Detection and enumeration of aromatic oxygenase genes by multiplex and real-time PCR

Our abilities to detect and enumerate pollutant-biodegrading microorganisms in the environment are rapidly advancing with the development of molecular genetic techniques. Techniques based on multiplex and real-time PCR amplification of aromatic oxygenase genes were developed to detect and quantify aromatic catabolic pathways, respectively. PCR primer sets were identified for the large subunits of aromatic oxygenases from alignments of known gene sequences and tested with genetically well-characterized strains. In all, primer sets which allowed amplification of naphthalene dioxygenase, biphenyl dioxygenase, toluene dioxygenase, xylene monooxygenase, phenol monooxygenase, and ring-hydroxylating toluene monooxygenase genes were identified. For each primer set, the length of the observed amplification product matched the length predicted from published sequences, and specificity was confirmed by hybridization. Primer sets were grouped according to the annealing temperature for multiplex PCR permitting simultaneous detection of various genotypes responsible for aromatic hydrocarbon biodegradation. Real-time PCR using SYBR green I was employed with the individual primer sets to determine the gene copy number. Optimum polymerization temperatures for real-time PCR were determined on the basis of the observed melting temperatures of the desired products. When a polymerization temperature of 4 to 5 degrees C below the melting temperature was used, background fluorescence signals were greatly reduced, allowing detection limits of 2 x 10(2) copies per reaction mixture. Improved in situ microbial characterization will provide more accurate assessment of pollutant biodegradation, enhance studies of the ecology of contaminated sites, and facilitate assessment of the impact of remediation technologies on indigenous microbial populations.     

Ankle-arm index versus angiography for the preassessment of the fibula free flap

Peripheral arterial occlusive disease or congenital anomalies of the major crural arteries may limit the use of the fibula free flap and should be detected preoperatively. Conventional selective angiography is the definitive standard imaging method for making this diagnosis, but it has drawbacks. A safer, cheaper, more accurate, and noninvasive alternative is desirable. The authors sought to test the hypothesis that the ankle-arm index of each of the three crural arteries, combined with pencil Doppler examination of the peroneal skin perforators, would provide adequate information to restrict the use of angiography to cases in which the outcomes of either or both of these options are insufficient. The ankle-arm index data of each of the three crural arteries, as well as pencil Doppler examination of the peroneal skin perforators of both legs of nine prospectively included patients and the nonoperated legs of 13 retrospectively included patients, were compared statistically in four different ways with the preoperative angiographic findings. A combined ankle-arm index and pencil Doppler examination is not accurate enough to detect legs or arteries with subclinical peripheral arterial occlusive disease or vascular variation and, hence, is not a sufficient basis on which to develop the surgical plan for a fibula free flap.     

Strict infection control measures do not prevent clonal spread of coagulase negative staphylococci colonizing central venous catheters in neutropenic hemato-oncologic patients

Coagulase negative staphylococci (CoNS) are a main cause of catheter related infections (CRI). Earlier studies (1994-1996) revealed a high incidence of CRI (6 per 1000 catheter days) among neutropenic hemato-oncologic patients in the Erasmus MC Hematology Department (Rotterdam, The Netherlands). This was mainly explained by expansion of two methicillin resistant Staphylococcus epidermidis (MRSE) clones (Nouwen et al., J. Clin. Microbiol. 36 (1998) 2696-2702). In a new, 16-bed unit in the same institution, we investigated the effect of strict clinical isolation measures on the incidence of CRI. During two 6-month screening periods (period I: April 1998-December 1998 and period II: April 1999-October 1999) all patients receiving a central venous catheter were prospectively monitored for the development of CRI. During period I every visitor of the cubicles had to wear hair caps, masks, gowns and gloves. During period II these procedures were abolished, but hands were cleansed using alcohol and masks were worn during both periods in case of coughing and sneezing. All CoNS strains isolated from blood cultures were genetically classifies by pulsed field gel electrophoresis (PFGE). The incidence of CRI during period I was 13.0 per 1000 catheter days, in comparison to 9.6 in period II (P=0.84). During this latter period, 19 CRI were diagnosed, 14 catheter related bacteremia episodes (CRB) and five local infections. Seventy-two percent (n=9) of CRB were due to a CoNS. The mean catheter survival until appearance of a CRI increased from 43 days during period I to 78 days in period II (P=0.39). The mean catheter survival until infection related removal was increased from 43 days to 133 days (P=0.12). During period I less experienced intervention radiologists introduced the catheters, which may have limited the efficacy of the strict hygiene measures. Thus, abolishing strict isolation precautions had no negative effect on the incidence of CRI. After genotyping of 38 MRSE strains isolated from blood and central venous catheter cultures of 12 patients in period II, eight PFGE types were found. Three types were found in more than one patient, but based on epidemiological data patient-to-patient spread could not be proven. No genotypic identity between patient and personnel CoNS isolates was shown and the two major clonal types that were present between 1994 and 1996 were not encountered. However, from December 1998 onwards new MRSE clones could be identified (types E and J). In conclusion, despite a constant rate of CRI and implementation of optimal patient care, clonal spread of MRSE strains was not prevented by strict hygiene measures.     

