Age-related changes of urinary nitrite/nitrate excretion in normal children.

We measured the urinary excretion of nitrite/nitrate, stable metabolites of nitric oxide, using the brucine method in 90 healthy normal children (47 boys and 43 girls), aged from 1.0 to 17.1 years, to establish the age-related normal range in children. The urinary nitrite/nitrate excretion was highest in the youngest children and decreased in an age-dependent manner to reach constant levels at about 12 years of age in both sexes. The data may be useful in identifying sick children with abnormal nitric oxide production.

Urinary leukotriene E4 after exercise challenge in children with asthma.

To assess the role of sulfidopeptide leukotrienes in the pathogenesis of exercise-induced asthma (EIA), the urinary levels of leukotriene E4 (LTE4), a metabolite of LTC4 and LTD4, were measured by RIA before and after exercise in 13 children with EIA and 10 healthy children. Mass spectrometry was used to confirm the presence of LTE4 in urine and the specificity of the RIA. There was no significant difference in the urinary LTE4 levels before exercise between the children with asthma and healthy children (109 [21 to 265] versus 122 [45 to 156] pg/mg of creatinine; median and range). Urinary LTE4 levels increased significantly after exercise in the children with EIA (from 109 [21 to 265] to 196 [40 to 655] pg/mg of creatinine; median and range; p less than 0.05) but not in the healthy children. The children with asthma demonstrated no significant correlation between the LTE4 level after exercise and the degree of bronchoconstriction, as revealed by the maximal percent fall in the peak expiratory flow rate. Taken together with a recent study that pretreatment with a potent and selective LTD4 antagonist markedly attenuated EIA, our findings suggest that sulfidopeptide leukotrienes may play some role in the pathogenesis of this type of asthma with other factors also being involved in determining the overall airway response.

Agranulocytosis following infectious mononucleosis.

A girl developed acute agranulocytosis (45/mm3), 37 days after the onset of infectious mononucleosis. The bone marrow showed myeloid hyperplasia with maturation arrest and erythroid hypoplasia. A normal amount of colony forming units of granulocytes and macrophages (CFU-GM) colonies with a relative high number of clusters was observed. Neither anti-neutrophil antibodies nor circulating inhibitors of colony growth were found in serum. Granulocyte and macrophage colony stimulating factor (GM-CSF) activity in the patient's serum rose at this time. The agranulocytosis lasted 5 days and her clinical state soon improved. These results suggested that agranulocytosis was presumably not due to serum factors, including auto-antibodies and/or suppressive substances, and that Epstein-Barr virus (EBV) had some direct or indirect effect on the marrow cells of the myeloid series.

Further studies on the biological activities of the CFU-S inhibitory tetrapeptide AcSDKP. II. Unresponsiveness of isolated adult rat hepatocytes, 3T3, FDC-P2, and K562 cell lines to AcSDKP. Possible involvement of intermediary cell(s) in the mechanism of AcSDKP action.

Because the molecular mechanisms of the tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP; an inhibitor of spleen colony-forming unit [CFU-S] DNA synthesis) are difficult to study on bone marrow due to the scarcity of CFU-S in this tissue, we sought a pure cell population responsive to the molecule in vitro. Although growth factor-stimulated DNA synthesis in primary culture of hepatocytes and Balb/c 3T3 cells can be inhibited by transforming growth factor beta (TGF beta) and interferon alpha/beta (IFN[alpha/beta], respectively, neither hepatocytes nor 3T3 cells were found to be sensitive to AcSDKP. DNA synthesis in stimulated murine FDC-P2 cell lines and in human K562 cell lines also remained unchanged after exposure to the tetrapeptide. The fact that hepatocytes do respond in vivo to AcSDKP implies the existence of intermediary cell(s) involved in AcSDKP action in vivo that are lacking in hepatocyte culture. Whether intermediary cell(s) are implicated in the inhibitory action of AcSDKP on CFU-S entry into DNA synthesis is now being investigated.

Radioprotection of hemopoiesis conferred by Acanthopanax senticosus Harms (Shigoka) administered before or after irradiation.

