Production of macrophage colony-stimulating factor by adult murine parenchymal liver cells (hepatocytes).

The activity of macrophage colony-stimulating factor (M-CSF) was found in the culture supernatant of mouse parenchymal liver cell fractions in a bone marrow colony-forming assay. The activity of an M-CSF-like substance purified by a four-step procedure was neutralized by goat anti-mouse M-CSF antiserum. M-CSF mRNA was detected in cellular RNA prepared from cultured parenchymal liver cell fractions by Northern blot analysis and also in cultured parenchymal liver cells by in situ hybridization. These results indicate that parenchymal liver cells have the capacity to produce M-CSF. We discuss the role of M-CSF in hematopoiesis, the immune response, and other biological phenomena.

Effects of recombinant granulocyte colony-stimulating factor (rG-CSF) and recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) on acute radiation hematopoietic injury in mice.

We have attempted to evaluate in vivo effects of granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on acute radiation hematopoietic injury in mice. BDF1 mice, irradiated with 7.5-Gy x-rays, were injected i.p. twice daily for 10 days with 10(5) U recombinant human G-CSF, 3.75 x 10(5) U recombinant murine GM-CSF, or a combination of both. G-CSF significantly enhanced the recovery of not only peripheral leukocytes but also platelets and hematocrit on days 14 and 21 after irradiation. GM-CSF significantly enhanced the recovery of platelets on day 14 and peripheral leukocytes on day 21. G-CSF markedly enhanced the recovery of spleen colony-forming units (CFU-S), colony-forming units in culture (CFU-C), erythroid burst-forming units (BFU-E), and megakaryocyte colony-forming units (CFU-Meg) both in bone marrow and in the spleen. GM-CSF significantly enhanced the recovery of CFU-Meg in bone marrow on day 14. We found synergistic effects between G-CSF and GM-CSF on CFU-S, CFU-C, and CFU-Meg in the spleen on day 14, although we found antagonistic effects between G-CSF and GM-CSF on CFU-S and CFU-C in bone marrow on day 7, and on platelet counts on day 7. These results indicate that the administration of recombinant G-CSF and GM-CSF may be useful in accelerating hematopoietic recovery in patients with acute radiation hematopoietic injuries.

Enhancement of the action of colony stimulating factor (CSF) by soluble component(s) of erythrocytes in mouse bone marrow cells cultures.

Rat erythrocytes, separated from other blood cells by SE-cellulose chromatography, were lysed by exposure to hypotonic solution, dialyzed and ultracentrifuged. The supernatant contained a substance which enhanced the activity of colony stimulating factor (CSF) in soft agar cultures of granulocyte-macrophage progenitor cells (CFU-C) from normal mouse bone marrow. The growth-enhancing effect of the erythrocyte factor was observed when mouse L-cell conditioned medium was used as the CSF source and also when serum from endotoxin-treated mice or from mice undergoing graft-versus-host reaction was used as the stimulant. The erythrocyte factor effect was also detected by 3H-thymidine uptake of bone marrow cells in liquid cultures. These results suggest that the effect of the erythrocyte factor on CSF is not restricted to CSF from a specific source nor to semi-solid agar cultures.

Radioprotection of mice by a single subcutaneous injection of heat-killed Lactobacillus casei after irradiation.

Treatment of whole-body gamma-irradiated mice with a preparation of Lactobacillus casei (LC 9018) immediately after irradiation caused a sustained increase in serum colony-stimulating activity which was followed by an enhanced repopulation of granulocyte-macrophage colony-forming cells in the femoral marrow and spleen. Numbers of blood leukocytes, erythrocytes, and platelets were increased earlier in the treated mice than in the controls, and the survival rate was elevated significantly. The radioprotective effect was dependent on the dose of LC 9018 as well as on the dose of radiation. These results demonstrate the value of LC 9018 for the treatment of myelosuppression after radiotherapy or radiation accidents.

Increased expression of intracisternal A-particle RNA in regenerated myeloid cells after X irradiation in C3H/He inbred mice.

Myeloid Cells after X Irradiation in C3H/He Inbred Mice. Myeloid leukemia cells were derived from regenerated hematopoietic cells damaged by sublethal doses of X radiation in C3H/He inbred mice. We previously found that within the genome of the myeloid leukemia cells, a retrotransposon, the intracisternal A-particle (IAP) element, is integrated. Levels of IAP RNA, the source of cDNA for the integration, were analyzed quantitatively in C3H mice. Higher levels of IAP transcripts were observed in normal cells, particularly in hematopoietic cells, from C3H/He mice, than in those from C57BL/6J and STS/A mice. In the C3H/He mice, an approximately twofold increase in IAP RNA was found in the regenerated spleen and bone marrow cells at 5 days and from 12 to 90 days after whole-body X irradiation. In addition, an increased level of IAP RNA was observed in all the myeloid leukemia cells derived from C3H/He mice. This suggests that the elevated levels of IAP RNA in the regenerated hematopoietic cells after irradiation contribute to the increase in retrotransposition of IAP found in myeloid leukemia cells from C3H/He mice.

