EGR-1 activation by EGF inhibits MMP-9 expression and lymphoma growth.

Progression of hematologic malignancies is strongly dependent on bidirectional interactions between tumor cells and stromal cells. Expression of members of the matrix metalloproteinase (MMP) family by stromal cells is a central event during these interactions. However, although several studies have focused on the mechanisms responsible for induction of MMP in stromal cells, the signals that negatively regulate their secretion of in these cells remain largely unknown. Here, we provide evidence that MMP-9 production by stromal cells is suppressed through activation of early growth response protein 1 (EGR-1), thereby inhibiting the growth of thymic lymphoma. We found that EGR-1 expression is induced in stromal cells after contact with lymphoma cells via epidermal growth factor (EGF). Moreover, development of thymic lymphoma was inhibited when induced by lymphoma cells overexpressing EGF compared with control lymphoma cells. Using transgenic mice containing MMP-9 promoter-driven luciferase transgene in its genome, we further demonstrated that EGF/EGR-1 repressed transcriptional activation of the MMP-9 gene by stromal cells. De novo expression of EGR-1 alone by gene transfer or exposure to recombinant human EGF also inhibited MMP-9 expression. Taken together, these results indicate that EGR-1 could be a source of novel targets for therapeutic intervention in lymphoid tumors in which MMP-9 plays a critical role.

Lymphoma cells contribute to the augmentation of plasma sL-selectins in the serum of lymphoma-bearing mice.

Like many integral membrane glycoproteins, the extracellular domain of L-selectin undergoes rapid shedding, which occurs on both resting and activated host leucocytes. Incubating normal or transformed leukocytes with phorbol esters can also artificially induce shedding of L-selectin, providing multiple possibilities for the source of soluble forms of L-selectin found in the serum of patients with hematological malignancies. Here, using genetically engineered L-selectin-deficient mouse models, we have measured the release of soluble circulating forms of L-selectin in the serum of lymphoma-bearing mice. We found that L-selectin-deficient lymphoma cells could not induce an elevation of circulating soluble forms of L-selectin in normal mice, as compared to lymphoma cells expressing L-selectin. Moreover, soluble forms of L-selectin were detected in the serum in mice bearing lymphoma induced by injection of T lymphoma cells expressing L-selectins. Interestingly, we also found that lymphoma cells that are unable to shed L-selectin in vitro following exposure to phorbol ester can generate soluble forms of serum L-selectin in vivo. Taken together, these results indicate that lymphoma cells are the major contributors to levels of soluble forms of L-selectins in lymphoma-bearing mice.

Role of selectins in the triggering, growth, and dissemination of T-lymphoma cells: implication of L-selectin in the growth of thymic lymphoma.

We previously showed that intercellular adhesion molecule-1 (ICAM-1) expression by the host is essential for lymphoma dissemination. Because selectins usually act in a coordinated fashion with ICAM-1 in the recruitment of circulating normal cells, we investigated their implication in lymphomagenesis and metastasis. Using selectin-deficient mice, we found that though the absence of E-, P-, or L-selectins did not affect the triggering of radiation-induced thymic lymphoma, the absence of L-selectin on lymphoma cells reduced their capacity to grow in the thymus. This defect, however, was overcome by altering the integrity of the L-selectin-mediated interactions in the thymus, as shown in L-selectin-deficient mice and by adoptive transfer experiments. We also found that lack of selectin expression by the host significantly delayed the dissemination of lymphomas to peripheral tissues. This resistance of selectin-deficient mice to lymphoma metastasis was dependent on the intrinsic properties of lymphoma cells because highly tumorigenic variants were insensitive to the absence of selectins. Observations that lymphoma cells disseminate with the same efficiency in normal and selectin-deficient mice suggest that selectins exert their influence at the posthoming stage of metastasis, as does ICAM-1. These results provide definitive evidence that selectins play a significant role at different steps of T-cell lymphoma development.

Phylogeny of the Enterobacteriaceae based on genes encoding elongation factor Tu and F-ATPase beta-subunit.