Chronic nicotine inhibits inflammation and promotes influenza infection

Epidemiological data suggest an association between smoking, respiratory infections, and impaired wound healing. Inflammation is critical in the body's defense against pathogens and in the wound-healing process. Although nicotine is used to treat some inflammatory conditions, the mechanism of this action is largely unknown. To determine how nicotine affects inflammation, rats and mice were exposed to nicotine via miniosmotic pumps, and the inflammatory response to turpentine or influenza virus was assessed. Results showed that while nicotine suppressed the migration of leukocytes to the inflammation/infection site, it increased the influenza titer in the lung. The decreased inflammation correlated with lower chemotaxis/chemokinesis of peripheral blood mononuclear cells (PBMC) toward formyl-methionyl-leucyl-phenylalanine and monocyte chemoattractant protein-1 without affecting the density of their respective receptors. However, nicotine suppressed the chemokine-induced Ca(2+) response in PBMC, indicating impaired chemokine signaling. Thus, because nicotine suppresses leukocyte migration, it might contribute to the delayed wound healing and increased incidence of respiratory infections among smokers.     

Production of cloned pigs by whole-cell intracytoplasmic microinjection

Cloning by somatic cell nuclear transfer has been successfully achieved by both fusing of a donor cell with and injecting an isolated donor cell nucleus into an enucleated oocyte. However, each of the above methods involves extended manipulation of either the oocytes (fusion) or the donor cells (nucleus isolation). Additionally, cloning efficiency can be reduced by low fusion rate of the cell fusion method, and specialized micromanipulation equipment and exacting nucleus isolation techniques are required for the nucleus injection method. Here we report a whole-cell injection technique for nuclear transfer in pigs and the production of cloned piglets with comparable, if not higher, efficiency than the other two nuclear transfer procedures. First, we tested the feasibility of this technique with three types of frequently used donor cells (cumulus, mural granulosa, and fibroblasts) and obtained the optimal nuclear reprogramming conditions for these cells. We further improved our protocol by avoiding ultraviolet exposure during enucleation and achieved a 37% blastocyst rate. We then conducted whole-cell injection using skin fibroblasts from the ear of a sow transgenic for two genes, the porcine lactoferrin and the human factor IX, and produced four live-born cloned transgenic piglets from three recipients. The present study demonstrated the applicability of producing normal, cloned piglets by the simple and less labor-intensive whole-cell intracytoplasmic injection.     

Immunoglobulin activation of T cell chemoattractant expression in fibroblasts from patients with Graves' disease is mediated through the insulin-like growth factor I receptor pathway

