T-cell Receptor-optimized Peptide Skewing of the T-cell Repertoire Can Enhance Antigen Targeting

Altered peptide antigens that enhance T-cell immunogenicity have been used to improve peptide-based vaccination for a range of diseases. Although this strategy can prime T-cell responses of greater magnitude, the efficacy of constituent T-cell clonotypes within the primed population can be poor. To overcome this limitation, we isolated a CD8⁺ T-cell clone (MEL5) with an enhanced ability to recognize the HLA A*0201-Melan A_27–35 (HLA A*0201-AAGIGILTV) antigen expressed on the surface of malignant melanoma cells. We used combinatorial peptide library screening to design an optimal peptide sequence that enhanced functional activation of the MEL5 clone, but not other CD8⁺ T-cell clones that recognized HLA A*0201-AAGIGILTV poorly. Structural analysis revealed the potential for new contacts between the MEL5 T-cell receptor and the optimized peptide. Furthermore, the optimized peptide was able to prime CD8⁺ T-cell populations in peripheral blood mononuclear cell isolates from multiple HLA A*0201⁺ individuals that were capable of efficient HLA A*0201⁺ melanoma cell destruction. This proof-of-concept study demonstrates that it is possible to design altered peptide antigens for the selection of superior T-cell clonotypes with enhanced antigen recognition properties.     

A comparative study of migratory behaviour and body mass as determinants of moult duration in passerines

Birds moult to maintain plumage function through life, but the factors that determine moult duration are poorly understood. In temperate areas, variation in moult duration could be largely associated with between-species differences in migratory behaviour (migrants have less time for moulting after breeding), and body mass (because the aerodynamic cost of rapid moult increases allometrically with body size). Moreover, if the energetic cost of transport favours a smaller body size in migratory species, then the effects of migratory behaviour and body mass on moult duration could be confounded. We conducted a comparative study of the duration of adult complete moult in 48 European passerine species, in relation to body mass and migratory behaviour (sedentary, short-distance migrants and long-distance migrants). Lighter and more migratory species moulted faster than heavier and more sedentary species, but migration was not associated with body mass. If accelerated moult compromises the success of migration, changes in the physiology or phenology of moult in migratory birds are better interpreted as adaptive responses to compensate for such costs.     

Relationships among timing of moult,moult duration and feather mass in long‐distance migratory passerines

Moult is a costly but necessary process in avian life, which displays two main temporal patterns within the annual cycle of birds (summer and winter moult). Timing of moult can affect its duration and consequently the amount of material invested in feathers, which could have a considerable influence on feather structure and functionality. In this study, we used two complementary approaches to test whether moult duration and feather mass vary in relation to the timing of moult. Firstly, we conducted a comparative study between a sample of long‐distance migratory passerine species which differ in moult pattern. Secondly, we took advantage of the willow warbler's Phylloscopus trochilus biannual moult, for which it is well‐known that winter moult takes longer than summer moult, to assess between‐moult variation in feather mass. Our comparative analysis showed that summer moulting species performed significantly shorter moults than winter moulters. We also detected that feathers produced in winter were comparatively heavier than those produced in summer, both in between‐species comparison and between moults of the willow warbler. These results suggest the existence of a trade‐off between moult speed and feather mass mediated by timing of moult, which could contribute to explain the diversity of moult patterns in passerines.     

