[Bacterial infection and the regulation of in vivo granulopoiesis].
D. HartmannM. A. EntringerWilliam A. RobinsonMichael L. VasilCarla DrebingNicholas M. MortonLarry True
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The mechanism which regulates granulopoiesis was investigated in C 57 BL mice injected i.p. with E.coli. The positive and negative feedback arm of the regulation was studied by correlating the number of bacteria, the number of granulocytes, serum CSF levels and the number of CFU-C in the bone marrow.Keywords:
Granulopoiesis
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Abstract. The effect of leucophoretic serum (LS), obtained from rats with polyvinylpyrrolidone (PVP)‐induced inflammation, on granulopoiesis in the bone marrow of normal CBA mice was studied. The following test systems were used: short term cultures (4 hr), diffusion chambers (8, 24, 48 and 72 hr) and in vivo assays (12, 24 and 48 hr). The results indicate that LS stimulates the proliferations of granulocytic cells by increasing the number of proliferative granulocytes in mitosis, as well as increasing the total number of proliferative granulocytes. LS did not appear to effect monocytes and other cell lines. It is concluded that a factor present in LS specifically stimulates the proliferation of granulocytic cells, both in vitro and in vivo .
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A uropathogenic strain of Proteus mirabilis was grown in vitro in human and mouse urine and brain-heart infusion broth (BHIB) and in vivo in subcutaneous open chambers (SOC) in mice, intraperitoneal diffusion chambers (IPC) in rats and by ascending urinary tract infection in mice in order to compare growth pattern, cellular differentiation and expression of virulence factors. Although the growth rate was slower in vivo than in vitro, the extent of growth was similar after 24 h. P. mirabilis differentiated into filamentous swarmer cells in all in-vitro culture conditions, but no filamentous cells were observed in either of the in-vivo chamber models. Transurethrally infected mice showed a rapid release or loss of filamentous cells and these could not be seen in kidney or bladder homogenates 7 days after infection. Bacteria showed increasing haemagglutination titres for fresh and tanned red blood cells after subculturing in BHIB, but bacteria grown in vivo did not show haemagglutination. An increasing resistance to normal serum was found when bacteria were grown in vivo. Significant haemolytic activity was detected with bacteria grown in BHIB and IPC, but almost no activity was found when bacteria had grown in urine. These findings improve the understanding of the role of P. mirabilis uropathogenic virulence factors in vivo.
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Differential counts of bone marrow smears and aspirated material from the spleen were compared in 17 cases of chronic myeloid leukaemia (CML). With increasing signs of relapse i.e. increasing WBC, an increasing proportion of granulopoietic precursor cells were found in the bone marrow. The proportion of granulopoietic precursor cells did not, however, change significantly in the spleen. It is suggested that the differences observed may reflect differences in granulopoiesis between bone marrow and extramedullary tissue in CML.
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β-glucosidase, β-glucuronidase and nitroreductase activities of in vitro cultures of intestinal bacteria were compared with those of the caecal contents of gnotobiotic mice monoassociated with the same bacteria. Although bacterial enzyme activities detected in vitro were also detected in the caecal contents of monoassociated mice with each bacterium, activities of each bacterial enzyme in vivo were not always parallel to those in vitro . The difference of β-glucosidase activity among bacterial strains was smaller in the in vivo assay than that in vitro . Three enzyme activities in the caecal contents of gnotobiotic mice monoassociated with Eubacterium nitrogenes became stable more than 1 wk after inoculation, and were also changed by diets with different protein contents. Keywords: nitroreductase, β-glucosidase, β-glucuronidase, gnotobiotic mice, intestinal bacteria, caecal content.
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Total and free endotoxin release in time from cultures of Escherichia coli by different antibiotics was studied in vitro for 4 h in relation to the antibiotic effect on viable counts and morphological features of the test cultures. The most rapid fall in viable counts was seen after treatment with imipenem or the combination of imipenem with tobramycin, accompanied by an early, but minimal increase (1.8-fold) of the total (free plus cell-bound) endotoxin level at 1 h. Total endotoxin levels increased approximately 5-fold upon incubation with ceftazidime, tobramycin or the combination of tobramycin with cefuroxime, whereas incubation with cefuroxime or aztreonam alone caused a late 22-and 49-fold increase in total endotoxin, respectively, at 4 h. In chloramphenicol treated cultures there was still an increase in viable counts during therapy, resulting in an ultimately 78-fold increase of mean levels of total endotoxin. Free endotoxin levels increased approximately 6-fold within 1 h upon treatment with imipenem, alone or in combination with tobramycin, or ceftazidime as the result of rapid lysis of bacteria. Treatment with cefuroxime or aztreonam induced a relatively late but much higher release of free endotoxin (118-and 222-fold, respectively), which was due to the formation of long filamentous structures during the first 2 h of incubation and eventually cell lysis. Both tobramycin and the combination of tobramycin with cefuroxime caused a more gradual rise in free endotoxin, with a +/- 15-fold increase in free endotoxin at 4 h. In chloramphenicol treated cultures, as in the control cultures, the level of free endotoxin remained proportional to the amount of viable organisms. We also studied plasma endotoxin levels in 20 patients with septic shock. 10 out of these 20 patients had a detectable endotoxemia (greater than 5 ng/l) on admission. We describe the patterns of plasma endotoxin in these patients during the first 24 h of antibiotic treatment. We conclude that, in the in-vitro study, values of total endotoxin, free endotoxin, and the rate of release of endotoxin varies with the antibiotic used. We also demonstrate that in patients under treatment for septic shock endotoxin release can be related to the administration of antibiotics.