Graves' disease (GD) is associated with T cell infiltration, but the mechanism for lymphocyte trafficking has remained uncertain. We reported previously that fibroblasts from patients with GD express IL-16, a CD4-specific chemoattractant, and RANTES, a C-C chemokine, in response to GD-specific IgG (GD-IgG). We unexpectedly found that these responses result from a functional interaction between GD-IgG and the insulin-like growth factor (IGF)-I receptor (IGF-IR). IGF-I and the IGF-IR-specific IGF-I analog, des(1-3), mimic the effects of GD-IgG. Neither GD-IgG nor IGF-I activates chemoattractant expression in control fibroblasts from donors without GD. Interrupting IGF-IR function with specific receptor-blocking Abs or by transiently transfecting fibroblasts with a dominant negative mutant IGF-IR completely attenuates signaling provoked by GD-IgG. Moreover, GD-IgG displaces specific (125)I-labeled IGF-I binding to fibroblasts and attenuates IGF-IR detection by flow cytometry. These findings identify a novel disease mechanism involving a functional GD-IgG/IGF-IR bridge, which potentially explains T cell infiltration in GD. Interrupting this pathway may constitute a specific therapeutic strategy.     

A general method for the covalent labeling of fusion proteins with small molecules in vivo

Characterizing the movement, interactions, and chemical microenvironment of a protein inside the living cell is crucial to a detailed understanding of its function. Most strategies aimed at realizing this objective are based on genetically fusing the protein of interest to a reporter protein that monitors changes in the environment of the coupled protein. Examples include fusions with fluorescent proteins, the yeast two-hybrid system, and split ubiquitin. However, these techniques have various limitations, and considerable effort is being devoted to specific labeling of proteins in vivo with small synthetic molecules capable of probing and modulating their function. These approaches are currently based on the noncovalent binding of a small molecule to a protein, the formation of stable complexes between biarsenical compounds and peptides containing cysteines, or the use of biotin acceptor domains. Here we describe a general method for the covalent labeling of fusion proteins in vivo that complements existing methods for noncovalent labeling of proteins and that may open up new ways of studying proteins in living cells.     

c-Jun is essential for organization of the epidermal leading edge

The migration of epithelial layers requires specific and coordinated organization of the cells at the leading edge of the sheet. Mice that are conditionally deleted for the c-jun protooncogene in epidermis are born at expected frequencies, but with open eyes and with defects in epidermal wound healing. Keratinocytes lacking c-Jun are unable to migrate or elongate properly in culture at the border of scratch assays. Histological analyses in vitro and in vivo demonstrate an inability to activate EGF receptor at the leading edge of wounds, and we demonstrate that this can be rescued by supplementation with conditioned medium or the EGF receptor ligand HB-EGF. Lack of c-Jun prevents EGF-induced expression of HB-EGF, indicating that c-jun controls formation of the epidermal leading edge through its control of an EGF receptor autocrine loop.     

The combination of a chemokine,cytokine and TCR-based T cell stimulus for effective gene therapy of cancer

Cytotoxic T cells can recognize and kill tumor cells that present peptides derived from tumor-associated antigens (TAA) on their surface when associated with major histocompatibility complex (MHC) class I molecules. However, immune responses to tumor-associated antigens are often suppressed by a tumor-induced state of immune anergy. Previous work has attempted to overcome tumor-induced T cell anergy by the direct injection of vectors carrying genes encoding one of a variety of cytokines. Polyclonal stimulation of T cells, preferably via the TCR complex, results in a cascade of cytokines associated with T cell activation and thus may be better able to overcome T cell anergy. We have previously reported the use of the highly attenuated MVA poxvirus to express on tumor cells, in vitro and in vivo, antibodies specific for the CD3epsilon chain (KT3). When injected into growing tumors, these constructs induce the activation of immune effector cells and result in rejection of the tumor. A variety of recombinant adenovirus (Ad) vectors expressing immunostimulatory and/or immunoattractant molecules have now been produced. With this collection of viruses, we have carried out in vivo analyses of combinations of vectors in tumor therapy experiments. For example, we have tested, in murine tumor models, the combination of MVA-KT3 with Ad expressing recently identified cytokines [for example interleukin-12 (IL-12), IL-18] as well as chemokines (e.g. RANTES, MIP1beta). One combination, MVA-KT3/Ad-IL-12/Ad-MIP1beta causes rejection of 100% of growing RENCA tumors. Much attention has been focused on cancer gene therapy using gene transfer of single agents. These data show that antigenic stimulation via the MHCI/TCR-CD3+cytokine+chemokine combination may provide a new and promising approach to cancer gene therapy which is more likely to bypass tumor immunosuppression mechanisms.