Conservation of a CD1 multigene family in the guinea pig.

CD1 is a family of cell-surface molecules capable of presenting microbial lipid Ags to specific T cells. Here we describe the CD1 gene family of the guinea pig (Cavia porcellus). Eight distinct cDNA clones corresponding to CD1 transcripts were isolated from a guinea pig thymocyte cDNA library and completely sequenced. The guinea pig CD1 proteins predicted by translation of the cDNAs included four that can be classified as homologues of human CD1b, three that were homologues of human CD1c, and a single CD1e homologue. These guinea pig CD1 protein sequences contain conserved amino acid residues and hydrophobic domains within the putative Ag binding pocket. A mAb specific for human CD1b cross-reacted with multiple guinea pig CD1 isoforms, thus allowing direct analysis of the structure and expression of at least a subset of guinea pig CD1 proteins. Cell-surface expression of CD1 was detected on cortical thymocytes, dermal dendritic cells in the skin, follicular dendritic cells of lymph nodes, and in the B cell regions within the lymph nodes and spleen. CD1 proteins were also detected on a subset of PBMCs consistent with expression on circulating B cells. This distribution of CD1 staining in guinea pig tissues was thus similar to that seen in other mammals. These data provide the foundation for the development of the guinea pig as an animal model to study the in vivo function of CD1.

Affinity and kinetics of the interactions between an alphabeta T-cell receptor and its superantigen and class II-MHC/peptide ligands.

Immune activation is mediated by a specific interaction between the T-cell receptor (TCR) and an antigenic peptide bound to the major histocompatibility complex (MHC). T-cell activation can also be stimulated by superantigens which bind to germline-encoded variable domain sequences of certain TCR beta-chains. We have used a surface plasmon resonance biosensor to characterize the molecular interactions between a class II-restricted alphabeta TCR and its superantigen and MHC/peptide ligands. The extracellular domains of the murine D10 TCR (Valpha2, Vbeta8.2) were expressed in insect cells and secreted as a disulfide-linked heterodimer. In the absence of MHC class II, purified soluble D10 TCR bound to Staphylococcus aureus enterotoxin C2 with an association rate of 1.69+/-0.12 x 10(4)M(-1) sec(-1) and a dissociation rate of 1.9+/-0.47 x 10(-2) sec(-1), giving a dissociation constant of 1.1 microM. Binding of the TCR to S. aureus enterotoxin B was barely detectable and could not be measured accurately due to the rapid dissociation rate. Soluble D10 TCR also bound to a soluble murine MHC class II I-A(k) molecule containing a fused antigenic conalbumin peptide and complementary leucine zipper sequences to facilitate efficient chain pairing. The purified I A(k) chimera specifically stimulated proliferation of the D10 T-cell clone, and bound to immobilized soluble D10 TCR with an association rate of 1.07+/-0.19 x 10(4)M(-1)sec(-1) and a dissociation rate of 2.2+/-0.65 x 10(-2) sec(-1), giving a dissociation constant of 2.1 microM.

Conformational integrity and ligand binding properties of a single chain T-cell receptor expressed in Escherichia coli.

We recently showed that a soluble, heterodimeric murine D10 T-cell receptor (TCR) (Valpha2Calpha, Vbeta8.2Cbeta) expressed in insect cells binds both Vbeta8.2-specific bacterial superantigen staphylococcal enterotoxin C2 (SEC2) and a soluble, heterodimeric major histocompatibility complex class II I-Ak.conalbumin peptide complex with a low micromolar affinity. To define further the structural requirements for the TCR/ligand interactions, we have produced in Escherichia coli a soluble, functional D10 single chain (sc) TCR molecule in which the Valpha and Vbeta domains are connected by a flexible peptide linker. Purified and refolded D10 scTCR bound to SEC2 and murine major histocompatibility complex class II I-Ak.conalbumin peptide complex with thermodynamic and kinetic binding constants similar to those measured for the baculovirus-derived heterodimeric D10 TCR suggesting that neither the TCR constant domains nor potential N- or O-linked carbohydrate moieties are necessary for ligand recognition and for expression and proper folding of the D10 scTCR. Purified D10 scTCR remained soluble at concentrations up to 1 mM. Circular dichroism and NMR spectroscopy indicated that D10 scTCR is stabilized predominantly by beta-sheet secondary structure, consistent with its native-like conformation. Because of its limited size, high solubility, and structural integrity, purified D10 scTCR appears to be suitable for structural studies by multidimensional NMR spectroscopy.

Cemental annulation enhancement: a technique for age determination in man.

Cemental annulations are easily countable in teeth from animals that have an exaggerated regular change of food intake from season to season. Although present in human teeth, cemental annulations are not always easy to count. A method for preparing human teeth for evaluation involving collection, identification, measuring, sectioning, cleaning, acid etching, staining, and mounting is reported. Sections 100-microns thick were stained with cresyl fast violet as a stain of choice and were photographed using standard light microscopic techniques as well as Nomarsky interference microscopy. Countability of annulations from photographic enlargements was evaluated.