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Interleukin-6 (IL-6), a pleiotropic cytokine, is postulated to be involved in the pathogenesis of sick euthyroid syndrome, although the direct in vitro effects of IL-6 on human thyroid function are controversial. Because IL-6 signal can be transduced when the complex of IL-6 and soluble IL-6 receptor (sIL-6R) binds to gp 130, an IL-6 signal transducer, we studied the effects of IL-6 and sIL-6R on thyroid function, using human thyroid follicles obtained from patients with Graves' disease. IL-6 alone had no inhibitory effect on TSH-induced thyroid function (125I incorporation and organic 125I release), even at supraphysiological concentrations. However, in the presence of physiological concentrations of sIL-6R (100 ng/ml), IL-6 inhibited thyroid function dose dependently and completely, accompanied with the decreased ratio of 125I-T3/125I-T4 not only in the thyroid follicles but also in the culture medium. Thyroid follicles did not secrete sIL-6R but produced IL-6 constitutively. Consistent with these findings, sIL-6R inhibited thyroid function slightly at high concentrations. Furthermore, RT-PCR analyses revealed that human thyroid follicles expressed the messenger RNAs for IL-6 and gp130 but scarcely messenger RNA for IL-6R. These in vitro findings suggest that IL-6 alone hardly affects thyroid function in thyroid follicles in which IL-6R gene is scarcely expressed. However, because sIL-6R is present abundantly in serum, IL-6 in vivo would be capable of inhibiting the synthesis and release of T4 and, to a greater extent, T3 from the thyroid gland. These in vitro findings are at least partly related to the development of sick euthyroid syndrome.

Interleukin-6 (IL-6) has been postulated as a possible mediator of bone loss after estrogen deficiency, and its signal is transduced via glycoprotein 130 (gp130) after binding IL-6 receptor (IL-6R) in the membrane of target cells. In this study, the expression of IL-6R and gp130 in bone marrow cells during osteoclast differentiation was investigated. Mouse bone marrow cells were isolated and cultured with or without 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]. During the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs), IL-6R and gp130 expression in the mononuclear cells, stromal cells, and TRAP-positive MNCs were quantitated, using a laser cytometer with a fluorescence confocal microscopy. With 1,25(OH)2D3 stimulus, the level of gp130 significantly increased, but that of IL-6R did not in the stromal cells. In contrast, the levels of both gp130 and IL-6R significantly increased in the mononuclear cells by the treatment with 1,25(OH)2D3. The high expression of both gp130 and IL-6R was observed in the TRAP-positive mononuclear cells. Moreover, both IL-6R and gp130 were expressed in the TRAP-positive MNCs and isolated murine osteoclasts. The treatment of TRAP-positive MNCs with IL-6 caused enhancement of the resorbing activity in a dose-dependent manner, and the effect was prevented by a neutralizing antibody against IL-6R. These data suggest that gp130 and IL-6R, as well as IL-6, are involved in the formation and activation of osteoclasts.

To clarify the biological behavior of micromegakaryocytes in myelodysplastic syndrome (MDS), the relationship between the morphological classification and the ploidy of megakaryocytes was studied in bone marrow aspirates obtained from patients with MDS and from normal controls. The morphology was determined according to Feinendegen's classification, which is considered to reflect megakaryocytic maturation, and the ploidy was determined by microcytofluorometry, using 4',6-diamidino-2-phenylindole (DAPI) staining after the removal of Wright-Giemsa stain. Most micromegakaryocytes (i.e., megakaryocytes20 microns in diameter) in MDS were morphologically mature, as were those in the normal controls. The peak micromegakaryocytic ploidy was 4N or 8N, whereas that of the megakaryocytes in normal controls was 16N. These findings indicated that the micromegakaryocytes in MDS were morphologically mature but had impaired polyploidization.

"In this paper I tried to show how the ageing of the population influences the change in the growth of employment, employment structure, the savings ratio, economic growth and the cost of social security [in Sweden]. In the latter part of the paper I suggested a close correlation between the average marriage age of women, the total fertility rate and the work participation ratio of women." (SUMMARY IN ENG)

The patient was a 76-year-old female who had been referred to our hospital because of fever of unknown origin on October 15, 1987. On admission, the body temperature was 38.6 degrees C and atonic palsy of the left upper limb was noted. Abnormal laboratory findings included CRP5+, an increase in LDH, Hb 7.9 g/dl. The cause of the fever could not be identified. The fever did not respond to various treatment. The patient developed DIC in late October and died on November 5. In autopsy histological examination revealed tumor cells in the vessels of the generalized organs. A diagnosis of neoplastic angioendotheliosis (NAE) and immunohistologically B lymphoma was made. We reviewed the literature on 37 Japanese cases of NAE. The cases, consisting of 19 males and 18 females, were aged 37-87 years with a median value of 60 years. The symptoms observed during the course were most frequently mental or neurological symptoms and fever, and rash was uncommon. Laboratory findings were non-specific and biopsy was needed for definitive diagnosis. By autopsy, lesions were noted more frequently in the brain, kidneys, and lungs, and the findings in the skin were indeterminate. These observations suggest that when NAE should be considered, kidney, lung or skin biopsy should be performed for definitive diagnosis.