A colony-stimulating factor produced by mouse L . P3 cells in the presence of tunicamycin or 2-deoxy-D-glucose.

L . P3 cells grown in serum-free synthetic medium produced a colony-stimulating factor (CSF: a sialoglycoprotein stimulating proliferation of granulocyte-macrophage progenitor cells). Addition of tunicamycin (2 microgram/ml) or 2-deoxy-D-glucose (10 mM) to the culture neither decreased the yield of CSF relative to the cell number grown nor induced heterogeneity of the produced CSF. However, CSF produced in the presence of tunicamycin (CSF-tm) was about 23 percent smaller in its molecular weight than normally produced CSF (CSF-normal). Similarly, the addition of 2-deoxy-D-glucose resulted in the formation of a CSF (CSF-dGlc) which was about 16 percent smaller than CSF-normal. However, both CSF-tm and CSF-dGlc seemed to have retained sialic acid residues, because they were focused respectively at pH 4.2 and pH 3.7 upon isoelectric focusing, and both were converted to a pH 5.2 species by treatment with neuraminidase. In addition, CSF-tm was significantly less heat-stable than CSF-normal, whereas CSF-dGlc was only slightly less stable. These results suggest that complete glycosylation of the factor is not necessary for its production nor for its stimulatory action on the proliferation of myeloid stem cells, but is necessary for its maximal stabilization.

Induction of the expression of the interleukin-1 beta gene in mouse spleen by ionizing radiation.

Spleen cells freshly isolated from normal mice were irradiated with 20 Gy X rays in culture. Northern blot hybridizations revealed that expression of the interleukin-1 beta (IL-1 beta) gene was induced immediately after irradiation and was increased for 2 h thereafter. Dibutyryl cyclic AMP also caused a persistent expression of the IL-1 beta gene, although it differed from X rays in that it coincidentally induced expression of the c-fos gene, which was not induced by X rays. Activation of either protein kinase C or calmodulin also induced early expression of both IL-1 beta and c-fos. Myeloid cells collected from the spleen of mice with granulocytic leukemia were X-irradiated in culture as above. The leukemia cells responded to X rays as well as to other stimuli in the same manner as the spleen cells, except that IL-1 beta mRNA was no longer detected 30 min after irradiation while c-fos was detectable for 2 h. When the leukemia cells were irradiated twice with a 3-h interval between irradiations, the second irradiation led to prolonged expression of IL-1 beta without inducing c-fos expression. These results suggest that ionizing radiation elicits early expression of the IL-1 beta gene through a mechanism that does not involve protein kinase C or A, or the transcription factor, c-fos. Whole-body irradiation of mice with 50 Gy 137Cs gamma rays also induced IL-1 beta expression in spleen but not in bone marrow or liver, although there was a delay of several hours before it was amply expressed. Furthermore, a delay as long as 24 or 72 h was observed when the radiation dose was reduced to 8.5 or 4 Gy. The results of this in vivo study suggest that the rapidity of expression of the IL-1 beta gene is dependent on the dose of radiation, and that the cells in the body cannot respond to radiation as rapidly as cells in culture.

Constitutive overexpression of the c-fos gene in radiation-induced granulocytic leukemia in mice.

Among various myeloid leukemias which were induced by X rays in C3H/He mice (Seki et al., Radiat. Res. 127, 146-149, 1991), the three most frequent types were analyzed for myeloperoxidase, c-myc, c-myb, and c-fos mRNAs. It was shown by in situ hybridization that all the component cells were positive for myeloperoxidase mRNA in granulocytic leukemia, whereas only half the cells were positive in myelomonocytic leukemia and none in monocytic leukemia. Granulocytic leukemia was also characterized by a persistently heightened expression of c-fos, while the other two types of leukemia showed negligibly low expression of the c-fos message. By contrast, both c-myc and c-myb were expressed to a similar extent in all three types of leukemia. When fresh granulocytic leukemia cells were transferred to culture in a medium containing 0.5% fetal calf serum, c-fos mRNA was decreased rapidly during incubation. The decay of c-fos mRNA was inhibited by cycloheximide markedly but was not changed significantly by actinomycin D. In the culture containing 10% fetal calf serum, the rate of decay of c-fos mRNA was slowed down significantly. Addition of dibutyryl cyclic AMP rapidly restored the c-fos expression and kept it elevated for at least 2 h in the cultured granulocytic leukemia cells. Phorbol ester (TPA) and calcium ionophore A23187 also caused a rapid but transient c-fos expression. A transient c-fos expression was inducible by TPA in the other two types of leukemia cells and in the granulocytic leukemia cells. The results suggest that the persistent expression of c-fos is distinguished from its transient expression and is characteristic for granulocytic leukemia cells as it is for normal mature granulocytes.

