Migration of stem cells and progenitors between marrow and spleen following thiamphenicol treatment of mice.

Recovery of hemopoiesis was studied after a 3-day treatment with the antibiotic thiamphenicol (TAP). A contrasting behavior of the spleen colony-forming units (CFU-S), granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), and erythroid colony-forming units (CFU-E) numbers in the bone marrow versus those in the spleen was found. Whereas the cell numbers reached nadirs in the marrow, they peaked 30 to 100-fold above control values in the spleen on day 4. Simultaneously the number of CFU-S, BFU-E, and CFU-GM, but not of CFU-E, increased drastically in the peripheral blood. The tritiated thymidine kill of the splenic CFU-S was too small to explain the endogenous splenic production of these cells. A quantitative analysis further revealed that an effective erythropoiesis was established in the spleen. As a consequence, the first part of a reticulocytosis was mainly due to the splenic contribution, whereas the second part predominantly originated from a delayed marrow erythropoiesis. In contrast, the CFU-GM of the spleen did not effectively differentiate into granuloid precursors. The bulk of the granuloid production occurred in the marrow. The best explanation for these results is a net migration of CFU-S, BFU-E, and CFU-GM from the marrow to the spleen during early recovery, and a back-migration of CFU-GM to the marrow later in the recovery phase. These observations indicate a link between migration of hemopoietic cells and their differentiation at the two hemopoietic sites.

Hemopoiesis during thiamphenicol treatment. I. Stimulation of stem cells during eradication of intermediate cell stages.

Continuous treatment of C57bl/6 mice for 4 days with the cytostatic antibiotic thiamphenicol revealed a dual response of hemopoietic cells. On one hand, morphologically recognizable erythroid precursors and late progenitors (erythroid colony-forming units; CFU-E) and, to a lesser extent, granuloid precursors were found substantially reduced. On the other hand, early granuloid (granulocyte-macrophage colony-forming units; CFU-GM) and erythroid (erythroid burst-forming units; BFU-E) progenitors increased on day 3 to 220%-240% and 120%-130% of the control value, respectively. This was accompanied by a decline of the initial spleen colony-forming units (CFU-S) (day 8) pool size to approximately 60%. These patterns were similar in the bone marrow and the spleen. In addition, the tritiated thymidine kill of femoral and splenic CFU-S rose significantly (p less than 0.05) from 16% +/- 3% to 38% +/- 2% and from 3% +/- 1% to 17% +/- 2%, respectively. A sudden decline of peripheral reticulocytes between days 2 and 3 from 2.8% +/- 0.3% to 0.6% +/- 0.2% was observed, whereas the hematocrit gradually decreased from day 1 to day 4 from 45.2% +/- 0.1% to 39.3% +/- 0.3%. The white blood cells were not affected. From these results we conclude that stem cells were stimulated as a consequence of the suppression of the intermediate cell stages. As analyzed in the accompanying paper, this confirms a prediction stated by a quantitative theoretical concept of in vivo stem cell regulation.

Hemopoiesis during thiamphenicol treatment. II. A theoretical analysis shows consistency of new data with a previously hypothesized model of stem cell regulation.

In a recent theoretical model of stem cell regulation, specific quantitative assumptions were made about an in vivo feedback process from erythroid and granuloid precursor cell stages to the spleen colony-forming units (CFU-S), erythroid burst-forming units (BFU-E), and granulocyte-macrophage colony-forming units (CFU-GM). Utilizing specific effects of the antibiotic thiamphenicol (TAP), new experiments have been performed to challenge this model. Here these data are treated in an analysis that implies three steps. First, model assumptions about TAP toxicity are justified. The toxic TAP effects on erythroid and granuloid precursors are quantified as a continuous reduction of the normal amplification coefficient for CFU-E (down to 1/250), proerythroblasts, basophilic erythroblasts, and proliferating granuloid precursors (down to 1/4). Second, the original model predictions for the behavior of CFU-S, CFU-GM, and BFU-E are compared with the corresponding data. Third, discrepancies are discussed and it is demonstrated that adjustment of one single parameter resolves most of them. Thus one can quantitatively explain the experimental results for CFU-S, BFU-E, and CFU-GM by an activation of the regulatory process postulated: the decline in erythroid (and granuloid) cell numbers enhances the cycling of CFU-S while their self-renewal probability is reduced; consequently CFU-S numbers decline; as more cells differentiate towards BFU-E and CFU-GM per unit time the cell numbers of these cell stages increase. Thus the new data on stem cell behavior during TAP treatment support the hypothesis of a feedback from erythroid and granuloid precursors to the stem cells.

