Allen R. Zeiger

Frank Stinchfield Award. Titanium surface with biologic activity against infection.

Despite immense improvements, periprosthetic infection continues to compromise the result of otherwise successful joint arthroplasty. There are various limitations in the treatment of periprosthetic infection, the most important of which is the inability to deliver antibiotics to the local tissue without the need for intravenous administration. We have developed a novel route to covalently tether vancomycin to a metal (titanium) surface, which showed effective bactericidal activity because of a vancomycin coupling. The chemistry of tethering does not affect the biological activity of the biofactors that are attached to the metal surface. This technology holds great promise for the manufacturing of “smart” implants that can be self protective against periprosthetic infection, or can be used for the treatment of periprosthetic infections when they occur.

A donor-acceptor substrate of the exocellular DD-carboxypeptidase-transpeptidase from streptomyces R61

North-Holland Publishing Company Amsterdam of glycine of another hexapeptide, so that dimers or trimers (or rarely higher polymers) are built up [ 1 ] . The location of the physiological transpeptidase appears to be the plasma membrane [4]. An exocellular enzyme has been isolated from Streptomyces R6 1 and shown to have DD-carboxypeptidase and transpeptidase activities [ S-81. The enzyme was shown to catalyse the formation of a D-Ala-Gly peptide bond, such as is found in the native, completed wall peptidoglycan. An example of such a reaction is the transformation of Aca-LLys-D-Ala-D-Ala and Gly-LAla into Aca-LLys-D-Ala-Gly-L-Ala and D-alanine. In this report, it is shown that the tetrapeptide, p-[ l4 C] Ac-NE-(Gly)LLys-D-Ala-D-Ala, can serve as both donor and acceptor substrate of this enzyme, yielding several products, including one containing more than one D-Ala-Gly interpeptide linkage.

Genetic control of two independently segregating loci in mice to the sequential polypeptide (tyr-ala-glu-gly)n.

Immune responses to the sequential helical polypeptide (Tyr-Ala-Glu-Gly)n [(T-A-G-Gly)n] in mice is under the control of at least two separate genes. One gene,Ir-(T-A-G-Gly)-1, which is linked, toH-2 haplotypesb, f, andr, controls the ability to respond and maps to theIA subregion. A non-H-2-linked locus,Ir-(T-A-G-Gly)-2. is responsible for the magnitude of the antibody response, which is expressed as a high, intermediate, or low level of antibody production. The antibody produced is of the IgG class, and does not crossreact even with the closely related sequential helical polymer (Tyr-Glu-Ala-Gly)n [(T-G-A-Gly)n]. Immune responsiveness is a dominant trait,i.e., the F1 generations of responder x nonresponder crosses are responders. However, the data obtained with both backcross populations are not easily interpretable. The contribution of the B-cell mitogenic activity of the sequential polymer to activation of suppressor T cells is considered as a possible explanation for the backcross results. The possible role of the Ia. W29 specificity present in the mouse strains responding to both (T-A-G-Gly)n and calf skin collagen type I in modulating responses to the polymers is discussed.

Changes in mu opioid receptors and rheological properties of erythrocytes among opioid abusers.