The phylogeny of enterobacterial species commonly found in clinical samples was analysed by comparing partial sequences of their elongation factor Tu gene (tuf) and of their F-ATPase beta-subunit gene (atpD). An 884 bp fragment for tuf and an 884 or 871 bp fragment for atpD were sequenced for 96 strains representing 78 species from 31 enterobacterial genera. The atpD sequence analysis exhibited an indel specific to Pantoea and Tatumella species, showing, for the first time, a tight phylogenetic affiliation between these two genera. Comprehensive tuf and atpD phylogenetic trees were constructed and are in agreement with each other. Monophyletic genera are Cedecea, Edwardsiella, Proteus, Providencia, Salmonella, Serratia, Raoultella and Yersinia. Analogous trees based on 16S rRNA gene sequences available from databases were also reconstructed. The tuf and atpD phylogenies are in agreement with the 16S rRNA gene sequence analysis, and distance comparisons revealed that the tuf and atpD genes provide better discrimination for pairs of species belonging to the family Enterobacteriaceae. In conclusion, phylogeny based on tuf and atpD conserved genes allows discrimination between species of the Enterobacteriaceae.

Rapid detection of Clostridium difficile in feces by real-time PCR.

Clostridium difficile is the major causative agent of nosocomial antibiotic-associated diarrhea, colitis, and pseudomembranous colitis. The pathogenicity of C. difficile is closely related to the production of toxins A and B. Toxigenic C. difficile detection by a tissue culture cytotoxin assay is often considered the "gold standard." However, this assay is time consuming, as it implies an incubation period of at least 24 h. We have developed a rapid real-time fluorescence-based multiplex PCR assay targeting the C. difficile toxin genes tcdA and tcdB, with the Smart Cycler. Two molecular beacons bearing different fluorophores were used as internal probes specific for each amplicon type. The analytical sensitivity of the assay was around 10 genome copies for all nine C. difficile strains tested, representing the 6 most common toxinotypes. The specificity was demonstrated by the absence of amplification with DNA purified from bacterial species other than C. difficile (n = 14), including Clostridium sordellii for which the lethal toxin gene sequence is closely related to the toxin genes of C. difficile. Following a rapid (15 min) and simple fecal sample preparation protocol, both tcdA and tcdB were efficiently amplified from 28 of 29 cytotoxin-positive feces samples. There was no amplification observed with all 27 cytotoxin-negative feces samples tested. This is the first real-time PCR assay for the detection of C. difficile. It is rapid, sensitive, and specific and allows detection of C. difficile directly from feces samples.

Rapid detection of Shiga toxin-producing bacteria in feces by multiplex PCR with molecular beacons on the smart cycler.

We have developed a rapid (1-h) real-time fluorescence-based PCR assay with the Smart Cycler thermal cycler (Cepheid, Sunnyvale, Calif.) for the detection of Shiga toxin-producing Escherichia coli (STEC), as well as other Shiga toxin-producing bacteria. Based on multiple-sequence alignments, we have designed two pairs of PCR primers that efficiently amplify all variants of the Shiga toxin genes stx(1) and stx(2), respectively. These primer pairs were combined for use in a multiplex assay. Two molecular beacons bearing different fluorophores were used as internal probes specific for each amplicon. Assays performed with purified genomic DNA from a variety of STEC strains (n = 23) from diverse geographic locations showed analytical sensitivities of about 10 genome copies per PCR. Non-STEC strains (n = 20) were also tested, and no amplification was observed. The PCR results correlated perfectly with the phenotypic characterization of toxin production in both STEC and non-STEC strains, thereby confirming the specificity of the assay. The assay was validated by testing 38 fecal samples obtained from 27 patients. Of these samples, 26 were PCR positive for stx(1) and/or stx(2). Compared with the culture results, both the sensitivity and the negative predictive value were 100%. The specificity was 92%, and the positive predictive value was 96%. Moreover, this assay detected STEC from a sample in which the STEC concentration was at the limit of detection of the conventional culture methods and from a sample in which STEC was not detected by the conventional culture methods. This real-time PCR assay is simple, rapid, sensitive, and specific and allows detection of all Shiga toxin-producing bacteria directly from fecal samples, irrespective of their serotypes.