CD1b Tetramers Bind αβ\alpha \beta T Cell Receptors to Identify a Mycobacterial Glycolipid-Reactive T Cell Repertoire in Humans

Microbial lipids activate T cells by binding directly to CD1 and T cell receptors (TCRs) or by indirect effects on antigen-presenting cells involving induction of lipid autoantigens, CD1 transcription, or cytokine release. To distinguish among direct and indirect mechanisms, we developed fluorescent human CD1b tetramers and measured T cell staining. CD1b tetramer staining of T cells requires glucose monomycolate (GMM) antigens, is specific for TCR structure, and is blocked by a recombinant clonotypic TCR comprised of TRAV17 and TRBV4-1, proving that CD1b-glycolipid complexes bind the TCR. GMM-loaded tetramers brightly stain a small subpopulation of blood-derived cells from humans infected with Mycobacterium tuberculosis, providing direct detection of a CD1b-reactive T cell repertoire. Polyclonal T cells from patients sorted with tetramers are activated by GMM antigens presented by CD1b. Whereas prior studies emphasized CD8$^+$ and CD4$^-$CD8$^-$ CD1b-restricted clones, CD1b tetramer-based studies show that nearly all cells express the CD4 co-receptor. These findings prove a cognate mechanism whereby CD1b-glycolipid complexes bind to TCRs. CD1b tetramers detect a natural CD1b-restricted T cell repertoire ex vivo with unexpected features, opening a new investigative path to study the human CD1 system

Bifunctional Anti-Huntingtin Proteasome-Directed Intrabodies Mediate Efficient Degradation of Mutant Huntingtin Exon 1 Protein Fragments

Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by a trinucleotide (CAG)n repeat expansion in the coding sequence of the huntingtin gene, and an expanded polyglutamine (>37Q) tract in the protein. This results in misfolding and accumulation of huntingtin protein (htt), formation of neuronal intranuclear and cytoplasmic inclusions, and neuronal dysfunction/degeneration. Single-chain Fv antibodies (scFvs), expressed as intrabodies that bind htt and prevent aggregation, show promise as immunotherapeutics for HD. Intrastriatal delivery of anti-N-terminal htt scFv-C4 using an adeno-associated virus vector (AAV2/1) significantly reduces the size and number of aggregates in HDR6/1 transgenic mice; however, this protective effect diminishes with age and time after injection. We therefore explored enhancing intrabody efficacy via fusions to heterologous functional domains. Proteins containing a PEST motif are often targeted for proteasomal degradation and generally have a short half life. In ST14A cells, fusion of the C-terminal PEST region of mouse ornithine decarboxylase (mODC) to scFv-C4 reduces htt exon 1 protein fragments with 72 glutamine repeats (httex1-72Q) by ∼80–90% when compared to scFv-C4 alone. Proteasomal targeting was verified by either scrambling the mODC-PEST motif, or via proteasomal inhibition with epoxomicin. For these constructs, the proteasomal degradation of the scFv intrabody proteins themselves was reduced<25% by the addition of the mODC-PEST motif, with or without antigens. The remaining intrabody levels were amply sufficient to target N-terminal httex1-72Q protein fragment turnover. Critically, scFv-C4-PEST prevents aggregation and toxicity of httex1-72Q fragments at significantly lower doses than scFv-C4. Fusion of the mODC-PEST motif to intrabodies is a valuable general approach to specifically target toxic antigens to the proteasome for degradation

Pros and cons of different therapeutic antibody formats for recombinant antivenom development.

Antibody technologies are being increasingly applied in the field of toxinology. Fuelled by the many advances in immunology, synthetic biology, and antibody research, different approaches and antibody formats are being investigated for the ability to neutralize animal toxins. These different molecular formats each have their own therapeutic characteristics. In this review, we provide an overview of the advances made in the development of toxin-targeting antibodies, and discuss the benefits and drawbacks of different antibody formats in relation to their ability to neutralize toxins, pharmacokinetic features, propensity to cause adverse reactions, formulation, and expression for research and development (R&D) purposes and large-scale manufacturing. A research trend seems to be emerging towards the use of human antibody formats as well as camelid heavy-domain antibody fragments due to their compatibility with the human immune system, beneficial therapeutic properties, and the ability to manufacture these molecules cost-effectively

Structural Basis for the Interconversion of Maltodextrins by MalQ, the Amylomaltase of Escherichia coli