Optimal erythroid cell production during erythropoietin treatment of mice occurs by exploiting the splenic microenvironment.

In this study, quantitative effects on erythroid cell production by a prolonged recombinant human erythropoietin (rhEpo) treatment of mice are presented. Epo treatments, given subcutaneously (s.c.) twice per day in doses of 0.5 to 500 U per day, were performed under steady-state production conditions. We found striking differences between the behavior of the different erythroid cell compartments (burst-forming unit erythroid [BFU-E], colony-forming unit erythroid [CFU-E] and erythroid precursors), as well as between the microenvironments of bone marrow and spleen. Whereas the total-body BFU-E was not changed by Epo, a redistribution of BFU-E from marrow to spleen occurred, resulting in decreasing marrow and increasing splenic BFU-E numbers. Splenic BFU-E produced CFU-E as much as 8 times more efficiently than marrow BFU-E at 50 U of Epo. At low Epo doses (to 1 U/day) no difference was found. The CFU-E in the spleen produced erythroblasts at a higher efficiency at all Epo doses (1.5 to 5 times). It seems as if this efficiency was higher at low Epo doses. Because of the migration phenomenon and the excellent microenvironment in the spleen, at the highest Epo concentrations nearly 70% of all erythroid cells reside in the spleen. Even at the highest Epo doses, granuloid cell production was not affected. Similar to the BFU-E, total-body granuloid cells remained constant (despite a shift of granulocyte-macrophage progenitors [CFU-GM]) from marrow to spleen; however, these cells did not flourish in the spleen. Under these conditions, 90% of the granuloid precursors were still localized in the marrow. Erythropoietin did not change the transit time of erythroid cells at high Epo doses.

Recovery of murine myelopoiesis after cytostatic reduction by Ara-C. Effect of bacitracin-induced changes in the intestinal microflora and influence of timing.

The influence of intestinal flora modulation by oral bacitracin on the recovery of myelopoiesis after Ara-C was studied in C3H/Law mice. Bacitracin resulted in a 3-5 log increase of Gram-negative bacteria and a 10-fold increase of the intestinal endotoxin concentration. Initiation of bacitracin before Ara-C stimulated the initial rebound increase of colony-forming units for granulocytes and macrophages (CFU-GM) from 23.2 +/- 1.3 to 28.4 +/- 1.4 x 10(3) per femur. Starting the bacitracin after Ara-C advanced the second phase of the rebound CFU-GM increase with 6 days. An important role in the recovery of myelopoiesis after cytostatic drugs in C3H/Law mice is suggested for the intestinal Gram-negative microflora, probably mediated by bacterial endotoxin.

Influence of high versus low intestinal concentration of gram-negative bacteria and endotoxin on the susceptibility of murine myelopoiesis in bone marrow and spleen to cytostatic treatment with Ara-C.

The haematopoietic recovery after i.v. cytarabine was studied in C3H/Law mice as a measure for stem cell susceptibility in relation to the intestinal Gram-negative bacteria (GNB) and endotoxin. Reduction or elevation of GNB and endotoxin was induced by either polymyxin or bacitracin, both non-absorbable antibiotics. Bacitracin caused less suppression of the splenic cellularity after cytarabine, and an advancement of the recovery of femoral nucleated cells. The femoral recovery of CFU-GM exhibited a biphasic pattern. The speed and height of the rebound increase of CFU-GMs were significantly affected by the antibiotics. Thus, (modulation of) the murine intestinal microflora influences the haematopoietic recovery after cytostatic drugs. The mechanisms involved are complex; intestinal endotoxin seems to play a role.

Osler-Rendu-Weber with liver involvement.

Osler-Rendu-Weber disease, or hereditary hemorrhagic telangiectasia, is a vascular disease with autosomal dominant transmission. The liver is involved in 31% of patients with hereditary hemorrhagic telangiectasia (1). It appears that the lesions uncommonly causes serious disease. We report the imaging findings and the therapy in a case with extensive telangiectasis in the liver.

Kinetics of endotoxin release by gram-negative bacteria in the intestinal tract of mice during oral administration of bacitracin and during in vitro growth.

