Decrypting the structure of major histocompatibility complex class I-restricted cytotoxic T lymphocyte epitopes with complex peptide libraries.

Complex synthetic peptide libraries with defined amino acids in one or more positions of the H-2Kb-restricted cytotoxic T lymphocyte (CTL) epitopes SIINFEKL and RGYVYQGL and mixtures of 19 amino acids in the remaining positions were used to analyze the structural requirements of peptide binding to MHC class I molecules and antigen recognition by CTLs. This approach provides means to assess semiquantitatively the contribution of every amino acid to the binding of peptides to major histocompatibility complex (MHC) molecules without biases introduced by naturally processed peptides. Primary and secondary anchor residues were defined for their major contribution to the binding efficiency of the peptides. In contrast to primary anchors, secondary anchor amino acids vary greatly in their side chains and position in the sequences. All amino acids in the octapeptide sequences were found to exhibit positive or negative influences on binding to the MHC molecules and on recognition of the resulting complexes by CTLs. Strong interdependence of the effects of the individual residues in the epitope sequences was demonstrated. CTL responses to peptide libraries were suppressed when residues were introduced; however, they were augmented when the critical residues for T cell recognition were fixed, suggesting a potential use of the peptide libraries for defining epitope sequences in general.

The structure and mechanical properties of articular cartilage are highly resilient towards transient dehydration.

Articular cartilage is a mechanically highly challenged material with very limited regenerative ability. In contrast to elastic cartilage, articular cartilage is exposed to recurring partial dehydration owing to ongoing compression but maintains its functionality over decades. To extend our current understanding of the material properties of articular cartilage, specifically the interaction between the fluid and solid phase, we here analyze the reversibility of tissue dehydration. We perform an artificial dehydration that extends beyond naturally occurring levels and quantify material recovery as a function of the ionic strength of the rehydration buffer. Mechanical (indentation, compression, shear, and friction) measurements are used to evaluate the influence of de- and rehydration on the viscoelastic properties of cartilage. The structure and composition of native and de/rehydrated cartilage are analyzed using histology, scanning electron microscopy, and atomic force microscopy along with a 1,9-dimethylmethylene blue (DMMB) assay. A broad range of mechanical and structural properties of cartilage can be restored after de- and rehydration provided that a physiological salt solution is used for rehydration. We detect only minor alterations in the microarchitecture of rehydrated cartilage in the superficial zone and find that these alterations do not interfere with the viscoelastic and tribological properties of the tissue. STATEMENT OF SIGNIFICANCE: We here demonstrate the sturdiness of articular cartilage towards changes in fluid content and show that articular cartilage recovers a broad range of its material properties after dehydration. We analyze the reversibility of tissue dehydration to extend our current understanding of how the material properties of cartilage are established, focusing on the interaction between the fluid and solid phase. Our findings suggest that the high resilience of the tissue minimizes the risk of irreversible material failure and thus compensates, at least in part, its poor regenerative abilities. Tissue engineering approaches should thus not only reproduce the correct tissue mechanics but also its pronounced sturdiness to guarantee a similar longevity.

Characterization of p-aminohippurate transport from rat kidney which is expressed after injection of size-selected mRNA into oocytes of Xenopus laevis.

First, the existence of an endogenous p-aminohippurate (PAH) transporter in oocytes of Xenopus laevis was demonstrated. When, however, the oocytes were injected with mRNA from rat kidney cortex, an expressed p-aminohippuric acid (PAH) uptake was seen which differed from the endogenous transporter. Both transport systems are saturated at high PAH concentrations, exhibit trans-stimulation by PAH and are partially inhibited by probenecid. The endogenous transport has a rather low affinity for PAH (Km = 0.57 mM) and is about 50% inhibited by probenecid (one apparent inhibition site with half maximal inhibition at 0.5 mM). The expressed PAH transport has a high affinity for PAH (Km = 60 microM) and can be inhibited 80% by probenecid (two apparent inhibition sites with half maximal inhibitions at 1 microM and 2 mM). Expression experiments with fractionated mRNA revealed that the PAH transport expressed from rat kidney cortex is encoded by an mRNA of 1.8 to 2.5 kb.

Determination of T cell epitopes with random peptide libraries.

A new approach to T cell epitope determination is presented. Critical amino acids for the induction of cytotoxic T cell responses were identified using synthetic peptide libraries with single defined sequence positions combined with randomized sequence positions. Sequences for potential T cell epitopes were deduced from scan profiles using combinations of the active amino acids. Highly potent epitopes for cytotoxic T lymphocytes were obtained. Epitopes defined by this approach are, as shown in this communication, not necessarily the natural epitopes and, therefore, were named synthetic epitopes. They can serve effectively for the development of vaccines or for the determination of T cell receptor antagonists.