The relation between polyploidy and morphological classification of human megakaryocytes was studied in bone marrow aspirates from five normal individuals. On a Wright-Giemsa stained smear, megakaryocytes were morphologically classified into four groups according to a modification of Feinendegen's classification which is considered to reflect megakaryocyte maturation. The DNA of the morphologically classified cell is measured by microcytofluorometry using DAPI (4',6-diamidino-2-phenylindole) staining after removing the Wright-Giemsa stain. Most of the normal megakaryocytes were classified into type III (mature megakaryocytes) and the maximum peak in population of the megakaryocyte ploidy was observed at 16N. In each individual, the ploidy showed a similar pattern regardless of the classification. These findings suggest that the development of ploidy depends on a factor different from the one that determined the megakaryocyte maturation of cytoplasm and the ploidy is determined at the level of a megakaryocyte precursor or the most juvenile megakaryocyte.

Hemoglobin (Hb) synthesis in colony-forming-unit erythroid (CFU-E) colonies from the bone marrow of 10 normal subjects and 6 patients with myelodysplastic syndromes (MDS) was studied using microcytofluorometry. Hb content was determined utilizing a photochemical reaction in which the intracellular Hb is converted into fluorescent porphyrin. Briefly, the CFU-E in plasma clots was assayed according to Tepperman's method. Bone-marrow mononuclear cells were then dispensed onto petri dishes containing the plasma clots. The cells in plasma clots were cultured for seven days and then air-dried. The samples were then fixed with pure methanol. Thereafter, the dishes were exposed to ultraviolet light (lambda = 405 nm) in the presence of an SH-donor (0.2 M mercaptoethylamine hydrochloride). The intensity of porphyrin fluorescence was measured using a microcytofluorometer. When the photochemical reaction was carried out for 50 min, the intensity of fluorescence was found to be proportional to the mean corpuscular Hb (MCH) level, suggesting that the intracellular Hb level can be determined as a function of the intensity of this fluorescence. The number of CFU-E colonies in MDS patients was smaller than that in normal subjects. In addition, intracolony Hb level was markedly lower in the MDS cases than in normal subjects. Our findings suggest that the maturation process of CFU-E is impaired in MDS patients.

Massive bone marrow necrosis was seen in a 42-year-old male with acute leukemia. In December, 1988, on admission, laboratory data revealed pancytopenia and a high level of serum LDH and ALKP. Bone marrow aspiration resulted in dry-tap and showed bone marrow necrosis in the bone marrow biopsy specimen. A bone marrow scintigraphy with 111In faintly visualized the bone marrow but visualized area was expanded in the extremities compared with normal subjects. The second bone marrow biopsy showed proliferation of blasts. In the middle of March, blasts began to appear in peripheral blood. The blasts were cytochemically negative for POX, Es, PAS, AcP, TdT and had surface markers CD3-, CD19-, CD33-, CD13-, LCA-, HLA-DR-. Even by investigation on rearrangement of the immunoglobulin heavy chain region, an origin of the blasts could not be determined. In April, the number of blasts in peripheral blood increased and hepatosplenomegaly developed rapidly. Therefore, he was put on the chemotherapy with vincristine and prednisolone, but he died of cerebral hemorrhage. The autopsy revealed widespread bone marrow necrosis. It has rarely been reported that massive bone marrow necrosis is found prior to the occurrence of acute unclassified leukemia.

4',6-Diamidino-2-phenylindole (DAPI) staining was applied to measuring nuclear DNA contents of single cells previously identified on a Wright-Giemsa stained smear. The smear was first photographed and the location of each cell was mapped. The smear was then destained with 50% acid ethanol and absolute methanol, and restained with DAPI (pH 7.4) at 4 degrees C. Blue fluorescence was observed in the nucleus, and the intensity of this fluorescence was proportional to the amount of DNA, with the lowest coefficient of variation of 3.6% in the case of staining the peripheral lymphocytes for 30 min. This method is useful for measuring DNA contents in heterogeneous cell populations.

