Model systems to study the parameters determining the success of phage antibody selections on complex antigens

Phage antibody display technology offers a powerful tool for the isolation of specific antibodies to defined target antigens. Most selection strategies described to date have relied on the availability of purified and often recombinant antigen, providing the possibility to perform selections on a well-defined antigen source. However, when the target antigen cannot be purified (e.g., an integral membrane protein), or if the antigen is unknown (e.g., when searching for novel markers on cells or tissues), panning of phage antibody libraries has to be performed on complex antigen sources such as cell surfaces or tissue sections, or even by in vivo selection methods. This provides a series of technical and experimental challenges. One focus of our research is to select antibodies directed to novel cancer-induced antigens expressed by tumours and by the tumour vasculature. To understand the parameters governing selection on complex antigen sources and to assess the efficiency of these phage library selections, we have set up two model selection systems in which both tumour cells and vascular endothelial cells serve as target "antigen". We describe a model based on phage antibodies directed to the tumour antigen epithelial glycoprotein-2, to compare phage antibody selections on a range of different antigen sources including purified and recombinant antigen, whole live cells, tissue cryosections and in vivo grown solid tumours. Secondly, we describe a model based on a phage antibody directed against the endothelial cell inducible adhesion molecule E-selectin. We compare selections on cultured cell monolayers with selections on cell suspensions immobilised on columns, to determine which selection approach is most suitable for the identification of novel tumour endothelial cell markers. Our data provide insight into the efficiency and thus potency of different selection strategies and show that there are very large differences in the recovery and enrichment of binding phage between the different methods tested. Our results further demonstrate the feasibility of phage antibody selections on whole, intact cells and show that these may sometimes compare favourably to selections on purified antigen. Selections on endothelial cells immobilised on columns compare favourably with selections on cell-monolayers; the most favourable conditions for both selection procedures are described. The implications of our data for phage antibody selections on these different complex antigen sources using either non-immune or immune phage antibody repertoires are discussed. The use of model systems such as the ones described here will help to determine optimal experimental conditions for phage library selections on complex antigens and aid in developing more powerful selection procedures for target discovery.     

The New Millennium Palliative Care Project (2000-2003): the impact of specialised GP advisors.

This study describes a novel type of support for GPs caring for patients dying at home: the establishment and evaluation of a telephone advisory service for GPs, run by GPs with a special interest in palliative care (GPwSIs) in the Netherlands 2000-2003. A growing number of GPs called for advice, 10% during out of hours. Prognosis of the patients was generally short (days to weeks in 70% of cases). Most advice sought by GPs concerned symptom management and on evaluation, 85% of the GPs followed the advice.     

Predictive testing for complex diseases using multiple genes: Fact or fiction?

PURPOSE: There is ongoing debate about whether testing low-risk genes at multiple loci will be useful in clinical care and public health. We investigated the usefulness of multiple genetic testing using simulated data. METHODS: Usefulness was evaluated by the area under the receiver-operating characteristic curve (AUC), which indicates the accuracy of genetic profiling in discriminating between future patients and nonpatients. The AUC was investigated in relation to the number of genes assumed to be involved, the risk allele frequency, the odds ratio of the risk genotypes, and to the proportion of variance explained by genetic factors as an approximation of the heritability of the disease. RESULTS: We demonstrated that a high (AUC > 0.80) to excellent discriminative accuracy (AUC > 0.95) can be obtained by simultaneously testing multiple susceptibility genes. A higher discriminative accuracy is obtained when genetic factors play a larger role in the disease, as indicated by the proportion of explained variance. The maximum discriminative accuracy of future genetic profiling can be estimated at present from the heritability and prevalence of disease. CONCLUSIONS: Genetic profiling may have the potential to identify individuals at higher risk of disease depending on the prevalence and heritability of the disease.     

Experimental infection of immunomodulated NOD/LtSz-SCID mice as a new model for Plasmodium falciparum erythrocytic stages

The main objective of this study was to determine whether a chemical immunomodulation protocol could reduce the resistance of NOD/LtSz-SCID mice to Plasmodium falciparum infection and provide an improved mouse model for screening the antimalarial activity of new compounds. This model was compared with the presently used immunodeficient Beige/Nude/Xid (BNX) mouse model, using the same protocol, in terms of percentage of infected mice, parasite development, leukocyte response and phagocytosis of P. falciparum infected cells in various organs. Our results show that the combination of the chemical immune modulation protocol with the genetic background of NOD/LtSz-SCID mice results in the development of long-lasting P. falciparum infection in a high percentage of mice. A comparison of the results obtained in the histological study for both mouse models suggests that the higher rate of success in NOD/LtSz-SCID mice could be related to the reduced macrophage recruitment developed in different tissues to remove the parasite from blood.     

Storage conditions of blood samples and primer selection affect the yield of cDNA polymerase chain reaction products of hepatitis C virus.

We have noticed that suboptimal specimen processing and storage conditions may cause false-negative results in the detection of hepatitis C virus (HCV) RNA in plasma or serum. To establish the influence of specimen handling in a serological laboratory on the rate of detection of HCV RNA by the cDNA polymerase chain reaction (cDNA-PCR), we tested routine serum samples and fresh-frozen plasma samples from the same bleeding from confirmed anti-HCV-positive blood donors. When primers from the NS3/NS4 region were used, HCV RNA was detected in fresh-frozen plasma from 67% of the donors, whereas positive results were obtained with only 50% of the serum samples that had been subjected to routine serological procedures. Analysis of the same samples with primers from the highly conserved 5'-terminal region (5'-TR) revealed an HCV RNA detection rate of 92% for both the routine and the fresh-frozen samples. However, the yield of the amplification product in routine samples was strongly reduced compared with that in fresh-frozen plasma. Comparison of both primer sets for cDNA-PCR showed that the 5'-TR primer set was 10- to 100-fold more effective in detecting HCV RNA. We also analyzed the effect of storage of whole EDTA-blood and serum at room temperature and at 4 degrees C on the yield of the amplification product. A rapid decline in detectable HCV RNA of 3 to 4 log units was observed within 14 days when whole blood and serum were stored at room temperature. By contrast, no perceptible reduction in the cDNA-PCR signal was found in freshly prepared serum stored at 4 degrees C.     