Promotion of neural cell adhesion by electrochemically generated and functionalized polymer films.

New strategies for spatially controllable cell adhesion have been developed for brain cells from embryonic chicken. They are based on electrochemically active phenol and pyrrole derivatives, and can be used for the selective coverage of electroconductive substrates. Besides mimicking standard laminin-related adhesion promoting mechanisms by means of an electroactive monomer-linked 18-peptide segment from laminin (SRARKQAASIKVAVSADR), electrochemically generated thin (6-30 nm) polymer films of 3-hydroxybenzyl-hydrazine (3HBH) and 2-(3-hydroxyphenyl)-ethanol (2(3HP)E) with and without mechanically entrapped or covalently linked D-lysine have proved to promote cell adhesion in serum-free medium on indium-doped tin oxide (ITO) substrates during the first 6 culturing days in vitro. The effectiveness of the peptide was strongly density-dependent. Unexpectedly, laminin itself or a combination of laminin and poly-D-lysine (PDL) did not promote cell adhesion and neuron differentiation in serum-free cultures on ITO. However, they worked perfectly well on regular polystyrene substrates in serum-free medium or on ITO when medium with serum was used. This finding might suggest that the adhesion efficiency of laminin does not depend only on the kind of medium supplement but also on the type of substrate. In contrast, the adhesion-promoting properties of "artificial" polymeric films seemed to be based on a more direct cell-film interaction, with the film masking the substrate properties.

Electropolymerization of a phenol-modified peptide for use in receptor-ligand interactions studied by surface plasmon resonance.

The combination of surface plasmon resonance (SPR) with an electrochemical method for surface modification is presented. The SLP1 sequence of the sodium channel protein of rat cardiac muscle cells was N-terminally modified with an electropolymerizable group and immobilized on a gold-coated glass slide by oxidative polymerization. The resulting peptide-functionalized substrate was incubated with a polyclonal-specific anti-SLP1 serum. Growth of the peptide layer and the immunological reaction between ligand and receptor were detected on-line by SPR. The applicability of this approach for the rapid and selective analysis of receptor-ligand interactions is demonstrated.

Expression of a rat renal sodium-dependent dicarboxylate transporter in Xenopus oocytes.

Microinjection of mRNA isolated from rat kidney cortex into Xenopus laevis oocytes resulted in the expression of a Na(+)-dependent dicarboxylate transporter, as detected by uptake measurements with [14C]succinate as substrate. The expressed transporter showed an S-shaped Na(+)-dependence with half-maximal activation at 19-21 mM Na+ and a Hill coefficient between 2 and 3. Endogenous succinate uptake was not Na(+)-dependent. Na(+)-stimulated succinate uptake in mRNA-injected oocytes exhibited a maximum at pH 7.5, whereas endogenous Na(+)-independent transporter was fastest at pH 8.5. The expressed dicarboxylate transporter also differed from the endogenous transporter in its sensitivity to citrate as well as dicarboxylates in trans and cis configurations. The expressed transporter resembled the renal basolateral transporter, especially with respect to affinity for succinate (Km 28 microM), activation by Na+, pH-dependence and substrate specificity. After injection of size-fractionated mRNA, succinate uptake was expressed by mRNA of 2-3 kb. Our results suggest expression of the basolateral Na(+)-dependent dicarboxylate transporter after injection of mRNA from rat kidney into Xenopus oocytes.

Specificity and degeneracy of minor histocompatibility antigen-specific MHC-restricted CTL.

Random peptide libraries were employed to investigate the specificity of Ag recognition by H-3-specific, H-2K(b)-restricted CTL clones. The peptide libraries consist of octapeptides with one defined sequence position and mixtures of 19 amino acids (all proteinogenic amino acids except for cysteine) in the remaining seven sequence positions. The complete set of 152 peptide libraries includes all octapeptides possible with these amino acids. Responses of the CTL clones to these peptide libraries reveal patterns of preferred epitope amino acids. Depending on the CTL clone tested, varying numbers of different amino acids were identified for the different sequence positions indicating degeneracy of Ag recognition. Sequences for synthetic epitopes active at low pM concentrations could be deduced from these patterns. They confirm that TCRs of CTL clones do not exhibit specificity for unique ligand structures but rather can interact with sets of ligands. The sequences of peptides recognized by a single clone exhibit great sequence heterogeneity.

Tolerance to amino acid variations in peptides binding to the major histocompatibility complex class I protein H-2Kb.

