Assembly of Protein Building Blocks Using a Short Synthetic Peptide

Combining proteins or their defined domains offers new enhanced functions. Conventionally, two proteins are either fused into a single polypeptide chain by recombinant means or chemically cross-linked. However, these strategies can have drawbacks such as poor expression (recombinant fusions) or aggregation and inactivation (chemical cross-linking), especially in the case of large multifunctional proteins. We developed a new linking method which allows site-oriented, noncovalent, yet irreversible stapling of modified proteins at neutral pH and ambient temperature. This method is based on two distinct polypeptide linkers which self-assemble in the presence of a specific peptide staple allowing on-demand and irreversible combination of protein domains. Here we show that linkers can either be expressed or be chemically conjugated to proteins of interest, depending on the source of the proteins. We also show that the peptide staple can be shortened to 24 amino acids still permitting an irreversible combination of functional proteins. The versatility of this modular technique is demonstrated by stapling a variety of proteins either in solution or to surfaces

How Many Thymocytes Audition for Selection?

T cell maturation requires the rearrangement of clonotypic T cell receptors (TCR) capable of interacting with major histocompatibility complex (MHC) ligands to initiate positive and negative selection. Only 3–5% of thymocytes mature to join the peripheral T cell pool. To investigate the basis for this low success rate, we have measured the frequency of preselection thymocytes capable of responding to MHC. As many as one in five MHC-naive thymocytes show upregulation of activation markers on exposure to MHC-expressing thymic stroma in short-term reaggregate culture. The majority of these cells display physiological changes consistent with entry into the selection process within 24 h. By exposing TCR transgenic thymocytes to a range of MHC–peptide complexes, we show that CD69 induction is indicative of thymocyte selection, positive or negative. Our data provide evidence that the fraction of thymocytes that qualify to enter the thymic selection process far exceeds the fraction that successfully complete it, and suggest that most MHC-reactive thymocytes are actively eliminated in the course of selection

The Efficiency of CD4 Recruitment to Ligand-engaged TCR Controls the Agonist/Partial Agonist Properties of Peptide–MHC Molecule Ligands

One hypothesis seeking to explain the signaling and biological properties of T cell receptor for antigen (TCR) partial agonists and antagonists is the coreceptor density/kinetic model, which proposes that the pharmacologic behavior of a TCR ligand is largely determined by the relative rates of (a) dissociation of ligand from an engaged TCR and (b) recruitment of lck-linked coreceptors to this ligand-engaged receptor. Using several approaches to prevent or reduce the association of CD4 with occupied TCR, we demonstrate that consistent with this hypothesis, the biological and biochemical consequence of limiting this interaction is to convert typical agonists into partial agonist stimuli. Thus, adding anti-CD4 antibody to T cells recognizing a wild-type peptide–MHC class II ligand leads to disproportionate inhibition of interleukin-2 (IL-2) relative to IL-3 production, the same pattern seen using a TCR partial agonist/antagonist. In addition, T cells exposed to wild-type ligand in the presence of anti-CD4 antibodies show a pattern of TCR signaling resembling that seen using partial agonists, with predominant accumulation of the p21 tyrosine-phosphorylated form of TCR-ζ, reduced tyrosine phosphorylation of CD3ε, and no detectable phosphorylation of ZAP-70. Similar results are obtained when the wild-type ligand is presented by mutant class II MHC molecules unable to bind CD4. Likewise, antibody coligation of CD3 and CD4 results in an agonist-like phosphorylation pattern, whereas bivalent engagement of CD3 alone gives a partial agonist-like pattern. Finally, in accord with data showing that partial agonists often induce T cell anergy, CD4 blockade during antigen exposure renders cloned T cells unable to produce IL-2 upon restimulation. These results demonstrate that the biochemical and functional responses to variant TCR ligands with partial agonist properties can be largely reproduced by inhibiting recruitment of CD4 to a TCR binding a wild-type ligand, consistent with the idea that the relative rates of TCR–ligand disengagement and of association of engaged TCR with CD4 may play a key role in determining the pharmacologic properties of peptide–MHC molecule ligands. Beyond this insight into signaling through the TCR, these results have implications for models of thymocyte selection and the use of anti-coreceptor antibodies in vivo for the establishment of immunological tolerance