Osteodystrophy in patients with chronic hepatitis and liver cirrhosis.

Bone mineral density (BMD) of the lumber vertebrae and factors related to bone metabolism were determined in patients with chronic viral hepatitis and patients with liver cirrhosis to clarify correlations between hepatic dysfunction, considered to be one of the causes of hepatic osteodystrophy, and decrease in bone mass. BMD of the second to fourth lumbar vertebrae was determined with a Lunar (Madison, WI, USA) DPX, a dual-energy X-ray absorptiometry diagnostic system. BMD was significantly lowest in patients with liver cirrhosis, followed by patients with chronic hepatitis, and healthy subjects, in this order. There was a significantly positive but weak correlation between albumin and BMD. Levels of 25(OH)D and 1,25(OH)2D were significantly lower in patients with liver cirrhosis than in those with chronic hepatitis. BMD and vitamin D were decreased in all patients whose cholinesterase (ChE) was below 0.3 delta pH. Urinary pyridinoline (Upyr) was significantly higher in the patients with liver cirrhosis, in whom bone mass was decreased, than in the patients with chronic hepatitis, whereas serum osteocalcin levels were distributed in the upper normal range in patients with chronic hepatitis and those with liver cirrhosis. There was a positive correlation between 25(OH)D and serum osteocalcin levels in patients with liver cirrhosis. These results indicate that osteogenesis is decreased and suggest that the decrease in BMD which occurs in viral liver cirrhosis, probably related to decreased, bone formation and slight promotion of bone resorption, reflects deranged hepatic function. This is the first report of Upyr and urinary deoxypyridinoline (UDpyr) determination in patients with liver cirrhosis and patients with chronic hepatitis. The negative correlation of Upyr and UDpyr with ChE is a novel finding.

Acceleration of granulocytic cell recovery in irradiated mice by a single subcutaneous injection of a heat-killed Lactobacillus casei preparation.

Flow cytometric analysis showed that the treatment of irradiated mice with a heat-killed Lactobacillus casei preparation (LC 9018) accelerated the recovery of granulocytic cell populations in peripheral blood, spleen and femur bone marrow. The recovery of the lymphocytic cell population was not accelerated while the recovery of the B-lymphocytic cell population was inhibited. Histological analysis also showed that the LC-9018 treatment markedly enhanced granulopoiesis in the spleen and bone marrow of irradiated mice. The same LC-9018 treatment significantly increased 30-day survival rates of athymic nude mice after lethal whole-body irradiation. The recovery of the granulocytic cell population in peripheral blood of irradiated athymic nude mice was also accelerated by LC-9018 treatment. Our results suggest that LC 9018 protected lethally irradiated mice from bone marrow death by enhancing granulopoiesis rather than lymphopoiesis and that the contribution of activated T lymphocytes to the enhancement of the granulopoiesis was small.

Timing in administration of a heat-killed Lactobacillus casei preparation for radioprotection in mice.

A single subcutaneous injection of a preparation of heat-killed Lactobacillus casei (LC 9018), given before or after irradiation, significantly increased the survival rate of mice that had received 8.5-Gy 137Cs whole-body gamma-irradiation. A similar radioprotective effect was observed when LC 9018 was administered within the period from 2 days before irradiation to 9 h after irradiation, the pre-irradiation treatment being slightly better than the post-irradiation treatment. Increases in the weight of the spleen and in the number of endogenous spleen colonies on days 8 and 12 after irradiation suggested that the radioprotective effect was based on enhanced recovery of hematopoietic tissues. The activity of macrophage colony-stimulating factor (M-CSF) in serum was rapidly increased by the treatment and was maintained at the elevated level for 13 days. At the same time, an increased level of M-CSF mRNA was detected in the livers of the treated mice. However, LC 9018 failed to save the lives of mice when administered 3 days after irradiation, although it increased serum M-CSF as effectively as noted above. The small advantage of the pre-irradiation over the post-irradiation treatment was not explained by the increases of metallothionein in the hematopoietic tissues of the treated mice.

Immediate-early, transient induction of the interleukin-1 beta gene in mouse spleen macrophages by ionizing radiation.

