Dipstick Test for Rapid Diagnosis of Shigella dysenteriae 1 in Bacterial Cultures and Its Potential Use on Stool Samples

International audienceBACKGROUND: We describe a test for rapid detection of S. dysenteriae 1 in bacterial cultures and in stools, at the bedside of patients. METHODOLOGY/PRINCIPAL FINDINGS: The test is based on the detection of S. dysenteriae 1 lipopolysaccharide (LPS) using serotype 1-specific monoclonal antibodies coupled to gold particles and displayed on a one-step immunochromatographic dipstick. A concentration as low as 15 ng/ml of LPS was detected in distilled water and in reconstituted stools in 10 minutes. In distilled water and in reconstituted stools, an unequivocal positive reaction was obtained with 1.6×10⁶ CFU/ml and 4.9×10⁶ CFU/ml of S. dysenteriae 1, respectively. Optimal conditions to read the test have been determined to limit the risk of ambiguous results due to appearance of a faint yellow test band in some negative samples. The specificity was 100% when tested with a battery of Shigella and unrelated strains in culture. When tested on 328 clinical samples in India, Vietnam, Senegal and France by laboratory technicians and in Democratic Republic of Congo by a field technician, the specificity (312/316) was 98.7% (95% CI:96.6-99.6%) and the sensitivity (11/12) was 91.7% (95% CI:59.8-99.6%). Stool cultures and the immunochromatographic test showed concordant results in 98.4 % of cases (323/328) in comparative studies. Positive and negative predictive values were 73.3% (95% CI:44.8-91.1%) and 99.7% (95% CI:98-100%). CONCLUSION: The initial findings presented here for a simple dipstick-based test to diagnose S. dysenteriae 1 demonstrates its promising potential to become a powerful tool for case management and epidemiological surveys

The N-terminus of PKR is responsible for the activation of the NF-kappaB signaling pathway by interacting with the IKK complex

The interferon-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) has been shown to activate NF-κB independently of its kinase function after interaction with the IKK complex. In order to investigate the mechanism of NF-κB activation by PKR, we identified the domain of PKR responsible for stimulating the NF-κB pathway in PKR-deficient fibroblasts using an NF-κB dependent reporter assay. The N-terminal 1–265 AA of PKR activates NF-κB, whereas the 1–180 AA N-terminus restricted to the two dsRNA Binding Domains (DRBD), the third basic domain alone (AA 181–265), or the C-terminus of PKR (AA 266–550) were unable to stimulate the expression of the NF-κB dependent reporter gene. Using confocal microscopy, we confirmed that PKR full length as well as PKR N-terminus colocalized with IKKβ. By GST-pulldown analysis, using different PKR domains, we then revealed the specific ability of the PKR N-terminus 1–265 to bind to and activate IKK and showed that this activation requires the integrity of the IKK complex. This activation is not only due to DRBDs since the DRBD fragment 1–180 failed to inhibit PKR 1–265 induced NF-κB activation. Our results therefore demonstrate that the ability of PKR to mediate NF-κB activation resides in its full N-terminus, and requires both DRBDs and the third basic domain

Transfer of yeast artificial chromosomes into mammalian cells and comparative study of their integrity

Yeast artificial chromosomes (YACs) from the CEPH MegaYAC library (Paris, France) ranging in size from 350 to 1600 kb and mapping to the q22.1 and q22.2 regions of human chromosome 21 were transferred into mammalian cells by spheroplast fusion. The integrity of the YACs from two adjacent parts of the region was compared after retrofitting and stable transfer into mammalian cells. We found that large YACs could easily be manipulated to allow transfer of the YAC material into mammalian cells and that the size of the YAC did not appear to be limiting for fusion. However, we show that there was great variability in the integrity of the YACs from the two regions, which was not related to the size of the YACs. Four YACs in region I from sequence-tagged site (STS) G51E05 up to STS LL103 showed, in general, no loss of material and correct gene transfer into mammalian cells. In contrast, the three YACs in the more centromeric region II (from STS G51B09 up to G51E05) frequently showed a loss o

