An efficient chemical approach to bispecific antibodies and antibodies of high valency

Irreversible chemical programming of monoclonal aldolase antibody (mAb) 38C2 has been accomplished with β-lactam equipped mono- and bifunctional targeting modules, including a cyclic-RGD peptide linked to either the peptide (d-Lys⁶)-LHRH or another cyclic RGD unit and a small-molecule integrin inhibitor SCS-873 conjugated to (d-Lys⁶)LHRH. We also prepared monofunctional targeting modules containing either cyclic RGD or (d-Lys⁶)-LHRH peptides. Binding of the chemically programmed antibodies to integrin receptors α(v)β(3) and α(v)β(5) and to the luteinizing hormone releasing hormone receptor were evaluated. The bifunctional and bivalent c-RGD/LHRH and SCS-783/LHRH, the monofunctional and tetravalent c-RGD/c-RGD, and the monofunctional bivalent c-RGD chemically programmed antibodies bound specifically to the isolated integrin receptor proteins as well as to integrins expressed on human melanoma M-21 cells. c-RGD/LHRH, SCS-783/LHRH, and LHRH chemically programmed antibodies bound specifically to the LHRH receptors expressed on human ovarian cancer cells. This approach provides an efficient, versatile, and economically viable route to high-valency therapeutic antibodies that target defined combinations of specific receptors. Additionally, this approach should be applicable to chemically programmed vaccines.     

Coexistence of Probe Conformations in Lipid Phases—A Polarized Fluorescence Microspectroscopy Study

Several well-established fluorescence methods depend on environment-sensitive probes that report about molecular properties of their local environment. For reliable interpretation of experiments, careful characterization of probes’ behavior is required. In this study, bleaching-corrected polarized fluorescence microspectroscopy with nanometer spectral peak position resolution was applied to characterize conformations of two alkyl chain-labeled 7-nitro-2-1,3-benzoxadiazol-4-yl phospholipids in three model membranes, representing the three main lipid phases. The combination of polarized and spectral detection revealed two main probe conformations with their preferential fluorophore dipole orientations roughly parallel and perpendicular to membrane normal. Their peak positions were separated by 2–6 nm because of different local polarities and depended on lipid environment. The relative populations of conformations, estimated by a numerical model, indicated a specific sensitivity of the two probes to molecular packing with cholesterol. The coexistence of probe conformations could be further exploited to investigate membrane organization below microscopy spatial resolution, such as lipid rafts. With the addition of polarized excitation or detection to any environment-sensitive fluorescence imaging technique, the conformational analysis can be directly applied to explore local membrane complexity.     

Electroencephalographic changes in the lateral septum complex following systemic administration of DES-TYR1 -α-endorphin,Des-Tyr1-γ-endorphin and haloperidol in rats

The influence of systemically administered Des-Tyr¹-α-endorphin (DTαE), Des-Tyr¹-γ-endorphin (DTγE) and haloperidol on electroencephalographic (EEG) activity of the lateral septum complex (LSC) and the frontal cortex was studied in male rats. DTαE (2 μg) significantly increased whereas DTγE (10 μg) significantly decreased the amounts of activity in the 5 Hz band. In addition, DTαE promoted production of 15 - 20 Hz activity, while DTγE decreased the amount of 20 - 30 Hz activity. EEG activity exhibited a marked variability which persisted throughout the recording session following administration of the peptides. Haloperidol markedly decreased the proportion of 10 - 15 Hz activity. The alterations in EEG of the frontal cortex were similar to those in LSC but less pronounced. The differences in the time course and frequency bands affected suggest that the effects of peptides and haloperidol on EEG activity of LSC are not mediated by the same mechanisms.     

Narratives of success among Irish and African Caribbean migrants

This paper compares the narratives of two men in midlife who migrated to the UK from Ireland and from the Caribbean as children, in the middle of the last century. We examine how success is narrated over the life course to show how migrants’ positioning of themselves differs from the ways in which they are positioned by outsiders, including in policy and public discourse. We conclude that while outsider narratives often polarise success and failure, insider understandings of success are dynamic and culturally and historically situated.     