Apoptosis assays with lymphoma cell lines: problems and pitfalls

Much attention has been focused on the manner in which tumour cells die after treatment with cytotoxic agents. The basic question is whether cells die via apoptosis or via direct damage from the toxic agent. Various assays have been used to make this distinction. However, we show herein that some of the widely used assays for apoptosis do not in fact distinguish between apoptosis and other forms of cell death. More specifically: (1) A sub-G1 DNA content, identified by propidium iodide staining, does not distinguish between apoptotic and necrotic cells; (2) loss of mitochondrial membrane potential does not distinguish between apoptotic and necrotic cells, unless combined with an assay for an intact cell membrane; (3) subcellular fragments that arise from dead cells or from apoptotic bodies can interfere with some assays for apoptosis such as annexin V staining, as they may be close to the size of intact cells, making it difficult to decide where to set the size threshold; (4) irradiated cells display a large increase in nonspecific Ab binding. This may be partly due to an increase in cell size, but, regardless of the cause, it can lead to a mistaken conclusion that there is an increase in a particular antigen if appropriate control reagents are not tested; and (5) experiments utilising Ab crosslinking have neglected the role of cell aggregation, which can cause multiple problems including death from mechanical stress when cells are handled. Consideration of these factors will improve our ability to determine the mode of cell death

Short‐ and longer‐term impacts of Child Friendly Space Interventions in Rwamwanja Refugee Settlement, Uganda

Alastair Ager - ORCID 0000-0002-9474-3563 https://orcid.org/0000-0002-9474-3563Karin Diaconu - ORCID 0000-0002-5810-9725 https://orcid.org/0000-0002-5810-9725Background The establishment of Child Friendly Spaces (CFSs) has become a widespread intervention targeting protection and support for displaced children in humanitarian contexts. There is a lack of evidence of impact of these interventions with respect to both short‐term outcomes and longer‐term developmental trajectories.Methods We collected data from caregivers of Congolese refugee children residing in Rwamwanja Refugee Settlement at three timepoints. To assess short‐term impact of CFSs, we compared indicators assessed shortly after refugees’ arrival (baseline, T1) and endline (T2, three to six months after CFS implementation) amongst 430 CFS attenders and 161 nonattenders. Follow‐up assessments after the end of CFS programming were conducted 18 months post‐baseline (T3) with caregivers of 249 previous CFS attenders and 77 CFS nonattenders.Results In the short‐term, attendance at CFSs was associated with better maintenance of psychosocial well‐being (PSWB; β = 2.093, p < .001, Cohen's d = .347) and greater increases in developmental assets (β = 2.517, p < .001, Cohen's d = .231), with significantly stronger impacts for girls. CFS interventions meeting higher programing quality criteria were associated with greater impact on both PSWB and development assets (β = 2.603 vs. β = 1.793 and β = 2.942 vs. β = 2.337 for attenders at higher and lower‐quality CFSs c.f. nonattenders, respectively). Amongst boys, benefits of program attendance were only indicated for those attending higher‐quality CFS (β = 2.084, p = .006 for PSWB). At follow‐up, however, there were no discernable impacts of prior CFS attendance on any measures. Age and school attendance were the only characteristics that predicted an outcome – developmental assets – at follow‐up.Conclusions Attendance at CFSs – particularly involving higher‐quality programming – supported children's well‐being and development. However, sustained impact beyond active CFS programming was not demonstrated. Intervention goals and strategies in humanitarian contexts need to address the challenge of connecting children to other resources to facilitate developmental progress in conditions of protracted displacement.This research was funded by World Vision International and Elrha's Research for Health in Humanitarian Crises (R2HC) Programme (elrha.org/r2hc), which aims to improve health outcomes by strengthening the evidence base for public health interventions in humanitarian crises. At the time of the award supporting this study, the R2HC program was funded equally by the Wellcome Trust and the UK Department for International Development.https://doi.org/10.1111/jcpp.1306960pubpub1