The release of endotoxin by gram-negative bacteria was studied during bacitracin-induced intestinal colonization in C3H/Law mice and during in vitro growth. The "free" endotoxin concentration was determined by the Limulus amebocyte lysate test in faecal and in culture supernatants, respectively. After oral administration of bacitracin for 2 days a significant (p less than 0.001) increase of the faecal concentration of gram-negative bacteria of 3-4 logs accompanied a significant (p less than 0.001) increase of the faecal endotoxin concentration from 10(1.8 +/- 0.2) to 10(3.3 +/- 0.2) micrograms endotoxin/g faeces. In vitro, however, an increase of the concentration of gram-negative bacteria of 3-4 logs resulted in a 3-4 log increase of the concentration of endotoxin during the exponential and early stationary growth phase. The faecal endotoxin level after 8 days of bacitracin treatment dropped to a value not significantly different from the initial value, regardless of a high faecal level of gram-negative bacteria. Endotoxin determination by the Limulus amebocyte lysate test appeared to be unaffected by the amount of bacitracin present in faecal preparations after oral administration of this antibiotic. In addition, the in vitro release of endotoxin was not influenced by bacitracin. From these results we conclude, that "free" endotoxin is a product of extensive proliferation of gram-negative bacteria. Therefore, the intestinal endotoxin level does not necessarily correlate with the level of gram-negative bacteria, but corresponds with the proliferative activity of these bacteria.

Myelopoiesis in experimentally contaminated specific-pathogen-free and germfree mice during oral administration of polymyxin.

Oral administration of polymyxin to specific-pathogen-free C3H/Law mice which with previously contaminated with gram-negative bacteria resulted in complete suppression of cecal gram-negative bacteria. Suppression of cecal gram-negative bacteria was accompanied by reduction of the cecal endotoxin concentration from 10 to 1 microgram/g of cecal content as measured with a microtechnique for the Limulus amebocyte lysate assay. Endotoxin determination by this assay appeared to be unaffected by the amount of polymyxin present in cecal preparations after oral administration of this antibiotic. In experimentally contaminated specific-pathogen-free mice, the femoral concentration of progenitor cells forming granulocyte-macrophage colonies in vitro (CFU-GM) decreased significantly (P less than 0.001) to 66% of the initial control after 4 days of polymyxin treatment. However, the femoral CFU-GM concentration in germfree mice and splenic CFU-GM concentration in experimentally contaminated specific-pathogen-free and germfree mice was not affected by polymyxin treatment. The kinetic behavior of femoral and splenic CFU-GM in experimentally contaminated specific-pathogen-free and germfree mice was expressed as the in vivo sensitivity to the S-phase-specific cytostatic drug hydroxyurea, i.e., the hydroxyurea kill. Administration of polymyxin to experimentally contaminated specific-pathogen-free mice significantly diminished the hydroxyurea kill of femoral CFU-GM from 29 to 13% (P less than 0.02) and of splenic CFU-GM from 53 to 27% (P less than 0.005). The hydroxyurea kill of femoral CFU-GM in germfree mice was not significantly affected by polymyxin treatment. On basis of these results we conclude that the effect of polymyxin treatment on myelopoiesis is most likely due to elimination of intestinal gram-negative bacteria and may indicate a significant role of intestinal gram-negative bacteria in the regulation of myelopoiesis.

Oral administration of antibiotics and intestinal flora associated endotoxin in mice.

The contribution of aerobic and anaerobic gram-negative intestinal bacteria to the release of endotoxin in the intestinal tract was investigated during oral administration of various nonabsorbable antimicrobial drugs to C3H/Law mice. The intestinal endotoxin release was studied by determination of the endotoxin concentration in faecal supernatants with the Limulus amebocyte lysate assay. Selective elimination of aerobic gram-negative bacteria by oral treatment with polymyxin, aztreonam or temocillin resulted in a reduction of the endotoxin concentration of faecal supernatants to 10% of the untreated control. Further decrease of the endotoxin concentration to 1% was achieved by total decontamination of the intestinal tract by oral cephalothin/neomycin treatment. Endotoxin determination with the Limulus amebocyte lysate assay appeared to be unaffected by the antibiotics present in the faecal supernatants after oral treatment. On basis of these experiments, it is concluded that in mice 90% of the faeces derived endotoxin can be ascribed to release of endotoxin by intestinal aerobic gram-negative bacteria.

