Quantitation of Pseudomonas aeruginosa in wound biopsy samples: from bacterial culture to rapid `real-time' polymerase chain reaction

We developed a real-time detection (RTD) polymerase chain reaction (PCR) with rapid thermal cycling to detect and quantify Pseudomonas aeruginosa in wound biopsy samples. This method produced a linear quantitative detection range of 7 logs, with a lower detection limit of 103 colony-forming units (CFU)/g tissue or a few copies per reaction. The time from sample collection to result was less than 1h. RTD-PCR has potential for rapid quantitative detection of pathogens in critical care patients, enabling early and individualized treatment.

A synthetic random basic copolymer with promiscuous binding to class II major histocompatibility complex molecules inhibits T-cell proliferative responses to major and minor histocompatibility antigens in vitro and confers the capacity to prevent murine graft-versus-host disease in vivo.

Graft-versus-host disease (GVHD) is a T-cell-mediated disease of transplanted donor T cells recognizing host alloantigens. Data presented in this report show, to our knowledge, for the first time that a synthetic copolymer of the amino acids L-Glu, L-Lys, L-Ala, and L-Tyr (molecular ratio, 1.9:6.0:4.7:1.0; Mr, 6000-8500) [corrected], termed GLAT, with promiscuous binding to multiple major histocompatibility complex class II alleles is capable of preventing lethal GVHD in the B10.D2 --> BALB/c model (both H-2d) across minor histocompatibility barriers. Administration of GLAT over a limited time after transplant significantly reduced the incidence, onset, and severity of disease. GLAT also improved long-term survival from lethal GVHD: 14/25 (56%) of experimental mice survived > 140 days after transplant compared to 2/26 of saline-treated or to 1/10 of hen egg lysozyme-treated control mice (P < 0.01). Long-term survivors were documented to be fully chimeric by PCR analysis of a polymorphic microsatellite region in the interleukin 1beta gene. In vitro, GLAT inhibited the mixed lymphocyte culture in a dose-dependent fashion across a variety of major barriers tested. Furthermore, GLAT inhibited the response of nylon wool-enriched T cells to syngeneic antigen-presenting cells presenting minor histocompatibility antigens. Prepulsing of the antigen-presenting cells with GLAT reduced the proliferative response, suggesting that GLAT inhibits antigen presentation.

Selection for in vivo regulators of bacterial virulence

We devised a noninvasive genetic selection strategy to identify positive regulators of bacterial virulence genes during actual infection of an intact animal host. This strategy combines random mutagenesis with a switch-like reporter of transcription that confers antibiotic resistance in the off state and sensitivity in the on state. Application of this technology to the human intestinal pathogen Vibrio cholerae identified several regulators of cholera toxin and a central virulence gene regulator that are operative during infection. These regulators function in chemotaxis, signaling pathways, transport across the cell envelope, biosynthesis, and adherence. We show that phenotypes that appear genetically independent in cell culture become interrelated in the host milieu.

Clonal analysis of stably transduced human epidermal stem cells in culture.

We have transduced normal human keratinocytes with retroviral constructs expressing a bacterial beta-galactosidase (beta-gal) gene or a human interleukin-6 (hIL-6) cDNA under control of a long terminal repeat. Efficiency of gene transfer averaged approximately 50% and 95% of clonogenic keratinocytes for beta-gal and hIL-6, respectively. Both genes were stably integrated and expressed for more than 150 generations. Clonal analysis showed that both holoclones and their transient amplifying progeny expressed the transgene permanently. Southern blot analysis on isolated clones showed that many keratinocyte stem cells integrated multiple proviral copies in their genome and that the synthesis of the exogenous gene product in vitro was proportional to the number of proviral integrations. When cohesive epidermal sheets prepared from stem cells transduced with hIL-6 were grafted on athymic animals, the serum levels of hIL-6 were strictly proportional to the rate of secretion in vitro and therefore to the number of proviral integrations. The possibility of specifying the level of transgene expression and its permanence in a homogeneous clone of stem cell origin opens new perspectives in the long-term treatment of genetic disorders.