The moonlighting protein fructose‐1, 6‐bisphosphate aldolase of Neisseria meningitidis: surface localization and role in host cell adhesion

Fructose‐1, 6‐bisphosphate aldolases (FBA) are cytoplasmic glycolytic enzymes, which despite lacking identifiable secretion signals, have also been found localized to the surface of several bacteria where they bind host molecules and exhibit non‐glycolytic functions. Neisseria meningitidis is an obligate human nasopharyngeal commensal, which has the capacity to cause life‐threatening meningitis and septicemia. Recombinant native N. meningitidis FBA was purified and used in a coupled enzymic assay confirming that it has fructose bisphosphate aldolase activity. Cell fractionation experiments showed that meningococcal FBA is localized both to the cytoplasm and the outer membrane. Flow cytometry demonstrated that outer membrane‐localized FBA was surface‐accessible to FBA‐specific antibodies. Mutational analysis and functional complementation was used to identify additional functions of FBA. An FBA‐deficient mutant was not affected in its ability to grow in vitro, but showed a significant reduction in adhesion to human brain microvascular endothelial and HEp‐2 cells compared to its isogenic parent and its complemented derivative. In summary, FBA is a highly conserved, surface exposed protein that is required for optimal adhesion of meningococci to human cells.     

Smooth muscle phosphatase is regulated in vivo by exclusion of phosphorylation of threonine 696 of MYPT1 by phosphorylation of Serine 695 in response to cyclic nucleotides

Regulation of smooth muscle myosin phosphatase (SMPP-1M) is thought to be a primary mechanism for explaining Ca(2+) sensitization/desensitization in smooth muscle. Ca(2+) sensitization induced by activation of G protein-coupled receptors acting through RhoA involves phosphorylation of Thr-696 (of the human isoform) of the myosin targeting subunit (MYPT1) of SMPP-1M inhibiting activity. In contrast, agonists that elevate intracellular cGMP and cAMP promote Ca(2+) desensitization in smooth muscle through apparent activation of SMPP-1M. We show that cGMP-dependent protein kinase (PKG)/cAMP-dependent protein kinase (PKA) efficiently phosphorylates MYPT1 in vitro at Ser-692, Ser-695, and Ser-852 (numbering for human isoform). Although phosphorylation of MYPT1 by PKA/PKG has no direct effect on SMPP-1M activity, a primary site of phosphorylation is Ser-695, which is immediately adjacent to the inactivating Thr-696. In vitro, phosphorylation of Ser-695 by PKA/PKG appeared to prevent phosphorylation of Thr-696 by MYPT1K. In ileum smooth muscle, Ser-695 showed a 3-fold increase in phosphorylation in response to 8-bromo-cGMP. Addition of constitutively active recombinant MYPT1K to permeabilized smooth muscles caused phosphorylation of Thr-696 and Ca(2+) sensitization; however, this phosphorylation was blocked by preincubation with 8-bromo-cGMP. These findings suggest a mechanism of Ca(2+) desensitization in smooth muscle that involves mutual exclusion of phosphorylation, whereby phosphorylation of Ser-695 prevents phosphorylation of Thr-696 and therefore inhibition of SMPP-1M.     

High avidity antigen-specific CTL identified by CD8-independent tetramer staining

Tetrameric MHC/peptide complexes are important tools for enumerating, phenotyping, and rapidly cloning Ag-specific T cells. It remains however unclear whether they can reliably distinguish between high and low avidity T cell clones. In this report, tetramers with mutated CD8 binding site selectively stain higher avidity human and murine CTL capable of recognizing physiological levels of Ag. Furthermore, we demonstrate that CD8 binding significantly enhances the avidity as well as the stability of interactions between CTL and cognate tetramers. The use of CD8-null tetramers to identify high avidity CTL provides a tool to compare vaccination strategies for their ability to enhance the frequency of high avidity CTL. Using this technique, we show that DNA priming and vaccinia boosting of HHD A2 transgenic mice fail to selectively expand large numbers of high avidity NY-ESO-1(157-165)-specific CTL, possibly due to the large amounts of antigenic peptide delivered by the vaccinia virus. Furthermore, development of a protocol for rapid identification of high avidity human and murine T cells using tetramers with impaired CD8 binding provides an opportunity not only to monitor expansion of high avidity T cell responses ex vivo, but also to sort high avidity CTL clones for adoptive T cell transfer therapy.     