Abstract Opioids, reported originally to bind to specific receptors in the brain, now also appear to bind to receptors on blood cells. The high prevalence of anemia among chronic opioid users leads us to propose that chronic opiate use results in elevated mu opioid receptor levels on human erythrocytes and that these receptor changes may affect erythrocyte membrane properties. Blood samples from 17 opioid‐dependent subjects (based on the Diagnostic and Statistical Manual of Mental Disorders, 4th edition or DSM‐IV) and 15 drug‐free controls were assayed for mu opioid receptors on erythrocytes using a flow cytometry immunoassay. Deformability and the hydration status of erythrocytes were studied by ektacytometry. Data were analyzed by independent t‐tests, tests of correlation, chi square and cluster analyses. As expected, the percentage of erythrocytes from opioiddependent subjects with opioid receptors (opioid receptor levels) was significantly higher (47.4 ± 38.3%) than controls (22.8 ± 30.1%) (t = 2.01, df = 30, p < 0.05). Also, the opioid‐dependent patients showed a wide variation in the percentage of erythrocytes bearing opioid receptors and data analyses of these patients showed two strongly defined clusters. One subgroup consisted of nine individuals with very high receptor levels (mean = 81.5%) while the other had eight patients with low receptor levels (mean = 9.1%) that were not significantly different than the receptor levels of controls. Ektacytometry of opioid dependent patients with high opioid receptor levels showed changes in rheological parameters of erythrocytes, such as deformability index and cellular hydration. For example, a positive correlation was observed between opioid receptor levels and deformability indices among opioid‐dependent patients (r = 0.74, p < 0.005). Our findings indicate that the mu opioid receptor is present on human erythrocytes, although with considerable variation in receptor levels, and that the levels of this receptor are significantly elevated with chronic opioid exposure. Moreover, erythrocytes with high opioid receptor levels from chronic opiate users seem to have high deformability. This study may offer clues to the biological properties of peripheral blood cells that may be mediated by mu opioid receptors and lead to a better understanding of some of the clinical effects of opioid use.

The direction of peptide trimer synthesis from the donor-acceptor substrate Nalpha-(acetyl)-Nepsilon-(glycyl)-L-lysyl-D-alanyl-D-alanine by the exocellular dd-carboxypeptidase-transpeptidase of Streptomyces R61

The D-Ala-Gly interpeptide bonds in the dimers and trimers formed were identical to those found in the native, completed wall peptidoglycan of Streptomyces R61 [2]. In the present report, we show that peptide trimer (I) is preferentially formed by addition of the tripeptide moiety [14C]Ac-LLys-D-Ala of the tetraGly---’ peptide monomer (IV) acting as donor (through its C-terminal D-Ala-D-Ala sequence) to a preformed peptide dimer (II or III) acting as acceptor (through its N-terminal glycine residue) rather than by dimer (II) acting as donor and monomer (IV or V) acting as acceptor. The reactions are illustrated in Scheme 1.

Interpretations of immune responses of mice to poly(Glu60Lys40), its modified derivatives, and the terpolymers poly(Glu55Lys37Leu8) and poly(Glu56Lys37Ser7)☆

Abstract Reinvestigations of the murine immune responses against poly(Glu 60 Lys 40 ) and its derivatives modified with covalently linked amino acids indicated that they all could be immunogenic at high concentrations in all inbred strains. In contrast restricted responsiveness was noted with terpolymers as follows: to poly(Glu 55 Lys 37 Leu 8 ) mice of H-2 a,d,k haplotypes, to poly(Glu 56 Lys 37 Ser 7 ) mice of H-2 a,k haplotypes. It is hypothesized that the increased hydrophobicity of the modified GL derivatives can lead to increased combining constants with the antigen-specific T-cell receptor (V H -Ia complexes?) which may be responsible for the enhanced immunogenicity and positive in vitro T-cell proliferative responses detected with the modified GL(T) 4.7 . A similar hypothesis involving increased reactions with V H idiotypes on T cells is advanced to account for some of the known unique strain distribution patterns of the glu lys terpolymers containing limited amounts of a third α-amino acid.

