Initiation of T-lymphocyte proliferation by mitogen or antigen involves a cascade of gene activation events. Thus, by the time mitogen-activated T cells have reached the G1/S interface, many genes that are transcriptionally silent in G0, like the c-myc, IL-2, IL-2 receptor (IL-2R) and transferrin receptor (TfR) genes, have been transcriptionally activated. To understand the role of the individual genes in the activation process, one must be able to interfere specifically with the expression or function of each particular gene product. In this way, by blocking the IL-2R with an antibody, it has been demonstrated that IL-2/IL-2R interaction is required to induce TfR expression in activated T cells. When the function or expression of intracellular proteins is to be blocked, however, the need to introduce antibodies into the cytoplasm of viable cells, although possible, is a limiting factor. We have taken another approach, namely the exogenous addition to bulk cell cultures of small antisense oligomers. Sequence-specific antisense oligodeoxyribonucleotides have been reported to inhibit intracellular viral replication without interfering with cellular protein synthesis. Similarly, rabbit globin mRNA translation in a cell-free system and in rabbit reticulocytes has been inhibited by oligomers complementary to the globin mRNA initiation codon region. Recently, a pentadecadeoxyribonucleotide complementary to the initiation codon and four downstream codons of human c-myc mRNA was reported to inhibit the proliferation of the human leukaemic cell line HL-60 specifically. We report here that the same c-myc complementary oligonucleotide inhibits mitogen-induced c-myc protein expression in human T lymphocytes and prevents S phase entry. Interestingly, c-myc antisense treatment did not inhibit G0 to G1 traversal as assessed by morphologic blast transformation, transcriptional activation of the IL-2R and TfR genes, or induction of 3H-uridine incorporation.
The human promyelocytic leukemia cell line HL-60 overexpresses the c-myc protooncogene. A calculated secondary structure for c-myc mRNA placed the initiation codon in a bulge of a weakly base-paired region. Treatment of HL-60 cells with 5' d(AACGTTGAGGGGCAT) 3', complementary to the initiation codon and the next four codons of c-myc mRNA, inhibited c-myc protein expression in a dosedependent manner. However, treatment of HL-60 cells with 5'
Peri-prosthetic infections are notoriously difficult to treat as the biomaterial implant is ideal for bacterial adhesion and biofilm formation, resulting in decreased antibiotic sensitivity. Previously, we reported that vancomycin covalently attached to a Ti alloy surface (Vanc-Ti) could prevent bacterial colonization. Herein we examine the effect of this Vanc-Ti surface on Staphylococci epidermidis, a Gram-positive organism prevalent in orthopaedic infections. By direct colony counting and fluorescent visualization of live bacteria, S. epidermidis colonization was significantly inhibited on Vanc-Ti implants. In contrast, the gram-negative organism Escherichia coli readily colonized the Vanc-Ti rod, suggesting retention of antibiotic specificity. By histochemical and SEM analysis, Vanc-Ti prevented S. epidermidis biofilm formation, even in the presence of serum. Furthermore, when challenged multiple times with S. epidermidis, Vanc-Ti rods resisted bacterial colonization. Finally, when S. epidermidis was continuously cultured in the presence of Vanc-Ti, the bacteria maintained a Vanc sensitivity equivalent to the parent strain. These findings indicate that antibiotic derivatization of implants can result in a surface that can resist bacterial colonization. This technology holds great promise for the prevention and treatment of periprosthetic infections. b s t r a c tPeri-prosthetic infections are notoriously difficult to treat as the biomaterial implant is ideal for bacterial adhesion and biofilm formation, resulting in decreased antibiotic sensitivity. Previously, we reported that vancomycin covalently attached to a Ti alloy surface (Vanc-Ti) could prevent bacterial colonization. Herein we examine the effect of this Vanc-Ti surface on Staphylococci epidermidis, a Gram-positive organism prevalent in orthopaedic infections. By direct colony counting and fluorescent visualization of live bacteria, S. epidermidis colonization was significantly inhibited on Vanc-Ti implants. In contrast, the gram-negative organism Escherichia coli readily colonized the Vanc-Ti rod, suggesting retention of antibiotic specificity. By histochemical and SEM analysis, Vanc-Ti prevented S. epidermidis biofilm formation, even in the presence of serum. Furthermore, when challenged multiple times with S. epidermidis, Vanc-Ti rods resisted bacterial colonization. Finally, when S. epidermidis was continuously cultured in the presence of Vanc-Ti, the bacteria maintained a Vanc sensitivity equivalent to the parent strain. These findings indicate that antibiotic derivatization of implants can result in a surface that can resist bacterial colonization. This technology holds great promise for the prevention and treatment of periprosthetic infections.
Periprosthetic infections are life-threatening complications that occur in about 6% of medical device insertions. Stringent sterile techniques have reduced the incidence of infections, but many implant patients are at high risk for infection, especially the elderly, diabetic, and immune compromised. Moreover, because of low vascularity at the site of the new implant, antibiotic prophylaxis is often not effective. To address this problem, we designed a covalent modification to titanium implant surfaces to render them bactericidal. Specifically, we aminopropylated titanium, a widely used implant material and extended a tether by solid phase coupling of ethylene glycol linkers, followed by solid phase coupling of vancomycin. Vancomycin covalently attached to titanium still bound soluble bacterial peptidoglycan, reduced Staphylococcus aureus colony-forming units by 88% +/- 16% over 2 hr, and retained antibacterial activity upon a repeated challenge.
Periprosthetic infection is a devastating consequence of implant insertion and can arise from hematogenous sources or surgical contamination. Microbes can preferentially colonize the implant surface and, by forming a biofilm, escape immune surveillance. We hypothesized that if an antibiotic can be tethered to a titanium alloy (Ti) surface, it will inhibit bacterial colonization, prevent biofilm formation, and avert late-stage infection. To test this hypothesis, a Ti rod was covalently derivatized with vancomycin. Reaction efficiencies were evaluated by colorimetric and spectrophotometric measurements. The vancomycin-modified surface was stable in aqueous solutions over extended time periods and maintained antibiotic coverage, even after pressfit insertion into a cadaverous rat femora. When evaluated using fluorescently labeled bacteria, or by direct colony counts, the surface-bound antibiotic prevented bacterial colonization in vitro after: (1) exposure to high levels of S. aureus; (2) extended incubation in physiological buffers; and (3) repeated bacterial challenges. Importantly, whereas the vancomycin-derivitized pins prevented bacterial colonization, S. aureus adhered to control pins, even in the presence of concentrations of vancomycin that exceeded the strain MIC. These results demonstrate that we have effectively engineered a stable, bactericidal Ti surface. This new surface holds great promise in terms of mitigating or preventing periprosthetic infection. ß
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