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The present study was conducted to examine the morphology and antigenicity of Photobacterium damselae subsp.piscicida by culturing the bacterium in vivo in the peritoneal cavity of sea bass (Dicentrarchus labrax) within dialysis bags with either a low molecular weight (LMW) cut-off of 25 kDa or a high molecular weight (HMW) cut-off of 300 kDa.Differences were observed in the growth rate between the bacteria cultured in vivo or in vitro.Bacteria cultured in vivo were smaller and produced a capsular layer, which was more prominent in bacteria cultured in the HMW bag.Antigenicity was examined by Western blot analysis using sera from sea bass injected with live Ph.d. subsp.piscicida.The sera recognised bands at 45 and 20 kDa in bacteria cultured in vivo in the LMW bag.Bacteria cultured in vivo in the HMW bag did not express the 45 kDa band when whole cell extracts were examined, although the antigen was present in their extracellular products.In addition, these bacteria had a band at 18 kDa rather than 20 kDa.Differences in glycoprotein were also evident between bacteria cultured in vitro and in vivo.Bacteria cultured in vitro in LMW and HMW bags displayed a single 26 kDa band.Bacteria cultured in the LMW bag in vivo displayed bands at 26 and 27 kDa, while bacteria cultured in vivo in the HMW bag possessed only the 27 kDa band.These bands may represent sialic acid.The significance of the changes observed in the bacterium's structure and antigenicity when cultured in vivo is discussed.
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The objectives of this study were to determine the effect of bacterial culture conditions on adherence of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain 86-24 in vivo to pig enterocytes and to compare the results with adherence in vitro to cultured HEp-2 and IPEC-J2 cells. Growth of O157:H7 in MacConkey broth (MB) resulted in almost no adherence to both HEp-2 and IPEC-J2 cells; prior exposure of the bacteria to pH 2.5 reduced adherence. There was greater adherence by bacteria from static cultures than by those from shaken cultures and by bacteria cultured in brain-heart infusion (BHI) plus NaHCO₃ (BHIN) than by bacteria cultured in BHI. In contrast, in pig ileal loops, bacteria cultured in MB adhered well to enterocytes, and prior exposure to pH 2.5 had no effect on adherence. Among several media tested for their effect on bacterial adherence in the pig intestine, MB and BHIN proved to be the best. Bacterial adherence was dose-dependent and was more extensive in the ileum than in the colon. This study demonstrated that there are remarkable differences between culture conditions that promote adherence of an EHEC O157:H7 strain in vitro and in vivo, that culture conditions profoundly affect adherence to epithelial cells in vitro and in vivo, and that pig ileal loops are better suited to adherence studies than are colon loops.
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Colony-stimulating factor (CSF) is a well-established specific promoter of the committed stem cell colony-forming unit in culture (CFU-C) in vitro and thought to be a true regulator of granulopoiesis in vivo as well. However, the mechanisms involved in the regulation of granulopoiesis are still controversial. Bacterial products, like endotoxin and live bacteria, enhance levels of serum CSF in vitro and in vivo both in animals and in man. Data from our laboratory in germ-free mice showed evidence that high levels of bacteria stimulate production of granulocytes by activating the CSF-CFU-C system and that inhibition of granulocyte production may be simply due to clearance of bacteria by granulocytes. This study has been undertaken to determine the role of bacterial infection in conventional mice in the regulation of granulopoiesis in vivo. C57/bl 6J mice were injected intraperitoneally (ip) with 1 × 104Escherichia coli (E. coli). The animals were evaluated for the following parameters: serum CSF, bone marrow CFU-C, the absolute number of granulocytes in the peripheral blood, and the number of bacteria in peripheral blood and abdominal cavity. Groups of five mice were investigated at multiple time points after injection. The results show peak levels of bacteria both in peripheral blood and abdominal cavity at the 1-hr time point. There is a steady and marked rise in serum CSF reaching a first peak 8 hr, a second peak 96 hr after the injection. The number of CFU-C is increased at the 48- and the 120-hr time point. A pronounced rise in the absolute number of granulocytes is seen 2 hr after infection, which seems to be independent of the activation of stem cells. These findings demonstrate a temporal relationship between the peak in the number of bacteria, release of mature granulocytes, rise in serum CSF, and subsequent activation of bone marrow CFU-C. Following rises in CFU-C there are no marked elevations in peripheral blood granulocytes.
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Colony-stimulating factor
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