Comparative efficiencies of bispecific F(ab′γ)2 and chimeric mouse/human IgG antibodies in recruiting cellular effectors for cytotoxicity via Fcγ receptors

Summary The three forms of Fc receptor carried by monocytes (FcRI, II) and natural killer (NK) cells (FcRIII) are all capable of mediating cell lysis. Here we compare the use of F(ab)₂ bispecific antibodies, specifically targetting individual FcR, and chimeric IgG mouse/human antibodies which are capable of targetting all FcR, for their ability to mediate target cell destruction. The derivatives are prepared by linking hinge sulphydryl residues via tandem thioether bonds, using a bismaleimide crosslinker: Fab from an anti-FcR mAb linked to Fab from a common anti-target mAb (BsAb), or Fab from the common anti-target mouse antibody linked to human Fc (FabFc or bisFabFc). All the derivatives targetting chick red blood cells gave efficient lysis, although different effector cell donors yielded differences in both the lytic levels achieved and the comparative efficiencies of derivatives. In contrast, significant lysis of the guinea pig lymphoblastic leukaemia, L₂C, regularly resulted only via the anti-FcRIII BsAb and the chimeric derivatives. These results suggest that the chimeric, Fc-containing derivatives mediate tumour cell lysis principally through FcRIII on NK cells. This is in contrast to the situation with the chick red blood cells where the chimeric derivatives appear capable of lysing erythrocytes by utilizing either monocytes or NK cells, because significant (50%) lysis occurred with effector cell populations magnetically depleted through either FcRII or FcRIII. A major difference between these two types of antibody derivative was their ability to function in the presence of high concentrations of normal human Fc. The lysis mediated by BsAb reactive with FcRI or II was unaffected by the presence of human Fc at 2.5 mg/ml (a concentration comparable with that yielded by IgG in plasma) whereas the BsAb recognizing FcRIII and all the Fc-containing derivatives were completely inhibited.This work has been supported by Tenovus, the Cancer Research Campaign, the Leukaemia Research Fund, Italfarmaco, Milano, Italy and the Imperial Cancer Research Fund     

Seroreactivity to HPV-16 proteins in women with early cervical neoplasia

Summary Although serological reactivity to human papillomavirus type 16 (HPV-16) proteins has been demonstrated in patients with invasive cervical carcinoma, the degree of seroreactivity to these proteins in women with preinvasive disease and its relationship to the HPV type associated with the disease are unclear. We obtained sera from 27 women undergoing cone biopsy for cervical precursor lesions and 22 controls and analyzed seroreactivity by Western blot to fusion proteins containing portions of the HPV-16 E4, L1 and L2 open-reading frames (ORFs). Positives were analyzed by scanning densitometry and intensity values for each case plotted relative to controls. Cervical biopsy specimens from patients were analyzed for HPV-16 nucleic acids by DNA · DNA in situ hybridization. Mean intensity values for seroreactivity to the pATH-E4 protein approached significance (P = 0.058) and a significantly higher proportion of cases vs controls registered values over 4.0 for pATH-E4 (26% vs 4.5%;P = 0.04) and pATH-L2 (48% vs 18%;P = 0.03) proteins. A significantly higher mean intensity value for E4 was observed for cases containing HPV-16 DNA vs HPV-16 negative cases or controls. Thus, seroreactivity to HPV-16-derived proteins may be more common in women with preinvasive cervical disease, and for some protein targets (E4) may indicate a relatively type-specific response.Supported in part by grants from the National Cancer Institute [CA 47676 (C.P.C.)], American Cancer Society [MV-395 (C.P.C.)] and an institutional support grant (J.K.R.). Dr. Crum is a recipient of a Physician Scientist Award from the National Institute of Allergy and Infectious Disease (AI00628)     

FMRFamide modulates the action of phase shifting agents on the ocular circadian pacemakers of Aplysia and Bulla