CIITA B-cell specific promoter suppression in MHC class II-silenced cell hybrids

In this study, various sets of somatic cell hybrids, generated by the fusion of epithelial cell lines with B-lymphoblastoid cell lines, were analyzed for the expression of major histocompatibility complex (MHC) class II antigens. We first demonstrate, in human and mouse intraspecies hybrids, the coordinate suppression of MHC class II, Ii (invariant chain) and HLA-DM gene transcription, and the release of the silencing by the addition of interferon gamma. Using interspecies hybrids, the segregation of human chromosomes allowed us to establish that MHC class II extinction is linked to the presence in the hybrids of the chromosomes from the epithelial fusion partner. Moreover, our data provide evidence that the expression pattern of MHC class II mRNA is correlated with that of the class II transactivator (CIITA), suggesting that CIITA is the actual target of the silencing. To gain further insight into the suppression phenomenon we performed luciferase assays which show that silencing affects the activity of the B-cell-specific promoter of CIITA. These results therefore demonstrate that the MHC class II gene silencing in somatic cell hybrids is due to an active suppression of one of the promoters of the CIITA gene, mediated by the epithelial cell fusion partner

Targeting lactate metabolism by inhibiting MCT1 or MCT4 impairs leukemic cell proliferation, induces two different related death-pathways and increases chemotherapeutic sensitivity of acute myeloid leukemia cells

Metabolism in acute myeloid leukemia (AML) cells is dependent primarily on oxidative phosphorylation. However, in order to sustain their high proliferation rate and metabolic demand, leukemic blasts use a number of metabolic strategies, including glycolytic metabolism. Understanding whether monocarboxylate transporters MCT1 and MCT4, which remove the excess of lactate produced by cancer cells, represent new hematological targets, and whether their respective inhibitors, AR-C155858 and syrosingopine, can be useful in leukemia therapy, may reveal a novel treatment strategy for patients with AML. We analyzed MCT1 and MCT4 expression and function in hematopoietic progenitor cells from healthy cord blood, in several leukemic cell lines and in primary leukemic blasts from patients with AML, and investigated the effects of AR-C155858 and syrosingopine, used alone or in combination with arabinosylcytosine, on leukemic cell proliferation. We found an inverse correlation between MCT1 and MCT4 expression levels in leukemic cells, and showed that MCT4 overexpression is associated with poor prognosis in AML patients. We also found that AR-C155858 and syrosingopine inhibit leukemic cell proliferation by activating two different cell-death related pathways, i.e., necrosis for AR-C155858 treatment and autophagy for syrosingopine, and showed that AR-C155858 and syrosingopine exert an anti-proliferative effect, additive to chemotherapy, by enhancing leukemic cells sensitivity to chemotherapeutic agents. Altogether, our study shows that inhibition of MCT1 or MCT4 impairs leukemic cell proliferation, suggesting that targeting lactate metabolism may be a new therapeutic strategy for AML, and points to MCT4 as a potential therapeutic target in AML patients and to syrosingopine as a new anti-proliferative drug and inducer of autophagy to be used in combination with conventional chemotherapeutic agents in AML treatment

Rapid diagnosis of diarrhea caused by Shigella sonnei using dipsticks; comparison of rectal swabs, direct stool and stool culture.

BACKGROUND: We evaluated a dipstick test for rapid detection of Shigella sonnei on bacterial colonies, directly on stools and from rectal swabs because in actual field situations, most pathologic specimens for diagnosis correspond to stool samples or rectal swabs. METHODOLOGY/PRINCIPAL FINDINGS: The test is based on the detection of S. sonnei lipopolysaccharide (LPS) O-side chains using phase I-specific monoclonal antibodies coupled to gold particles, and displayed on a one-step immunochromatographic dipstick. A concentration as low as 5 ng/ml of LPS was detected in distilled water and in reconstituted stools in 6 minutes. This is the optimal time for lecture to avoid errors of interpretation. In distilled water and in reconstituted stools, an unequivocal positive reaction was obtained with 4 x 10(6) CFU/ml of S. sonnei. The specificity was 100% when tested with a battery of Shigella and different unrelated strains. When tested on 342 rectal swabs in Chile, specificity (281/295) was 95.3% (95% CI: 92.9% - 97.7%) and sensitivity (47/47) was 100%. Stool cultures and the immunochromatographic test showed concordant results in 95.5 % of cases (328/342) in comparative studies. Positive and negative predictive values were 77% (95% CI: 65% - 86.5%) and 100% respectively. When tested on 219 stools in Chile, Vietnam, India and France, specificity (190/198) was 96% (95% CI 92%-98%) and sensitivity (21/21) was 100%. Stool cultures and the immunochromatographic test showed concordant results in 96.3 % of cases (211/219) in comparative studies. Positive and negative predictive values were 72.4% (95% CI 56.1%-88.6%) and 100 %, respectively. CONCLUSION: This one-step dipstick test performed well for diagnosis of S. sonnei both on stools and on rectal swabs. These data confirm a preliminary study done in Chile