ABSTRACT
Genetic typing of the short tandem repeat (STR) polymorphism HUMTHO1 has been performed using capillary array electrophoresis and energy-transfer fluorescent dye-labeled polymerase chain reaction primers. Target alleles were amplified by use of primers labeled with one fluorescein at the 5' end and another fluorescein at the position of the 15th (modified) base to produce fragments that fluoresce in the green (lambda max = 525 nm). Unknown alleles were electrophoretically separated together with a standard ladder made up of alleles having 6, 7, 8, and 9 four-base pair repeats, each of which was amplified with an energy-transfer primer having a donor fluorescein at the 5' end and a rhodamine acceptor at the position of the 7th (modified) base to produce standard fragments fluorescing in the red (> 590 nm). Separations were performed on arrays of hollow fused-silica capillaries filled with a replaceable sieving matrix consisting of 0.8% hydroxyethyl cellulose plus 1 microM 9-aminoacridine to enhance the resolution. The labeled DNA fragments were excited at 488 nm, and the fluorescence was detected with a two-color confocal fluorescence scanner. Separations are complete in less than 20 min and allow sizing with an average absolute error or accuracy of less than 0.4 base pair and an average standard deviation of approximately 0.5 base pair with no correction for mobility shift and cross-talk between the fluorescence channels. This work establishes the feasibility of high-speed, high-throughput STR typing of double-stranded DNA fragments using capillary array electrophoresis.
Subject(s)
Alleles , Electrophoresis , Repetitive Sequences, Nucleic Acid , Tyrosine 3-Monooxygenase/genetics , Aminacrine , Base Sequence , DNA/analysis , DNA Primers , Electrophoresis/methods , Fluoresceins , Fluorescent Dyes , Humans , Molecular Sequence Data , Particle Size , Polymerase Chain Reaction/methods , Staining and Labeling/methodsABSTRACT
We have shown previously that expression of the costimulatory ligand B7.1 by the UV-induced melanoma K1735 leads to rejection of the tumor by syngeneic hosts and the induction of immunity to challenge by the parental B7-negative tumor. Here we extend our analysis of the effectiveness of B7-positive tumor cells as vaccines to additional tumor models and analyze the protective immunity in detail. We have found that the immunity induced by K1735 is not restricted to the parental tumor cells but is effective against an additional melanoma line and an unrelated fibrosarcoma as well. This immunity is, however, relatively short-lived, and no significant protection is observed after 90 days. Depletion of CD4+ T cells prior to rechallenge has no significant effect on the subsequent rejection of B7-negative tumor cells. EL-4 thymoma cells transfected with B7.1 are also effectively rejected, and mice which have rejected B7 + EL-4 cells are immune to challenge with not only EL-4, but also reject an unrelated thymoma, C6VL. In contrast to the short-lived immunity observed in the melanoma model, mice are effectively protected against challenge with EL-4 for longer than 90 days after rejection of B7 + EL-4. Finally, we show that irradiation severely diminishes the effectiveness of B7-positive tumor cells as immunogens. This work has implications for the use of B7-positive cells as tumor vaccines.
Subject(s)
B7-1 Antigen/immunology , Fibrosarcoma/immunology , Graft Rejection/immunology , Immunotherapy/methods , Melanoma/immunology , Thymoma/immunology , Animals , B7-1 Antigen/radiation effects , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Carcinoma, Squamous Cell/immunology , Carcinoma, Squamous Cell/therapy , Colorectal Neoplasms/immunology , Female , Fibrosarcoma/therapy , Histocompatibility Antigens Class II/immunology , Male , Mammary Neoplasms, Experimental/immunology , Mast-Cell Sarcoma/immunology , Melanoma/therapy , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Nude , Neoplasm Transplantation/immunology , Thymoma/therapyABSTRACT
A total of 118 epidemiologically related Staphylococcus epidermidis strains from hospital patients, staff, and fomites were examined with a provisional set of 18 typing phages. Seventy (59.3%) of these strains were typed using phage concentrations of 100 times routine test dilution. The remainder were nontypable. Thirty-six (30.5%) of the strains were of related phage types, 71/108/275A/459 and 71/108/275A. These latter strains were associated with clinical S. epidermidis endocarditis in patients with prosthetic valve replacements. Ninety-eight strains were characterized by the Baird-Parket biotyping schema. Eighty-three (84.7%) were biotype 1, and the majority (68.4%) of these were resistant to penicillin, ampicillin, methicillin, cephalothin, erythromycin, and clindamycin. Type 71, 71/108/275A/459, 71/108/275A and 71/108/275/459 strains were generally resistant to penicillin, ampicillin, erythromycin, and methicillin, whereas a less consistent resistance pattern was noted among miscellaneous and nontypable strains.