Effect of intestinal flora modulation by oral polymyxin treatment on hemopoietic stem cell kinetics in mice.

After oral treatment with polymyxin for only 1 day, fecal aerobic gram-negative bacteria were found completely suppressed in C3H/Law mice. Complete suppression of aerobic gram-negative bacteria was accompanied by a reduction of the fecal endotoxin concentration from 100 to 10 micrograms endotoxin per gram of feces as measured with the Limulus amebocyte lysate assay. Oral administration of polymyxin affected hemopoietic stem cell kinetics at different stages. The kinetic behavior of hemopoietic stem cells was determined as the in vivo sensitivity to the S phase specific cytostatic drug hydroxyurea. The hydroxyurea kill of bone marrow spleen colony-forming cells diminished not significantly (p less than 0.10) from 14 to 4% after 2 days of polymyxin treatment. Already after 1 day of treatment the hydroxyurea kill of bone marrow progenitor cells forming granulocyte-macrophage colonies in vitro decreased from 29 to 7% (p less than 0.05). It took 8 days of treatment before the hydroxyurea kill of splenic granulocyte-macrophage colonies was found reduced from 53 to 14% (p less than 0.001). The decreased susceptibility of hemopoietic stem cells to hydroxyurea during polymyxin treatment appears to argue for a role of intestinal aerobic gram-negative bacteria in the regulation of hemopoiesis, probably mediated by endotoxin.

Differential effects of recombinant human colony stimulating factor (rh G-CSF) on stem cells in marrow, spleen and peripheral blood in mice.

Previously it has been hypothesized that the granulopoietic and erythropoietic lineages may compete for differentiating stem cells. According to this hypothesis one would expect that a stimulation of granulopoiesis by G-CSF administration would lead to a reduction of the stem cell pool and be followed by a decline of erythropoietic progenitor numbers. In addition one would expect an enhanced response of granulopoiesis if G-CSF administration were combined with suppression of erythropoiesis by red cell transfusion. To evaluate whether this hypothesis holds true C57bl mice were injected subcutaneously for 6 d with 3.75 micrograms rh G-CSF/mouse/d (150 micrograms G-CSF/kg body weight/d). Marrow CFU-S numbers showed an increase to 160% on day 2, followed by a decrease to 50% of control on day 6. Splenic and peripheral blood CFU-S increased 20-fold and 10-fold, respectively. Marrow CFU-E declined to 40% of the control value. Splenic CFU-E increased 10-fold. The increase in marrow CFU-GM numbers ranged between 140% and 180%. CFU-GM obtained from the spleen and the peripheral blood increased 60-fold and 15-fold, respectively. Regarding the CFU-S and CFU-GM a similar pattern of response was found in an experiment where rh G-CSF administration was combined with an additional red cell transfusion. These data do not provide convincing evidence for an exhaustion of haemopoietic stem cells during treatment with G-CSF. They rather suggest that an important side effect of G-CSF treatment is a release of CFU-S and progenitors from the marrow to the peripheral blood and a reseeding in the spleen.

Toxicity of liposomal 3'-5'-O-dipalmitoyl-5-fluoro-2'-deoxyuridine in mice.

Toxicities of 5-fluoro-2'-deoxyuridine (FUdR) and its liposome incorporated dipalmitoyl derivative (FUdR-dipalmitate) to mouse bone marrow, spleen, liver and ileum were compared after treatment for 6 consecutive days. The applied doses of the two formulations, which were shown earlier to have equal antitumor activity in mouse tumor models, were 600 and 2 mumol/kg respectively. When applied in these doses, toxicity to the hemopoietic system, measured as a decreases in progenitor and precursor cells of the erythroid and granuloid/macrophage lineage in bone marrow and spleen, was more severe for FUdR than for liposomal FUdR-dipalmitate. In the liver, mitotic figures, as indicators of cell division, were absent for both drugs while in control livers the number of cells in mitosis was approximately 2%. Toxicity to the ileum was more severe for liposomal FUdR-dipalmitate than for FUdR and was manifested by granulocyte infiltration, the presence of cell debris, loss of columnar epithelial cells and enlarged nuclei with prominent nucleoli in these cells. Thus, by prolonging the retention time of FUdR in vivo, using liposomes as a vehicle and FUdR-dipalmitate as a lipophilic prodrug, the dose-limiting toxicity appears to shift from bone marrow to the gastrointestinal tract.