A 35-year-old man was admitted to our hospital because of lumbago on March 25, 1988. On admission white blood count was 1,200/microliters with neutrophils of 9% and lymphocytes of 91%, hemoglobin level was 11.2g/dl and platelet count was 55 x 10(3)/microliters. Bone marrow smear showed 77% leukemic cell including non-specific or specific esterase-positive cells. Chest X-rays showed the presence of mediastinal tumor and diffuse reticular shadows. A diagnosis of ANLL was made and a hematological remission was obtained after one course of combination chemotherapy consisting of BH-AC, daunorubicin and prednisolone, but the enlarged mediastinal tumor and pulmonary infiltration worsened rapidly followed by marked dyspnea. This radiographic abnormal shadow was confirmed to be leukemic infiltration from the finding of transbronchial lung biopsy. We hesitated to give systemic chemotherapy because he also had had liver abscess. Accordingly we performed BAI of ACNU at a dosage of 150 mg which led to a dramatic improvement in dyspnea. 60Co therapy was performed on the mediastinal tumor. On May 30, when he had a relapse, he was unsuccessfully treated with systemic chemotherapy. The leukemic cells invaded most of the organs and the patient died on July 19, 1988. It is likely that BAI of ACNU for leukemic pulmonary infiltration was effective.

A 53-year-old woman was admitted to our hospital on Nov. 16, 1987, because of general fatigue. On admission, she had hepatosplenomegaly and her peripheral blood profile showed a white blood cell count (WBC) of 309 x 10(3)/microliters with immature neutrophils, a hemoglobin level (Hb) of 7.6 g/dl, platelet count (PLT) of 536 x 10(3)/microliters, neutrophilic alkaline phosphatase (NAP) score of 44. Both Vitamin B12 and LDH levels were high. The bone marrow showed marked myeloid hyperplasia. In a cytogenetic study, Ph1 was found in 3 of 8 metaphases and Ph1 with an additional abnormality of 8 trisomy was noted in 5 of 8 metaphases. She was diagnosed as having chronic myelogenous leukemia (CML) and treated by i.m. injection of interferon (IFN)-alpha at a daily dose of 6 x 10(6) U. Administration of IFN-alpha induced fever for a few days. WBC, PLT count and LDH level gradually decreased, and the NAP score and hepatosplenomegaly improved. She achieved remission in February, 1988. Administration of IFN-alpha was stopped in April, 1988, when the bone marrow showed hypocellularity and normal karyotype. She was treated with 20 mg of prednisolone daily from May until August, because of progressive pancytopenia. She had received no treatment until July, 1989. In May, 1989, the bone marrow again showed myeloid hyperplasia and Ph1 was found in all cells analyzed. Therefore, we resumed IFN-alpha treatment. It is interesting that remission of CML continues for more than one year after discontinuation of IFN-alpha in this case.

Reported is the second documented case in Japan of acute leukemia with Klinefelter's syndrome. The patient, a 52-year-old male, was admitted to hospital on July 9, 1983, after complaining of a fever and general malaise. The hematological examinations revealed a WBC count of 14,300/cmm with 32% being leukemic cells. Bone marrow aspiration disclosed a nucleated cell count of 687,000/cmm with 41.6% being leukemic cells consisting of myeloblasts. The endocrinic laboratory data showed a high level of urine gonadotropin and chromosomal studies revealed a 47,XXY karyotype. A diagnosis of acute myelogenous leukemia (M1) with Klinefelter's syndrome thus was derived from these findings.

A 61 year old woman was admitted to our hospital suffering from severe pancytopenia. Bone marrow aspiration revealed marked hypoplasia with 24.8% plasma cells. There was a small amount of IgA type M protein in serum. Multiple myeloma was ruled out on the basis that 1) the amount of serum IgA-M protein was minimal and no increase was observed, 2) no indication of monoclonality of bone marrow plasma cells was shown by immunofluorescent staining of immunoglobulins, 3) neither bone lesion nor other symptoms related to multiple myeloma were observed. Therefore, a diagnosis of aplastic anemia was made, and bolus methylprednisolone therapy (1,000 mg/day x 3 days) was performed, which brought the patient to complete remission. An oral dose of 15 mg of prednisolone is administered daily now and the remission state is being maintained. Bolus methylprednisolone therapy is performed quite often for the treatment of aplastic anemia. However, there are few reports in which remission was reached with the use of glucocorticoids alone, most having used androgens or other supportive drugs in conjunction. Here, we will discuss this case of aplastic anemia which responded to repeated bolus methylprednisolone therapy, with minimal side effects, and discuss its effectiveness. In addition, aplastic anemia associated with idiopathic monoclonal gammopathy is not reported, so this case is interesting immunologically.