Amylomaltase MalQ is essential for the metabolism of maltose and maltodextrins in Escherichia coli. It catalyzes transglycosylation/disproportionation reactions in which glycosyl or dextrinyl units are transferred among linear maltodextrins of various lengths. To elucidate the molecular basis of transglycosylation by MalQ, we have determined three crystal structures of this enzyme, i.e. the apo-form, its complex with maltose, and an inhibitor complex with the transition state analog acarviosine-glucose-acarbose, at resolutions down to 2.1 Å. MalQ represents the first example of a mesophilic bacterial amylomaltase with known structure and exhibits an N-terminal extension of about 140 residues, in contrast with previously described thermophilic enzymes. This moiety seems unique to amylomaltases from Enterobacteriaceae and folds into two distinct subdomains that associate with different parts of the catalytic core. Intriguingly, the three MalQ crystal structures appear to correspond to distinct states of this enzyme, revealing considerable conformational changes during the catalytic cycle. In particular, the inhibitor complex highlights the requirement of both a 3-OH group and a 4-OH group (or α1–4-glycosidic bond) at the acceptor subsite +1 for the catalytically competent orientation of the acid/base catalyst Glu-496. Using an HPLC-based MalQ enzyme assay, we could demonstrate that the equilibrium concentration of maltodextrin products depends on the length of the initial substrate; with increasing numbers of glycosidic bonds, less glucose is formed. Thus, both structural and enzymatic data are consistent with the extremely low hydrolysis rates observed for amylomaltases and underline the importance of MalQ for the metabolism of maltodextrins in E. coli

Hearing non-signers use their gestures to predict iconic form-meaning mappings at first exposure to sign

The sign languages of deaf communities and the gestures produced by hearing people are communicative systems that exploit the manual-visual modality as means of expression. Despite their striking differences they share the property of iconicity, understood as the direct relationship between a symbol and its referent. Here we investigate whether non-signing hearing adults exploit their implicit knowledge of gestures to bootstrap accurate understanding of the meaning of iconic signs they have never seen before. In Study 1 we show that for some concepts gestures exhibit systematic forms across participants, and share different degrees of form overlap with the signs for the same concepts (full, partial, and no overlap). In Study 2 we found that signs with stronger resemblance with signs are more accurately guessed and are assigned higher iconicity ratings by non-signers than signs with low overlap. In addition, when more people produced a systematic gesture resembling a sign, they assigned higher iconicity ratings to that sign. Furthermore, participants had a bias to assume that signs represent actions and not objects. The similarities between some signs and gestures could be explained by deaf signers and hearing gesturers sharing a conceptual substrate that is rooted in our embodied experiences with the world. The finding that gestural knowledge can ease the interpretation of the meaning of novel signs and predicts iconicity ratings is in line with embodied accounts of cognition and the influence of prior knowledge to acquire new schemas. Through these mechanisms we propose that iconic gestures that overlap in form with signs may serve as some type of 'manual cognates' that help non-signing adults to break into a new language at first exposure

Hearing non-signers use their gestures to predict iconic form-meaning mappings at first exposure to sign

The sign languages of deaf communities and the gestures produced by hearing people are communicative systems that exploit the manual-visual modality as means of expression. Despite their striking differences they share the property of iconicity, understood as the direct relationship between a symbol and its referent. Here we investigate whether non-signing hearing adults exploit their implicit knowledge of gestures to bootstrap accurate understanding of the meaning of iconic signs they have never seen before. In Study 1 we show that for some concepts gestures exhibit systematic forms across participants, and share different degrees of form overlap with the signs for the same concepts (full, partial, and no overlap). In Study 2 we found that signs with stronger resemblance with signs are more accurately guessed and are assigned higher iconicity ratings by non-signers than signs with low overlap. In addition, when more people produced a systematic gesture resembling a sign, they assigned higher iconicity ratings to that sign. Furthermore, participants had a bias to assume that signs represent actions and not objects. The similarities between some signs and gestures could be explained by deaf signers and hearing gesturers sharing a conceptual substrate that is rooted in our embodied experiences with the world. The finding that gestural knowledge can ease the interpretation of the meaning of novel signs and predicts iconicity ratings is in line with embodied accounts of cognition and the influence of prior knowledge to acquire new schemas. Through these mechanisms we propose that iconic gestures that overlap in form with signs may serve as some type of ‘manual cognates’ that help non-signing adults to break into a new language at first exposure