In murine spleen cells, x ray irradiation induces the expression of the IL-1 beta gene at multiple phases of the peak time. We analyzed the immediate-early phase of IL-1 beta mRNA accumulation. To determine the lineage of cells that showed the immediate response to irradiation, normal spleen cells were analyzed by Northern blotting and in situ hybridization after separation by magnetic antibodies against specific cell-surface antigens. Although most of the spleen macrophages continuously expressed a low level of IL-1 beta mRNA, a portion of the macrophage population transiently accumulated large amounts of IL-1 beta message immediately after irradiation. A macrophage-like leukemia cell line that resembles these inducible macrophages was identified. A similar immediate-early and transient increase in the IL-1 beta mRNA level occurred when cultured spleen cells were irradiated with a low dose (3 Gy) of x rays. In contrast, the x ray-inducible expression of the IL-1 beta gene was immediate and continuous, not transient, in spleen cells from whole-body irradiated mice. Results of the run-on transcription assay and the determination of the decrease in the message using cultured spleen and macrophage-like leukemia cells indicated that x ray irradiation appears to activate the transcription of the IL-1 beta gene and partially stabilize the message. The results show that the x ray-induced immediate-early accumulation of IL-1 beta mRNA is regulated at both the transcriptional and post-transcriptional levels in an as yet unidentified population of spleen macrophages.

Expression of IL-1 beta mRNA in mice after whole body X-irradiation.

IL-1 beta is a stimulator of hematopoietic and inflammatory systems, and also acts as a radioprotector. After whole-body exposure to sublethal doses of ionizing radiation, the IL-1 beta mRNA level in spleen cells increases for a short time prior to regeneration of the spleen. We analyzed spleen cells of C3H/He mice after whole-body irradiation with 3 Gy x-rays to determine the cause of this short-term increase in the transcription level. An increase in the level of the message in spleen cells, found by Northern blot hybridization, reached its peak 5 to 7 days after irradiation. There was a low correlation between the curves of the mRNA level and the ratio of monocyte/macrophage lineage cells; a typical source of the message. Spleen macrophages that produce a large amount of the message were found 7 days after irradiation in an in situ hybridization experiment in which heterogeneous spleen cell populations were used. In contrast, spleen cells had no detectable levels of macrophages rich in IL-1 beta mRNA before and 17 days after irradiation. Additionally, the population of message-rich cells was 9.4% of the total number of monocytes/macrophages in the spleen. These results suggest that the short-term increase in IL-1 beta mRNA is a result of the heterogeneous differentiation of a subpopulation of spleen macrophages before regeneration of the spleen.

Rhabdomyolysis and myocardial damage induced by palytoxin, a toxin of blue humphead parrotfish.

A 55-year-old man had rhabdomyolysis and myocardial damage induced by palytoxin. Weakness and myalgia of four extremities occurred five hours after eating a fish. Rhabdomyolysis developed and the serum creatine phosphokinase (CK) was elevated to 40,000 IU/l on the 3rd day. Gastric lavage with activated charcoal and forced mannitol-alkaline diuresis therapy were performed. The patient recovered with no complication such as renal failure. In this case, palytoxin was suggested to induce myocardial damage which was demonstrated by an elevation of the myosin light chain level and a change in electrocardiogram.

[Mass screening for osteoporosis in Nansei--the Nansei Study (the second report)].

We performed screening for osteoporosis for the early detection of a decrease in the bone mineral density. The subjects consisted of 852 inhabitants (308 males and 544 females) aged 40 years or more in Nansei (population, 12, 107) in Mie Prefecture. Interviews, measurement of height and body weight, blood examination, and determination of the bone mineral density by the MD method were performed. In addition, a questionnaire on diet was carried out. The bone mineral density was decreased in 76 subjects (8.9%), of whom females were the majority. Height and body weight were significantly lower in the group with decreased bone mineral density than in the group with normal bone mineral density. The serum calcium (Ca), alkaline phosphatase (ALP), and inorganic phosphorus (P) levels were similar in the two groups. Concerning the family profile, the percentage of subjects living alone was significantly higher (p < 0.05) in the group with decreased bone mineral density (13.5%) than in the group with normal bone mineral density (3.6%). Screening for osteoporosis is still in the trial stage and involves various problems that require further studies. As subjects for screening, females before, during, and immediately after menopause are important.

Efficacy of sucralfate in the prevention of recurrence of peptic ulcer--double blind multicenter study with cimetidine.

A double blind, multicenter, six-month maintenance study was performed to assess the efficacy and safety of sucralfate (S), cimetidine (C), and their combination (S + C) in the prevention of peptic ulcer recurrence in a six-month observation period. 127 patients with gastric ulcer (GU) (group S: 39, group S + C: 48, group C:40) and 103 patients with duodenal ulcer (DU) (group S: 35, group S + C: 36, group C:32) were available for statistical analysis. Six to 12 months after healing of GU, the cumulative recurrence prevention rates by Kaplan-Meier method were 89.7----80.3% in group S, 97.6----72.6% in group S + C, 84.5----44.6% in group C. In duodenal ulcer they were 75.9----41.9% in group S, 87.8----47.7% in group S + C, 80.8----40.5% in group C. These results indicate that maintenance therapies of S and S + C are effective and safe for the prevention of gastric ulcer recurrence.