Natural killing target antigens as inducers of interferon: studies with an immunoselected, natural killing-resistant human T lymphoblastoid cell line

The human T lymphoblastoid cell line CEM was subjected to immunoselection by co-culture with peripheral blood mononuclear cells (PBMC) for resistance to natural killer (NK) cell-mediated lysis. The NK susceptibility of the resulting subline, CEM.NKR, was 8.4 to 20.6% of that of CEM when PBMC or adherent cell-depleted PBMC were used as effector cells, and -7.1 to 12.1% of that of CEM when Percoll gradient-enriched large granular lymphocytes (LGL) were used. However, CEM and CEM.NKR exhibited comparable sensitivity to antibody-dependent cellular cytotoxicity. Unlabeled CEM was eight- to 32-fold more effective than unlabeled CEM.NKR in inhibiting the NK lysis of labeled CEM target cells, and CEM bound 1.9 to 3.9-fold more Percoll gradient-enriched LGL than CEM.NKR in single cell-binding assays, suggesting that the NK-resistant variant has lost the expression of NK target antigens. However, CEM.NKR was comparable to CEM in its ability to induce interferon (IFN)-alpha production by PBMC in vitro, and the NK-resistant variant maintained its susceptibility to the antiproliferative effects of IFN-alpha, indicating that these phenomena may be mediated by molecules other than NK target structures. Comparison of CEM and CEM.NKR by indirect immunofluorescence with monoclonal antibodies specific for leukocyte antigens and the transferrin receptor, and by microcytotoxicity typing for HLA-A and B specificities, revealed no major differences.     

Peptide-induced modulation of target cell sensitivity to natural killing.

We have previously shown that the capacity of class I molecules to confer resistance to NK in transfected target cells maps to the Ag-binding site (ABS) of the HLA class I structure. Here we examine the effect of peptide (reagents specific for the ABS) pretreatment on the NK sensitivity of class I+ target cells. Synthetic peptides (10-17 amino acids in length) were used to pretreat C1R target cells expressing either no serologically detectable HLA-A, B class I molecules, or C1R transfectants expressing individual HLA-A or -B locus class I molecules. In each case in which the class I allele had previously been shown to directly bind a given peptide, peptide-pulsing of target cells resulted in increased sensitivity to NK-mediated conjugation and cytolysis. The NK susceptibility of C1R target cells expressing no HLA-A, B class I molecules or the nonprotective HLA-A2.1 or HLA-A2M70 mutant class I molecules was unaffected by pretreatment with HLA-A2-binding peptides. These results support the intimate involvement of the HLA class I ABS and potentially ABS-bound peptides in determining target cell sensitivity to NK. Furthermore, these findings form the basis of an effective screening procedure for discerning peptide class I allele-specific interactions.     

Managing human disturbance: factors influencing flight-initiation distance of birds in a West African nature reserve

Escape behaviour in response to perceived predators can be employed as a guide when designating protected areas around sensitive bird species to minimise the impact of human disturbance. A key measure of escape response is flight-initiation distance (FID), the distance at which a prey animal initiates its escape when approached by a potential predator. We tested the predictions of optimal escape theory by determining the factors that influence FID of bird species in a Nigerian reserved area and its surrounding habitats, and so the potential utility of FID in managing human disturbance on birds, for the first time within a West African context. We tested how FID varied with group size, proximity to vegetation acting as protective cover, levels of human use, and survival rate, and whether these relationships varied by species. We collected 504 FIDs for seven bird species in Amurum Forest Reserve and its surrounding habitats (Jos, Nigeria). The FID was lower in larger groups and when species were closer to protective cover. The FID was lower outside of the protected area because animals in sites with higher levels of human presence and use may become habituated. The FID was higher for species with higher survival, being consistent with predictions from life history theory. Overall, birds perceived humans as a potential threat and responded in accordance to the predictions of optimal escape theory, with FID increasing with increased cost of staying. Reserve managers in Africa could use species- and context-specific FIDs to designate buffer distances for the protection of wildlife from human disturbance.     

Molecular detection of streptomycin-producing streptomycetes in Brazilian soils.