Acanthopanax senticosus Harms (Shigoka) extract has been observed to have radioprotective effects on hemopoiesis of irradiated mice (CBA/olac) when administered before or after irradiation by 60Co. The mechanisms of action were explored by studying the following parameters: 1) survival after lethal doses of irradiation; 2) recovery of spleen colony-forming units (CFU-S); 3) protective effect on endogenous CFU-S; and 4) effect on self-renewal of CFU-S. 1) The 30-day mortality due to bone marrow failure after irradiation was significantly reduced in Shigoka-treated mice. The maximum effect of the extract (80% survival) at a dose of 5 mg was observed when given intraperitoneally 24 h before a lethal dose of irradiation (9.5 Gy) and it was still effective when administered as late as 12 h after irradiation (30% survival). The radioprotective effect of Shigoka extract given before irradiation was not due to an increase in the total number of CFU-S but probably due to quiescent CFU-S that entered DNA synthesis (S-phase) 24 h after the injection of Shigoka extract. 2) Recovery of CFU-S in mice given the extract within 15 min after 4.75 Gy of whole body irradiation was also enhanced, especially in the spleen. The number of CFU-S in the bone marrow 24 h after irradiation showed a marked decrease. It returned to normal values in the bone marrow at day 11 in Shigoka-treated mice and at day 15 in nontreated irradiated mice. CFU-S in the spleen recovered more rapidly with an overshoot from days 7 to 21 in the Shigoka-treated mice, whereas in control irradiated mice it did not reach the normal value until day 21. 3) Nine days after sublethal doses of irradiation (7.0 Gy) the number of endogenous spleen colonies (endogenous CFU-S) was highest in mice given the extract 24 h before irradiation. The extract administered as late as 24 h after irradiation also resulted in an increase of the endogenous CFU-S. 4) The number of CFU-S in each 9-day endogenous CFU-S was 73.9 +/- 7.2 per nodule in treated mice and 0.2 +/- 0.1 per nodule in irradiated control mice, which suggests increased self-renewal of CFU-S in Shigoka-treated groups. These results suggest that the radioprotection conferred by Shigoka extract results from enhanced stimulation of CFU-S not only toward proliferation but also toward CFU-S self-renewal. These two phenomena could explain the protective effects of Shigoka extract administered even after irradiation.

Establishment of a common acute lymphoblastic leukemia cell line (LC4-1) and effects of phorbol myristate acetate (PMA) on the surface antigen expression of the cell line.

A common acute lymphoblastic leukemia (ALL) cell line, designated LC4-1, was established from peripheral blood mononuclear cells of a patient with acute non-T-cell ALL. LC4-1 cells were characteristically positive for Ia, B4, and common ALL antigens (CALLA), but negative for B2, Tac, T3, T4, T8, T11, and M1 antigens and E-rosette formation. Approximately 30% of LC4-1 cells expressed detectable amounts of B1 antigens. LC4-1 cells expressed neither Epstein-Barr-virus-associated nuclear antigen (EBNA), cytoplasmic immunoglobulins (cIg) nor surface immunoglobulins (sIg). Gene rearrangements had already occurred in LC4-1 in the D-J region of immunoglobulin heavy chain genes, but not in T-cell receptor (beta-chain) genes, suggesting that LC4-1 is a progenitor cell line of B-cell lineage earlier than pre-B-cells. The expression of cell surface antigens of LC4-1 was changed by treatment with 4-phorbol 12-myristate 13-acetate (PMA) (0.1 ng/ml) for 2 days. Before treatment with PMA, about 98% of LC4-1 cells were positive for B4, CALLA, and Ia. However, following treatment they lost CALLA expression without any change in expression of Ia and B4. There was no change in B1-positive population. The change in surface antigens on LC4-1 cells seems to be due to differentiation induced in the cells by PMA. These results support the hypothesis that CALLA is a differentiation antigen and suggest one possible differentiation pathway for pre-B-cells.

Colony-stimulating factor and leukemia cell-growth factor produced by a murine endothelial cell line, MKM-O.

