four temperatures, whereas Cercozoan and Euglenozoan types predominated in the BBT system incubated at 37 and 30°C, respectively. The Amoebozoan Arachnula sp. was identified as candidate host for L. pneumophila at 42°C, but in vitro studies with this organism are needed for confirmation. These observations indicate that H. vermiformis, the predominating protozoan host for Legionella spp., is ubiquitous in water supplies and other engineered water installations. This organism can grow at temperatures ranging from less than 20 to 42°C. Proliferation is limited at active biomass concentrations </p
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