Murine responses to (Tyr-Glu-Ala-Gly)n

A series of known sequential polypeptides is being synthesized and used in our laboratory to study the contribution of antigen structure, i. e., amino acid sequence and conformation in antigen recognition and specificity of the immune response. The capacity to respond to one such α-helical polypeptide (T-G-A-Gly)n, is T-cell dependent and restricted to mice of theH- 2b haplotype. The response is controlled by anIr gene mapping to theK region and/or theIA subregion which allows the animal to make both a T-cell mediated response, as well as a humoral response to the polypeptide. The response of three mutant strains at theK end of the major histocompatibility locus (MHC) need not differ from that of the responder parental haplotype.PETLES obtained from mice possessing a responder haplotype proliferate when cultured in vitro with (T-G-A-Gly)n. The antibody level of individual inbred mice of a given strain at a given time differs significantly (from 80% binding to less than 10% antigen bound in 3 out of 57 mice). There is also great individual variability in time of appearance of the antibody response and where peak optimal levels are seen. Possible explanations for the variation in the antibody expression include: (a) the polymer is a weak immunogen, (b) the presence of modifier gene(s) outside of the major histocompatibility complex controlling the magnitude of the antibody level, (c) the possible effect of the polymer which is a B cell mitogen as a generator of suppressor T cells and, (d) a feedback mechanism effect on B cells controlling the antibody level.

The immunochemistry of (L-Ala-D-Glu-L-Lys-D-Ala-Gly)n: antibodies of restricted specificity.

Abstract The specificity of the antibody elicited by the sequential polypeptide, ( l - Ala - d - Glu - l - Lys - d - Ala-Gly ) n , which may exist in the pleated sheet conformation at physiological pH, was studied by a radioimmunoassay, using related haptens as inhibitors. The immunodominant region was X-Gly- l -Ala- d -Glu- l -Lys, where X was Boc or Boc- d -Ala. The presence of d -Glu in the haptens was essential for any inhibitory activity. Haptens with other amino acids substituted for d and l -Ala, l -Lys, and Gly were able to inhibit significantly the binding of the polymer to its antibody. In addition, amino acids of the opposite optical isomer could be substituted for l -lysine and d -alanine. Either d or l amino acids could be substituted for glycine. The results indicated that the biological recognition of the immunogen is highly selective of the amino acid sequence, recognizing only one out of the five possible sequences, and that the determinant recognized was located internally in the amino acid sequence of the polypeptide.

The immunochemistry of a linear polymer and a branched copolymer containing the sequence Glu-Lys-Ala-Gly

Abstract The antibody specificity to a randomly coiled polypeptide of known sequence, poly (glutamyllysyl-alanyl-glycine), and to a branched copolymer were studies in rabbits bo inhibition of homologous precipitin reactions. The reactions of both polymers with their homologous antisera were substantially inhibited by the tetrapeptide, Glu-Lys-Ala-Gly. Inhibition of the precipitation of the sequential polypeptide with its antiserum increased with the size of the oligomers, up to the tetramer, (Glu-Lys-Ala-Gly) 4 . From ultracentrifugal and chromatographic studies of the reaction of this antibody with the oligomers as well as passive cutaneous anaphylaxis (PCA) studies in guinea-pigs, it was concluded that the tetramer, but not the trimer, possessed two antigenic determinants. Inhibition of the reaction of the branched copolymer with its antiserum on a molar basis was independent of the length of the oligomer. The immunodominant region of the branched copolymer appeared to include the amino terminus of the tetrapeptide. The immune response to the linear polymer, on the other hand, was consistent with the presence in the latter of internal determinants.

IMMUNE RESPONSES OF MICE AGAINST MODIFIED RANDOM POLYMERS OF AMINO ACIDS, AND IMPLICATIONS IN GENE CONTROL

Abstract The introduction of limited amounts (about 5–10%) of amino acids into the nonimmunogenic random copolymer poly(Glu60Lys40), GL, produces conjugates that exhibit variable immunogenicity in all strains of mice. The modified GL derivatives are T cell independent antigens, elicit IgM responses, and are B cell mitogens. Similar modification of the polymers GA, GAL10, GLT5 and GLOS, the responses against which are under Ir gene(s) control, did not alter the strain distribution patterns (SDP) so as to convert nonresponder mice to responders. The above observations and the known SDP against a number of GL terpolymers are discussed with speculations to help explain: 1) the immunogenicity of the amino acid modified GL polymer; 2) the nonimmunogenicity of GL in mice; 3) the unique SDP against the GL terpolymers; 4) the divergent immune response data obtained with the same polymer in different laboratories.