Lymphoid and non-lymphoid cells in the adenoid of children with otitis media with effusion: a comparative study

We characterized on immuno- and enzymecytochemical level the lymphoid and non-lymphoid cells in the adenoid of children with upper respiratory tract infections (URI) and otitis media with effusion (OME) and compared these with the adenoid of children with URI without OME and with the adenoid of 'healthy' children and adults. Besides macrophages and dendritic cells we also showed the presence of MHC class II positive, ciliated, epithelial cells. These non-lymphoid cells were present in all adenoids. However, their number was less than 1% of all cells. We found no difference in lymphocyte subsets from children with URI + OME compared with those from children with URI alone. These two groups showed a significant decrease of CD8-positive (suppressor/cytotoxic) cells and a slight increase in CD22-positive B cells in comparison to 'healthy' children. No difference was found in percentages of CD4-positive (helper/inducer) cells. The localization of the lymphoid subsets in adenoids of children with URI and/or OME did not differ from those of 'healthy' children and adults.     

Production of fimbrial adhesins K99 and F41 by enterotoxigenic Escherichia coli as a function of growth-rate domain.

The production of fimbrial adhesins K99 and F41 by enterotoxigenic Escherichia coli has been measured in steady-state chemostat experiments at various specific growth rates (microseconds) and in a recycling fermentor across a range of mu values falling to less than 0.004 h-1. It has been demonstrated that the production of K99 and F41 fimbriae is correlated with mu both in aerobic and anaerobic chemostat experiments. A significant production of fimbriae was only detected at mu values higher than 0.2 h-1. This behavior was further examined by culturing the bacteria in a recycling fermentor with complete biomass retention. It could be shown that the production of K99 and F41 fimbriae only occurred during balanced growth, with a high biomass yield at mu values higher than 0.04 h-1 corresponding to mass doubling times (td) of less than 17 h. The production of both fimbriae halted during balanced growth with a lower biomass yield (at mu values between 0.012 and 0.04 h-1 corresponding to td values between 17 and 58 h) and unbalanced stringent growth (at mu values lower than 0.012 h-1 or td values higher than 58 h). The external pH of the medium greatly influenced the production of both K99 and F41 fimbriae. At pH values lower than 7, the production of fimbriae was strongly inhibited. Also, at pH values higher than 7, a decrease in production was observed. The consequences of the observed phenomena for the pathogenic behavior of this E. coli strain are discussed.     

Isolation,characterization, phosphorylation and site of synthesis of Spinacia chloroplast ribosomal proteins

Summary We have characterized the ribosomal proteins from Spinacia chloroplasts using two-dimensional gel electrophoresis. The 30S and 50S subunits contain 23–25 and 36 ribosomal proteins, respectively. In contrast to prokaryotic ribosomes, chloroplast ribosomes contain at least one (and possibly two) phosphorylated ribosomal proteins. Isolated chloroplasts synthesize in the presence of (³⁵S) labeled methionine and cysteine at least seven 30S and thirteen 50S ribosomal proteins which are assembled into (pre)ribosomes. This suggests that about one third of the chloroplast ribosomal proteins is encoded by the chloroplast DNA itself. The identity of several labeled proteins in the two-dimensional gel electrophoretic patterns which did not comigrate with stained chloroplast ribosomal proteins is discussed.Abbreviations CBB Coomassie Brilliant Blue - CHI cycloheximide - cp chloroplast - DTT dithiotreitol - EDTA ethylene diamine tetraacetate - EGTA ethylene glycol-bis (-amino ethyl ether) N,N-tetraacetic acid - kD kilodalton - LHCP light harvesting chlorophyll a/b protein - PMSF phenyl methyl sulfonyl fluoride - RuBPCase ribulose-1,5-bisphosphate carboxylase - SDS sodiumdodecylsulphate     

Seasonal abundance of Anopheles farauti (Diptera: Culicidae) sibling species in far north Queensland, Australia

In the Cairns area of far north Queensland, Australia, the seasonal abundance of Anopheles farauti Laveran sibling species was studied at 6 locations, representing 3 habitat types, between August 1995 and September 1997. A total of 45,401 An. farauti s.l. was collected using CO2 + octenol baited CDC light traps, and consisted of 29,565 An. farauti No. 2, 14,214 An. farauti No. 3, and 1,622 An. farauti s.s. The relative abundance of all 3 species differed significantly by season and location. An. farauti No.2 was the dominant species except in Cairns, where An. farauti s.s. was most abundant, and at Ninds Creek, where An. farauti No. 3 predominated. The dominant species at each location was present year round, although peaks in seasonal abundance were observed. An. farauti s.s. populations were highest during the wet season (January-April). In lowland freshwater swamp habitats and 1 brackish location, An. farauti No. 2 was more abundant during the wet season. However, at the highland freshwater swamp habitat, populations of An. farauti No. 2 were highest during the late dry season and early wet season (October-December). There was a significant positive correlation of both temperature and rainfall with An. farauti s.s. and An. farauti No. 2 trap collections. There was a negative correlation between An. farauti No. 3 and temperature, indicating that this species may be more abundant during cool weather. Although there were significant relationships among weather variables and An. farauti s.l. collections, correlation values were generally low, indicating that other factors may contribute to variability among An. farauti sibling species trap collections.