Major histocompatibility complex (MHC) class I molecules are cell-surface glycoproteins that bind peptides and present them to T cells. The formation of a peptide-MHC complex is the initial step in specific, T cell-mediated immune responses. But, unlike other receptor-ligand systems, peptides are essential for a stable conformation of the MHC proteins. To investigate the contribution of every amino acid of octapeptides to the stability and antigenic integrity of MHC proteins, complex octapeptide libraries with one defined amino acid and mixtures of 19 amino acids in the remaining seven positions were synthesized and tested for their capacity to stabilize the conformation of the mouse MHC class I molecule H-2Kb. Peptide transporter-deficient RMA-S cells were employed in this study. Amino acid preferences found for the eight sequence positions reveal constitutional, volumetric, and steric constraints that govern peptide selection by MHC molecules. The pattern of amino acid preferences indicates that the peptides behave as integral parts of the MHC proteins and follow rules established for the interrelationship of primary sequence and the conformation and stability of proteins in general.

[Liver transplantation: diagnostic imaging in the preoperative assessment]. / Il trapianto del fegato: la diagnostica per immagini nel bilancio preoperatorio.

The correct selection of patients for liver transplantation, which is essential for surgical success, requires thorough radiological evaluation. The authors present their experience on 94 pretransplant adult patients that underwent a total of 251 diagnostic exams (Doppler US, CT, angiography and cholangiography) and interventional radiology maneuvers (biopsy, chemoembolization, biliary drainage). Three sclerosing cholangitis, 3 Budd-Chiari syndromes and 20 hepatocellular carcinomas in cirrhotic patients were identified; venous collaterals were present in 62.7% of the cases, 12.8%, of which had important spontaneous porto-systemic shunts; 6 patients had portal thrombosis; 20 arterial variations were found. Interventional maneuvers were useful and free of complications. US, CT and angiographic findings of each patient were compared. Integrating informations from different exams allowed a significant increase in the accuracy of diagnostic conclusions. Thanks to interventional maneuvers 5 patients could be selected for transplantation (hepatic arterial lipiodolization stopped the growth of 4 hepatic neoplasms; 2 infected fluid collections were sterilized by percutaneous US-guided drainage and topic therapy.

Self-MHC-restricted peptides recognized by an alloreactive T lymphocyte clone.

Alloreactive T lymphocytes are readily detected in unprimed animals although they have never encountered the alloantigen before. This well-established phenomenon is usually explained with the assumption that a self-MHC molecule complexed with a defined peptide resembles the allo-MHC molecule with another peptide and induces the corresponding T cell specificities. Here, for the first time and in support of this hypothesis, self-MHC-restricted peptides are described for a T cell clone that was induced with allo-MHC. The allo-MHC-specific CTL clone 2C was derived from a H-2b mouse and recognizes H-2Ld complexed with the naturally occurring endogenous peptide LSPFPFDL. H-2Kb was shown to be involved in positive selection of its TCR, and peptides associated with this MHC molecule are implicated in the process. To identify such peptides, positional scanning with random peptide libraries combined with an iterative approach was employed. Several active peptides were found and the most efficient, SIYRYYGL, was chosen for further studies. Recognition by 2C of the two MHC-peptide adducts H-2Ld + LSPFPFDL and H-2Kb + SIYRYYGL is mediated by the same TCR and appears to be similarly efficient as concluded from inhibition experiments with an Id-specific Ab. CTLs from SIYRYYGL-primed H-2b mice respond to H-2Ld + LSPFPFDL. This reciprocal cross-reactivity suggests that structural features are shared by the two MHC-peptide complexes.

Recognition principle of the TAP transporter disclosed by combinatorial peptide libraries.

Transport of peptides across the membrane of the endoplasmic reticulum for assembly with MHC class I molecules is an essential step in antigen presentation to cytotoxic T cells. This task is performed by the major histocompatibility complex-encoded transporter associated with antigen processing (TAP). Using a combinatorial approach we have analyzed the substrate specificity of human TAP at high resolution and in the absence of any given sequence context, revealing the contribution of each peptide residue in stabilizing binding to TAP. Human TAP was found to be highly selective with peptide affinities covering at least three orders of magnitude. Interestingly, the selectivity is not equally distributed over the substrate. Only the N-terminal three positions and the C-terminal residue are critical, whereas effects from other peptide positions are negligible. A major influence from the peptide backbone was uncovered by peptide scans and libraries containing D amino acids. Again, independent of peptide length, critical positions were clustered near the peptide termini. These approaches demonstrate that human TAP is selective, with residues determining the affinity located in distinct regions, and point to the role of the peptide backbone in binding to TAP. This binding mode of TAP has implications in an optimized repertoire selection and in a coevolution with the major histocompatibility complex/T cell receptor complex.