Actinomycetes were isolated from soybean rhizosphere soil collected as two field sites in Brazil. All the isolates were identified as Streptomyces species and were screened for streptomycin production and the presence of two genes, strA and strB1, known to be involved in streptomycin biosynthesis in Streptomyces griseus. Antibiotic resistance profiles were determined for 53 isolates from cultivated and uncultivated sites, and approximately half the strains were streptomycin resistance. Clustering by the unweighted pair group method with averages indicated the presence of two major clusters, with the majority of resistant strains from cultivated sites being placed in cluster 1. Only representatives from this cluster contained strA. Streptomycetes containing strA and strB1 were phenotypically diverse, and only half could be assigned to known species. Sequence comparison of 16S rRNA and trpBA (tryptophan synthetase) genes revealed that streptomycin- producing streptomycetes were phylogenetically diverse. It appeared that a population of streptomycetes had colonized the rhizosphere and that a proportion of these were capable of streptomycin production.     

Interactive effects of pesticide exposure and pathogen infection on bee health – a critical analysis

Bees are fundamentally important for pollination services and declines in populations could have significant economic and environmental implications. Pesticide exposure and pathogen infection are recognised as potential stressors impacting upon bee populations and recently there has been a surge in research on pesticide–disease interactions to reflect environmentally realistic scenarios better. We critically analyse the findings on pesticide–disease interactions, including effects on the survival, pathogen loads and immunity of bees, and assess the suitability of various endpoints to inform our mechanistic understanding of these interactions. We show that pesticide exposure and pathogen infection have not yet been found to interact to affect worker survival under field‐realistic scenarios. Colony‐level implications of pesticide effects on Nosema infections, viral loads and honey bee immunity remain unclear as these effects have been observed in a laboratory setting only using a small range of pesticide exposures, generally exceeding those likely to occur in the natural environment, and assessing a highly selected series of immune‐related endpoints. Future research priorities include the need for a better understanding of pesticide effects on the antimicrobial peptide (AMP) component of an individual's immune response and on social defence behaviours. Interactions between pesticide exposure and bacterial and fungal infections have yet to be addressed. The paucity of studies in non‐Apis bee species is a further major knowledge gap.     

Purification of papain-solubilized histocompatibility antigens from a cultured human lymphoblastoid line, RPMI 4265.

HL-A antigens having specificities HL-A2, HL-A7, HL-A12 have been solubilized by papain treatment of membrane preparations from the cultured human lymphoblastoid cell line RPMI 4265 and purified about 80-fold by chromatography on carboxymethylcellulose, Sephadex G-150, and diethylaminoethylcellulose columns. Separation of HL-A2 from a mixture of HL-A7 and HL-A12 was achieved on the final DEAE-cellulose column. The yield was about 1 mg of protein of each antigen preparation per 100 g of packed, frozen cells. On sodium dodecyl sulfate gel electrophoresis both preparations showed two polypeptide bands. The smaller subunit of 12,000 daltons is common to all HL-A preparations and has been shown to be identical with beta2-microglobulin. The larger subunit is a glycopeptide and in the HL-A7, 12 preparation was resolved into a duplex of 34,000 and 37,000 daltons. The HL-A2 preparation showed a single band at 34,000 daltons. On isoelectric focusing under nondenaturing conditions, the preparation showed multiple bands, all of which contained both subunits and retained antigenic activity. On isoelectric focusing in the presence of 6 M urea a single band for beta2-microglobulin and multiple bands for the larger subunit were seen. This charge heterogeneity of the larger subunit has been shown to be due to variable amounts of sialic acid. When HL-A antigen preparations were subjected to Sephadex G-100 chromatography in the presence of 3 M KCl, no separation of the two subunits was observed.     

Preparation, properties and substrate-exclusion effects of an insoluble pronase derivative

1. A diazotized co-polymer of leucine and p-aminophenylalanine was used to prepare insoluble pronase. 2. The product was similar to the native enzyme in pH optimum, temperature optimum and broad specificity. 3. Exclusion effects were observed that appear related to the molecular weight of the substrate being hydrolysed. 4. The effects are explained on the basis of impedance of substrate access to the catalytic site by the supporting solid matrix.