Summary The eye of the marine mollusk Aplysia californica contains a photo-entrainable circadian pacemaker that drives an overt circadian rhythm of spontaneous compound action potentials in the optic nerve. Both light and serotonin are known to influence the phase of this ocular rhythm. The current study evaluated the effect of FMRFamide on both light and serotonin induced phase shifts of this rhythm. The application of FMRFamide was found to block serotonin induced phase shifts but, by itself, FMRFamide did not cause significant phase shifts. Furthermore, the effects of FMRFamide on light-induced phase shifts appeared to be phase dependent (i.e., the application of FMRFamide inhibited light-induced phase delays but actually enhanced the magnitude of phase advances). As in Aplysia, the eye of Bulla gouldiana also contains a circadian pacemaker. In Bulla, FMRFamide prevented light-induced phase advances and delays. Although FMRFamide alone generated phase dependent phase shifts, it did not cause phase shifts at the phases where it blocked the effects of light. These data demonstrate that FMRFamide can have pronounced modulatory effects on phase shifting inputs to the ocular pacemakers of both Aplysia and Bulla.Abbreviations ASW artificial seawater - CAP compound action potential - CT circadian time - 5-HT serotonin     

Molecular mimicry and the autoimmune response to the peripheral nerve myelin PO glycoprotein

In the Lewis rat immunisation with the myelin PO glycoprotein can induce an inflammatory demyelinating disease of the peripheral nervous system, experimental allergic neuritis (EAN), which has many clinical and histopathological parallels with the human disease the Guillain-Barre syndrome. In view of the reported association of GBS with a number of infectious agents we have investigated whether molecular mimicry may occur between microbial antigens and the PO protein that could possibly trigger a similar pathogenic autoimmune response in man. A computer search of the available protein sequence data bases identified several absolute sequence homologies between PO and viral proteins that involve five or more consecutive amino acid residues. Four of these sequence homologies involved viral pathogens previously associated with the Guillain-Barre syndrome, namely Epstein-Barr virus (EBV), cytomegalovirus (CMV), Varicella zoster virus (VZV) and human immunodeficiency virus I (HIV I). Although, sequence homologies were also found between viral peptides and the neuritogenic determinants of PO, residues 56–71 and 180–199, these homologies proved incapable of eliciting EAN in the Lewis rat. These observations are discussed with reference to the role that molecular mimicry between T cell epitopes on pathogen derived antigens and the PO protein may play in the pathogenesis of the Guillain-Barre syndrome.Abbreviations EAN Experimental allergic neuritis - EAE experimental allergic encephalomyelitis - PNS peripheral nervous system - CNS central nervous system - MBP myelin basic protein - GBS Guillain Barre syndrome - CFA complete Freund's Adjuvant - LPC lysophosphatidyl choline - VZV Varicella zoster virus - CMV cytomegalovirus - EBV Epstein Barr virus - HIV I human immunodeficiency virus I Special issue dedicated to Dr. Alan N. Davison     

Sequence of the 5-aminolevulinic acid dehydratase-encoding gene from the hyperthermophilic methanogen, Methanothermus sociabilis.

We report on the sequence of the Methanothermus sociabilis aladh gene, which encodes the 5'-aminolevulinic acid dehydratase. The identity of the enzyme was determined by sequence comparison and by expression in Escherichia coli.     

Expression, isolation and purification of antibody fragments fused to maltose-binding protein in Escherichia coli]

We have fused the variable domains of a mouse antibody to the C-terminal end of the maltose-binding protein (malE), at the genetic level. The hybrid proteins were expressed in E. coli under control of the malEp promoter, and exported to the periplasm, at low temperature. They were purified by affinity chromatography on cross-linked amylose. When the two variable domains were fused together through a peptide link, the hybrid displayed similar affinity and specificity to the antigen as the native antibody.     