A murine cultured cell line (MKM-O) was established from a tumor of a BALB/C (nu/nu) mouse that had been subcutaneously inoculated with human hepatoma tissue fragments in the same location. The MKM-O cell line was proven to be of endothelial origin by morphological examinations and positive staining with fluorescein-labeled antibody against factor VIII antigen. MKM-O cells grown in vitro produced a colony-stimulating factor (CSF) and a leukemia cell-growth factor (LC-GF). CSF from MKM-O acted on both human and murine bone marrow-derived granulocytes and macrophage colony-forming units (CFU-GM). The activity of LC-GF on human leukemia cell lines (HL-60, HSB-2, CEM, DAUDI and K562) was demonstrated both in conditioned medium (CM) of MKM-O and of rat vascular endothelial cells. The CM of MKM-O also demonstrated a limited growth promoting activity against the mononuclear cells from human cord blood and improved their survival, suggesting that endothelial cells play an important role in the proliferation and differentiation of blood cells.

Low burst promoting activity production by phytohemagglutinin-stimulated mononuclear cells of cord blood.

Burst-promoting activity (BPA) produced by phytohemagglutinin(PHA)-stimulated cord blood mononuclear cells (MNC) was examined using the two-stage cell culture assays. Burst-promoting activity was measured as the increase in the number of early erythroid progenitor cells in 2-day incubation of peripheral blood MNC with or without the conditioned medium of PHA-stimulated MNC (PHA-LCM). Burst-promoting activity in PHA-LCM of cord blood was significantly lower than that of adult blood (37 +/- 13 versus 105 +/- 19%, mean +/- SD, p less than 0.01). No elevation of inhibitors to the erythroid colony growth was noted in PHA-LCM of cord blood. In contrary, the response of cord blood MNC to PHA was similar to that of adult blood MNC, as determined by colony-stimulating activity production and cell proliferation. These results showed that burst-promoting activity production by PHA-stimulated MNC of cord blood was lower than that of adult blood.

Effect of colony-stimulating factor-producing tumor on hemopoiesis in splenectomized mice.

The colony-stimulating factor-producing BMA-1 tumor was transplanted into splenectomized mice. These mice showed as much granulocytosis as non-splenectomized mice bearing the tumor. The increase in the number of peripheral leukocytes induced per tumor weight was similar in the two groups. The hemopoietic cell population in the central bone marrow (tibia) was not increased by splenectomy. The total numbers of spleen colony-forming cells and granulocyte-macrophage colony-forming cells in the whole body of tumor-bearing splenectomized mice were 29% and 58% of those in the tumor-bearing non-splenectomized mice, respectively. The expansion of hemopoiesis to the peripheral bone marrow in tumor-bearing splenectomized mice was confirmed by histological examination of the tail bone. These results suggest that the lack of increase in the bone marrow cellularity is due to the space limitation, and that this is well compensated for by a great expansion of hemopoietic marrow to the peripheral bones in splenectomized mice.

Enhanced granulopoiesis in mice transplanted with colony-stimulating factor-producing BMA1 tumor.

Inoculation of BMA1 cells into BALB/c nude mice formed tumors (BMA1 tumor) that were transplantable into ddY mice, and induced marked granulopoiesis in vivo. Histological study revealed that the tumor was a fibrosarcoma, some parts of which were calcified, and consisted of hemopoietic foci surrounded by adipose tissue. This tumor was regarded as producing CSF in vivo as well as in vitro, since CSF activity was detected in sera of the tumor-bearing mice and tumor extract. Granulopoiesis and splenomegaly developed, associated with an increase of stem cells in the spleen. The number of CFUc and CFUs in the spleen increased to about 91 times and 21 times those of control mice, respectively, whereas the number of stem cells in the tibia did not change significantly. The number of peripheral leukocytes increased to 15 times that of normal mice and amounted to 78% of matured granulocytes. After tumor resection these hematological changes were reversed. The findings suggest that the granulopoiesis in BMA1 tumor-bearing mice may be induced by CSF produced by BMA1 tumor and that the spleen may be a direct target organ of the excessive amount of CSF.

Effects of bacterial lipopolysaccharide on the production of colony-stimulating activity in C3H/HeJ mouse long-term bone marrow cultures.