Amino acid preferences in the octapeptide subunit of the major histocompatibility complex class I heterotrimer H-2Ld.

Major histocompatibility complex class I (MHC-I) molecules are heterotrimers composed of polymorphic alpha-chains, monomorphic beta-chains, and peptides of eight or nine amino acids. The peptides are derived from various intracellularly occurring proteins and are very heterogeneous. They are essential for a stable conformation of the MHC-I protein at physiological temperature. This study presents results from stabilization experiments that were designed to determine the impact of the amino acids in every sequence position of octapeptides on the thermal stability of the mouse MHC-I molecule H2-Ld. OX7 octapeptide libraries with one defined and seven randomized positions were employed as they allow the effects of individual amino acids to be determined. The results confirm the importance of the motif amino acids proline and leucine for positions 2 and 8, respectively, of octapeptides. They are among the most efficient amino acids for these positions. However, with a few exceptions, all amino acids are permitted in all eight sequence positions. Hydrophobic amino acids are generally favored. Charged amino acids, especially aspartic acid and glutamic acid, are disfavored. Stabilization indices were defined as measures for the MHC stabilization power of the amino acids. These indices can serve to predict the efficiency of peptide binding to H-2Ld and can guide the design of T-cell epitopes.

Requirements for peptide binding to the human transporter associated with antigen processing revealed by peptide scans and complex peptide libraries.

Antigenic peptides are translocated into the lumen of the endoplasmic reticulum by the action of the transporter associated with antigen processing (TAP), where they are subsequently needed for the correct assembly of major histocompatibility complex molecules. The transport function was reconstituted in insect cells by expression of both TAP genes. On the basis of this over-expression system, substrate selection was analyzed in detail by a direct biomolecular peptide binding assay. Competition assays with peptide variants, including substitutions of residues with alanine or structurally related amino acids, underline the broad peptide specificity of the human TAP complex. Steric requirements of the substrate-binding pocket were mapped using elongated peptides and scans with bulky, hydrophobic amino acids. Complex nonapeptide libraries were used to determine the contribution of each residue to stabilize peptide-TAP complexes. For the first time, this approach lets us directly evaluate the importance of peptide selection for the overall process of antigen presentation on the level of the peptide transporter.

Modification of glassy carbon surfaces with synthetic laminin-derived peptides for nerve cell attachment and neurite growth.

Interactions between cultured nerve cells and surfaces are of importance for the implantation of biocompatible electrode materials such as glassy carbon (GC). Since implants serve as recording sensors in prosthetic neuroscience, we investigated whether coating electrodes with certain laminin derivatives containing the peptide sequences SIKVAV, CDPGYIGSR, PDSGR, YFQRYLI, and RNIAEIIKDA influences neuronal adhesion and neurite outgrowth in vitro. The coating of GC was performed by electrochemical polymerization and, for comparison, by adsorption or covalent coupling. Electrochemical polymerization is suitable for the coupling of peptides to GC, as shown by amino acid analysis and sequencing. Embryonic chicken retinal ganglion cells and brain cells (days E7 or E17) were used for both attachment and growth studies. Surfaces made by electrochemical polymerization of peptides were more efficient than those made by adsorption or covalent coupling of peptides. Synthetic cyclic peptide derivatives of CDPGYIGSR and 18-mer SIKVAV were found to be more efficient than the linear peptides. Competitive effects that resulted in a decreased cell attachment could be found upon application of soluble peptides. Nevertheless, irrespective of the method of coating, peptides were less efficient compared with the whole laminin molecule, as expected from its multiple adhesion sites. When small GC pins were implanted into the brain of E17 chicken after coating with the 18-mer SIKVAV peptide, nerve cell attachment was observed in vivo. The results suggest that chronically implantable materials may exert a higher neurocompatibility when coated with synthetic peptides.

An automated prediction of MHC class I-binding peptides based on positional scanning with peptide libraries.

Specificities of three mouse major histocompatibility complex (MHC) class I molecules, Kb, Db, and Ld, were analyzed by positional scanning using combinatorial peptide libraries. The result of the analysis was used to create a scoring program to predict MHC-binding peptides in proteins. The capacity of the scoring was then challenged with a number of peptides by comparing the prediction with the experimental binding. The score and the experimental binding exhibited a linear correlation but with substantial deviations of data points. Statistically, for approximately 80% of randomly chosen peptides, MHC-binding capacity could be predicted within one log concentration of peptides for a half-maximal binding. Known cytotoxic T-lymphocyte epitope peptides could be predicted, with a few exceptions. In addition, frequent findings of MHC-binding peptides with incomplete or no anchor amino acid(s) suggested a substantial bias introduced by natural antigen processing in peptide selection by MHC class I molecules.