Copepod succession in two South African estuaries

The seasonal succession of copepod species was studied fromNovember 1976 through October 1978 in the Swartkops and Sundaysriver estuaries. South Africa. Acartia natalensis appeared inthe plankton in spring and reaches maximum abundance duringsummer and autumn. It was replaced by A. longipatella in lateautumn which reached maximum abundance in winter and spring.Cycles of dominance are regulated by the interaction of temperature,salinity and competition between the two species. A natalensis a more tolerant of low salinity and the replacementof A. longipatella by A. natalensis starts in the upper estuaryand spreads seawards. Maximum abundance of A. natalensis isattained in water of lower salinity than in the case of A. longipatella.In the Sundays estuary A. natalensis appeared briefly in thelatter half of the study and in the upper and middle estuaryonly. In the lower estuary A. longipatella was present duringall seasons. This was due to the absence of competition fromA. natalensis, high salinity and lower summer temperatures dueto marine influence. A third species of copepod, Pseudodiaptomushessei functions as a pioneer species, exploiting "new water"after flooding or strong fresh water inflow. High abundancemay therefore occur during any season and no competition betweenP. hessei and either species of Acartia was observed.     

The geriatric institution as a therapeutic modality.

Geriatric centres complete with day-hospital facilities are essential for good care of the elderly. Institutions for the ill elderly should be upgraded to provide these people with the full range of services required as their diseases wax and wane. Within this community the resident could move as his needs altered, from minimal support in lodge-style accomodation, to continual "heavy" nursing in a hospital setting providing long-term care. Such a concept accepts that, in the elderly, as they age, new diseases develop that are often difficult to diagnose, and that they require diagnostic and therapeutic services of much greater range than is presently considered adequate in most institutions providing long-term care.     

Serum alters the uptake and biologic activity of CpG oligodeoxynucleotides in B cell chronic lymphocytic leukemia

Immunostimulatory CpG-containing oligodeoxynucleotides (CpG ODN) have a number of effects on B cells, including upregulation of immunogenic molecules, and, therefore, appear attractive as potential components of immunotherapy for B cell chronic lymphocytic leukemia (B-CLL). Previous in vitro studies investigating the effect of CpG ODN on B-CLL cells used serum-low conditions and did not account for the longer-half life of CpG ODN in vitro. The present study was designed to explore how the presence of serum and exposure time affect CpG ODN-mediated changes on B-CLL cells. The optimal concentration for CpG ODN-mediated effects in the presence of 100% serum or plasma was higher (10-20 microg/ml) than for serum-low conditions. Maximal CpG ODN-mediated effects required the presence of ODN for no longer than 3 hours. The inhibition of CpG ODN-mediated effects by serum correlated with lower uptake of ODN into B-CLL cells in the presence of serum. A threshold effect on biologic response was observed, with a given amount of ODN internalized, resulting in phenotypic changes. In conclusion, systemic short-term application of CpG ODN appears to be sufficient to induce phenotypic changes, but higher doses of CpG ODN than previously thought may be necessary because of inhibition of their uptake by serum.     

Doxomysis algoaensis, a new mysid species (Crustacea: Mysidacea) from Algoa Bay (South Africa)

Doxomysis algoaensis sp.nov. is described from Algoa Bay, South Africa where it is common in nearshore marine waters just beyond the breaker line. D. algoaensis sp.nov. is morphologically similar to D. australiensis, but can readily be distinguished by the shape and armature of the telson and the length of the exopod of the fourth male pleopod. The apical cleft is one fifth the telson length in the former species and one third the length in D. australiensis. The telson apex on each side of the cleft is also armed with five and four stout spines in the two species, respectively. In D. australiensis, the exopod of the fourth male pleopod is almost three times the length of the endopod; in D. algoaensis sp.nov., the exopod is only slightly longer than the endopod. Other distinctive features of D. algoaensis sp.nov. include the maxillary palp, which is only slightly broader than long and the greater number of spines on the endopod of the uropod.