Bacterial lipopolysaccharide (LPS)-induced colony-stimulating activity (CSA) in murine long-term bone marrow culture system was investigated. Bone marrow culture cells of LPS-nonresponsive C3H/HeJ mice responded to LPS in terms of CSA production as efficiently as bone marrow culture cells of LPS-responsive C3H/slc mice. On the other hand, both peritoneal macrophages and bone marrow macrophages from C3H/HeJ mice did not produce CSA in vitro after treatment with LPS. Percoll density gradient separation of adherent layer cells in bone marrow cultures showed that two cell populations were present. One population was nonspecific esterase positive, productive of high CSA to LPS stimulation and light density cells, the other population was nonspecific esterase negative, productive of low CSA to LPS stimulation and high density cells, and CSA production stimulated by LPS in C3H/HeJ mice bone marrow culture cells was mainly attributed to the latter population of cells. These results suggest that CSA production stimulated by LPS in C3H/HeJ mice is regulated by different cell populations, respectively in vivo and in vitro.

Survival and repopulation of irradiated 'pre-CFU-c' in mice.

Supernatants of murine bone-marrow cultures contain a colony-promoting factor (CPF) which increases the number of granulocyte and macrophage colonies in semi-solid agar cultures in the presence of colony-stimulating factor (CSF). Incubation of bone-marrow cells with CPF results in an increase in the number of granulocyte/macrophage progenitor cells (CFU-c) and the CPF-responsive cells may be younger than the CFU-c. We have investigated the radiosensitivity and the pattern of the recovery after irradiation of CPF-responsive cells. We found that the radiosensitivity of CPF-responsive cells was significantly lower than those of CFU-c, burst-forming units-erythroid (BFU-e) and pluripotent stem cells in vivo (CFU-s) and in vitro (CFU-mix). The CPF-responsive cells remained subnormal even at 28 days after irradiation of the mice, a time when the CFU-s and CFU-c had recovered completely. Therefore the CPF-responsive cells may constitute a separate compartment, namely 'pre-CFU-c', in the maturation sequence of granulopoiesis, and this maturation of the 'pre-CFU-c' to CFU-c seems to be highly stimulated after irradiation to counterbalance the influx from CFU-s.

Effect of carbon particles on the recovery of bone marrow stem cells after irradiation in LPS-resistant C3H/HeJ mice.

Effect of RES-blockade on bone marrow cells was studied serially after irradiation in LPS-resistant mice. Injection of carbon particles reduced damage and accelerated recovery of marrow hemopoietic stem cells, indicating that LPS-resistant mice can react normally to RES-blockade.

Colony promoting activity (CPA) produced in long-term culture of murine bone marrow cells.

The differences between colony promoting activity (CPA) and colony stimulating activity (CSA) in the culture media of murine long-term bone marrow cultures (LTBMC) were demonstrated and the role of adherent cells and nonadherent cells in the production of CPA was studied in this culture system. Supernatant harvested from intact continuous marrow cultures showed high CPA but contained no CSA. Assayable CSA was detected in concentrated supernatant. However, there was no significant relationship between levels of CPA and CSA in the supernatant. When adherent cells and nonadherent cells from LTBMC were separately cultured, CPA was detected in the conditioned medium of adherent cells but not in that of nonadherent cells. The CPA level in LTBMC was related inversely to the number of nonadherent cells and addition of nonadherent cells to adherent cell cultures reduced the level of CPA. Conditioned medium of nonadherent cells showed no inhibitory activity of CPA. These results indicate that CPA is produced by bone marrow adherent cells and that it may be consumed by myeloid progenitor cells in nonadherent cells.

Effects of bacterial lipopolysaccharide and X-irradiation on the production of colony-stimulating factor and the maintenance of granulopoiesis in bone marrow culture.

Effects of bacterial lipopolysaccharide (LPS) and X-irradiation on CSF production and granulopoiesis in long-term bone marrow cultures were studied. Levels of colony-stimulating factor (CSF) increased soon after the refeeding of the culture, but the activity was undetectable at day 7. Addition of LPS induced a significant increase in CSF levels in the culture, followed by an elevated granulopoiesis. The increase in CSF levels was suppressed when culture medium that had been harvested at refeeding on day 7 was added. Although irradiation did not increase CSF production, granulopoiesis was markedly stimulated shortly after irradiation. Thus granulopoiesis in long-term bone marrow culture may also be regulated by humoral factors such as CSF, and the culture system may represent the in vivo response to haemopoietic stimuli.