Genetic interaction between Non-MHC T- and B-cell alloantigens in response to rous sarcomas in chickens

Chickens of Regional Poultry Research Laboratory (RPRL) inbred line 6₃ regress sarcomas induced by Bryan high-titer Rous sarcoma virus to a greater extent than chickens of line RPRL 100, although these lines are identical for the major histocompatibility B complex. They differ, however, at three independent autosomal loci: Ly-4 and Th-1 determine the surface alloantigens of partly overlapping subsets of T lymphocytes, and Bu-1 determines a surface alloantigen of B lymphocytes. The association of genotypes at these loci with quantitative variation in their ability to regress Rous sarcomas was tested in segregating F₄ generation progeny derived from crosses of lines 100 and 6₃. The Ly-4 and Bu-1 genotypes showed association with Rous sarcoma regression, but the Th-1 genotype did not. Chickens of the Ly-4 ^a/Ly-4 ^a, Bu-1 ^b/Bu-1 ^b and Ly-4 ^b/Ly-4 ^b, Bu-1 ^a/Bu-1 ^a genotypes had a significantly higher regressor ability than the other two double homozygous genotypes. These results indicate that higher regression is associated with (1) interaction between the Ly-4 and Bu-1 loci, and (2) complementation between either the line 6 Ly-4 ^a allele and the line 100 Bu-1 ^b allele, or the line 100 Ly-4 ^b allele and the line 6 Bu-1 ^a allele.     

Purified Fc epsilon R+ bone marrow and splenic non-B, non-T cells are highly enriched in the capacity to produce IL-4 in response to immobilized IgE, IgG2a, or ionomycin.

Non-B, non-T cells from spleen and bone marrow cells produce IL-4 in response to cross-linkage of high affinity receptors for Fc epsilon R or Fc gamma RII, and to treatment with calcium ionophores. Cells bearing high affinity Fc epsilon R constituted 1 to 2% of non-B, non-T cells of spleen and of total bone marrow cells from naive donors. In mice whose immune systems had been polyclonally activated by injection with anti-IgD antibodies or had been infected with Nippostrongylus brasiliensis larvae, the frequency of Fc epsilon R+ cells in splenic non-B, non-T cells was also 1 to 2% but in bone marrow from anti-IgD-injected mice donors the frequency was approximately 5%. Cell sorting experiments revealed that all of the capacity to produce IL-4 in response to immobilized IgE or IgG2a or to ionomycin was found in the Fc epsilon R+ fraction. Among the Fc epsilon R+ spleen cells from naive donors, the frequency of IL-4-producing cells was 1/20 to 1/40 whereas in mice that had been injected with anti-IgD or infected with N. brasiliensis, the frequency of IL-4 producing cells in the Fc epsilon R+ population was approximately 1/5.     

Analysis of Human RSV Immunity at the Molecular Level: Learning from the Past and Present

Human RSV is one of the most prevalent viral pathogens of early childhood for which no vaccine is available. Herein we provide an analysis of RSV epitope data to examine its application to vaccine design and development. Our objective was to provide an overview of antigenic coverage, identify critical antibody and T cell determinants, and then analyze the cumulative RSV epitope data from the standpoint of functional responses using a combinational approach to characterize antigenic structure and epitope location. A review of the cumulative data revealed, not surprisingly, that the vast majority of epitopes have been defined for the two major surface antigens, F and G. Antibody and T cell determinants have been reported from multiple hosts, including those from human subjects following natural infection, however human data represent a minority of the data. A structural analysis of the major surface antigen, F, showed that the majority of epitopes defined for functional antibodies (neutralizing and/or protective) were either shown to bind pre-F or to be accessible in both pre- and post-F forms. This finding may have has implications for on-going vaccine design and development. These interpretations are in agreement with previous work and can be applied in the larger context of functional epitopes on the F protein. It is our hope that this work will provide the basis for further RSV-specific epitope discovery and investigation into the nature of antigen conformation in immunogenicity.