The Yop virulon enables extracellularly located Yersinia, in close contact with a eukaryotic target cell, to inject bacterial toxic proteins directly into the cytosol of this cell. Several Ysc proteins, forming the Yop secretion apparatus, display homology with proteins of the flagellar basal body. To determine whether this relationship could extend to the regulatory pathways, we analyzed the influence of flhDC, the master regulatory operon of the flagellum, on the yop regulon. In an flhDC mutant, the yop regulon was up-regulated. The transcription of virF and the steady-state level of the transcriptional activator VirF were enhanced. yop transcription was increased at 37°C and could also be detected at a low temperature. Yop secretion was increased at 37°C and occurred even at a low temperature. The Ysc secretion machinery was thus functional at room temperature in the absence of flagella, implying that in wild-type bacteria, FlhD and/or FlhC, or the product of a gene downstream of flhDC, represses the yop regulon. In agreement with this notion, increased expression of flhDC in wild-type bacteria resulted in the oversecretion of flagellins at room temperature and in decreased Yop secretion at 37°C.The Yop virulon enables extracellularly located Yersinia, in close contact with a eukaryotic target cell, to inject directly into the cytosol of this cell bacterial toxic proteins called Yop effectors. This type III system allows the three Yersinia species that are pathogenic for humans (Yersinia pestis, Y. enterocolitica, and Y. pseudotuberculosis) to resist the nonspecific immune response of the host, in particular by inhibiting phagocytosis and by inducing macrophage apoptosis (3,8). The Yop virulon, encoded by a large virulence plasmid called pYV in Y. enterocolitica, consists basically of a dozen secreted proteins, the Yop effectors and their translocators, as well as a secretion apparatus composed of the Ysc proteins (6). Yop secretion occurs at 37°C in vivo after contact with a eukaryotic target cell and in vitro when the bacteria are placed in a rich medium deprived of Ca 2ϩ ions. Transcription of the ysc and yop genes is strongly thermoinduced; the thermoregulation results from the interplay between a transcriptional activator, VirF (5), and the chromatin structure (31). The expression of the yop virulon is controlled first by temperature, but the expression of some of its genes is reinforced by the action of VirF (21), whose synthesis is also thermoregulated (5). Temperature, by modifying the structure of the chromatin, is thought to dislodge a repressor, presumably the histone-like protein YmoA (7), bound on promoter regions of VirF-sensitive genes and of some other thermoregulated genes (31).Y. enterocolitica organisms are peritrichously flagellated bacteria that are motile only when the temperature is below 30°C, so that once again, temperature is a key environmental factor for the Yersinia lifestyle. With regard to motility, the temperature-sensitive regulation of various flagellar genes has been report...
Controlled-rate freezing and storage in vapour phase nitrogen are used by most transplantation teams for the cryopreservation and storage of peripheral blood haematopoietic stem cells (PBSC). In this study, we analysed 666 autologous PBSC transplants after uncontrolled freezing and storage of PBSC at − 80°C. Statistical analysis showed that neutrophil recovery was associated with both the infused CD34 + cell dose (P = 0.01) and the post transplantation use of growth factors (P o 0.001) and that platelet recovery was associated with the infused CD34 + cell dose (P o0.001) and with the diagnosis (P = 0.02). We analysed three groups according to the duration of the cryopreservation period (less than 6 months, between 6 and 12 months or more than 1 year). Haematopoietic recovery was not found to be adversely affected by longer storage at − 80°C. The haematopoietic recoveries of 50 pairs of sequential transplantations from the same PBSC mobilization were analysed. Despite prolonged cryopreservation, there were no statistically significant differences in neutrophil (P = 0.09) or platelet (P = 0.22) recovery in the second compared with the first transplant. In conclusion, the long-term storage of PBSC at − 80°C after uncontrolled-rate freezing is an easy and comparatively inexpensive cryopreservation method that leads to successful haematopoietic recovery even after prolonged storage.
Introduction Automated slidemakers and stainers and digital microscopes are coupled with haematology analysers to achieve better efficiency and cost‐effectiveness. This study evaluates the integrated performance of slidemakers and digital microscopes commonly available on the market. Methods We compared the percentage of neutrophils for five slidemakers (two Siemens Advia Autoslides, a SysmexSP‐10 and SP‐50 and an Abbot Alinity hs) and a Horiba Hemaprep to the corresponding haematology analyser data (Siemens Advia 2120i, Sysmex XN and Abbot Alinity hq). Differential leucocyte counting (DLC) was performed on three different CellaVision digital microscopes (DM96, DM1200 and DI‐60) and manually. The quality of the smears was assessed using a CellaVision SmearChecker. Results We observed a significant positive absolute bias (P < .05) for the percentage of neutrophils with the Autoslide and Alinity hs smears on the digital microscopes, but not when DLC was performed manually. The SP‐10 and SP‐50 showed no bias regardless of the DLC method. No bias was observed for the Hemaprep smears. All the smears had an acceptable monolayer quality, stain intensity and colour. All smears, except those from Sp‐10, were of an acceptable length. Conclusion Users should be aware of a potential lack of accuracy that can be encountered when using some slidemakers and digital microscopes. All laboratories should validate or verify the differential counts from slidemakers and digital microscope with automated cell differential counters. Manual count validation should only be considered if a significant proportion of clinically relevant abnormal cells are present. Otherwise, haematology analyser results should be favoured.
This new phenotypic approach offers an accurate (sensitivity and specificity of 93% and 96%, respectively), fast and low sample consuming method for the diagnosis of FL.
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