Interactions between freshwater snails and tadpoles: competition and facilitation

Summary Freshwater snails and anuran tadpoles have been suggested to have their highest population densities in ponds of intermediate size where abiotic disturbance (e.g. desiccation) is low and large predators absent. Both snails and tadpoles feed on periphytic algae and, thus, there should be a large potential for competitive interactions to occur between these two distantly related taxa. In a field experiment we examined the relative strength of competition between two closely related snail species, Lymnaea stagnalis and L. peregra, and between L. stagnalis and tadpoles of the common frog, Rana temporaria. Snail growth and egg production and tadpole size at and time to metamorphosis were determined. Effects on the common food source, periphyton, were monitored with the aid of artificial substrates. Periphyton dry weight was dramatically reduced in the presence of snails and/or tadpoles. There were no competitive effects on growth or egg production of the two snail species when they were coexisting. Mortality of L. peregra was high (95%) after reproduction, but independent of treatment. Growth of L. stagnalis was reduced only at the highest tadpole densities, whereas egg production was reduced both by intraspecific competition and by competition with tadpoles. Differences in egg production were retained after tadpole metamorphosis. Tadpole larval period increased, weight of metamorphosing frogs decreased and growth rate was reduced as a function of increasing tadpole density. However, contrary to expectation, snails had a positive effect on tadpole larval period, weight and growth rate. Further, in experimental containers without snails there was a dense growth of the filamentous green alga Cladophora sp. We suggest that the facilitative effects of snails on tadpoles are due to an indirect mutualistic mechanism, involving competition between food sources of different quality (microalgae and Cladophora sp.) and tadpoles being competitively dominant over snails for the preferred food source (microalgae). In the presence of tadpoles snails will be forced to feed on low-quality Cladophora, increasing nutrient turnover rates, which results in enhanced productivity of microalgae, increasing tadpole food resources. Thus, tadpoles have a negative effect on snails through resource depression, while snails facilitate tadpole growth through an indirect enhancement of food availability.     

The effect of a single colony of the red wood ant,Formica polyctena,on the spider fauna (Araneae) of a beech forest floor

Summary The predation on spiders in a forest ecosystem by a colony of red wood ants, Formica polyctena, was estimated using a barrier to isolate the colony. Of the ants' total prey, 4.6% were spiders. In order to estimate the effect of F. polyctena within their hunting area on the spider population, the spiders' population density was studied inside and outside the hunting area. Samples of the forest floor were taken, spider webs were counted and pitfall traps were used. No significant difference was found in density or composition of the spider fauna inside and outside the hunting area.     

An integrated approach to oilseed rape cultivar selection using phenotypic stability

Summary Various methods of evaluating phenotypic stability have been proposed; however, no single method can adequately describe cultivar performance. The objectives of this study were to integrate a number of methods of evaluating stability and to use this approach for cultivar selection. These objectives were considered in the context of the broad-based oilseed rape cultivar (Brassica napus spp. oleifera) evaluation system currently used in western Canada. Regression analysis was used to assess cultivar response to environments. Cluster analysis was used to assemble cultivars into groups with similar regression coefficients (b _i ) and mean yield. Three parametric stability parameters, years within locations mean square (MS; Y/L), Shukla's stability variance ( _i ² ), and Francis and Kannenberg's coefficient of variability (CV _i ), were compared to determine which method would be most suitable for selection of oilseed rape cultivars from within clustered groups. Yield data from three cultivars and six breeding lines that had been tested for 2 years at 26 locations in the Western Canola Cooperative Test A were used for all calculations. The cluster analysis was successful in identifying commercially acceptable breeding lines. The parameter MS _i Y/L was considered to be more appropriate than either CV _i or _i ² , because it measured only the unpredictable portion of the genotype x environment interaction and was independent of the other cultivars in the test. The use of cluster analysis to group entries with similar b _i values and mean yields, followed by selection for stability within groups, was advocated.Contribution No. 846 of the Plant Science Department, University of Manitoba