A 77-year-old male, who had been treated with carboquone and busulfan for polycythemia vera (PV), developed myelodysplastic syndrome (MDS) 8 years later. On admission the peripheral blood revealed pancytopenia, but blastoid cells were not noted. The bone marrow showed hypercellularity, and functional and morphological abnormalities in trilineages of hemocytes. Cytogenetic study showed complex abnormalities involving chromosomes 5 and 7. We diagnosed this case as secondary MDS to alkylating agents. He was treated with 1, 25 (OH)2 vitamin D3. However, it was not effective and the percentage of myeloblasts increased to 14.4%. In spite of supportive therapy, he died of sepsis due to urinary tract infection.

The author considers the impact of demographic aging in Japan on the social security system and on economic growth. It is argued that "First of all, as the cost of social security (including social services) increases remarkably at the earlier stage of ageing, the disposable (after tax) income and private consumption of the present labour force generation tend to increase at a lower growth rate than that of the GNP....Secondly if pension systems are based on terminal funding schemes, the ageing of the population increases savings (net increase of the amount of the pension funds) at the earlier stage of the ageing of the population. Thirdly, there is a time lag between the increase of social security benefits and the decrease in the personal savings ratio. The high ratio of savings and the shortage of aggregate demand as well as the high pressure for export in...recent Japan can partly be attributed to the above factors." Possible future economic scenarios as demographic ageing in Japan proceeds are described, and policies to avert anticipated problems are outlined. (SUMMARY IN JPN)

A 42-year-old woman was diagnosed as having acute myelogenous leukemia (M 2 of FAB classification) in May, 1985. Complete remission was achieved with the BHAC-DP therapy (BHAC, daunomycin and prednisolone). Whole skull irradiation and intra thecal chemotherapy were performed by way of prevention. In 1986, she developed her visual disturbance and paraplegia. Myelography and metrizamide CT indicate intraspinal and meningeal infiltration of leukemic cells in the upper thoracic spine. Leukemic cells were found in the cerebrospinal fluid but not in the bone marrow. Complete remission was obtained by irradiation and intrathecal chemotherapy. In Dec. 1986, Ommaya tubing was repeated. Hematological and neurological examinations showed no evidence of malignancy for 3 years. In Oct. 1988, she admitted to our hospital for the maintenance of the remission state, and died of sepsis. The result of autopsy revealed no leukemic cells in her CNS. It is said the intraspinal infiltration of leukemic cells is rather rare. As it is said the intrathecal injection of anti-leukemic agents is not effective for the intraspinal infiltration, irradiation should be needed. Therefore it is very important to critically differentiated intraspinal from meningeal infiltration. Furthermore, Ommaya tubing is effective in order to maintain remission in a patient with CNS leukemia.

We have developed a method to microfluorometrically determine the amount of DNA in immunologically identified micromegakaryocytes on bone marrow smears. Bone marrow smears were fixed with acetone-formalin buffer and immunostained with a monoclonal anti-GPIIb/IIIa antibody, followed by FITC conjugated anti-mouse IgG. After the smears were re-fixed with methanol, DAPI (4', 6-diamidino-2-phenylindole) staining was performed. Using an automatic Digital-Microfluorometer (Olympus MMSP-FR-II), megakaryocytes on the smears were identifies by the GPIIb/IIIa immunofluorescence and, after changing the barrier filters, their nuclear DNA content was measured by the intensity of DAPI fluorescence, which is proportional to the amount of DNA. Using this method, we found that the DNA histogram of the megakaryocytes from a patient with myelodysplastic syndrome showed a shift to small ploidy compared with normal controls. This method may be valuable in the measurement of the megakaryocyte DNA content.

In the myelodysplastic syndrome (MDS) patients the ploidy distribution of megakaryocyte DNA was rarely reported. We applied DAPI (4',6-diamidino-2-phenylindole) staining for measuring nuclear DNA content in megakaryocytes which have been morphologically identified on the Wright-Giemsa stained smear in 8 normal subjects and 12 MDS patients. Briefly, megakaryocytes morphologically examined on a Wright-Giemsa stained smear were photographed, and were located. We then removed the Wright-Giemsa stains by immersing it in 50% ethanol, 37 degrees C for 1 hour and 100% methanol, 37 degrees C for 1 hour. The DAPI staining was performed in DAPI solution (DAPI 0.01 mg/ml, pH 7.4 Tris-EDTA-2 Na buffer solution and 0.01 mol 2-mercaptoethylamine hydrochloride were mixed at the ratio of 0.5: 98.5: 1.0) for more than 30 min. The amount of nuclear DNA in the megakaryocyte previously identified was measured by cytofluorometry. The population of megakaryocyte in the normal subjects was the largest in the 16N, and in the 10 cases of the 12 MDS patients was the largest in the 8N, in the other 2 cases was the largest in the 4N. These results represent the development of the megakaryocyte nucleus in the MDS patients may be disturbed.

The effects of N4-behenoyl-1-beta-D-arabinofuranosylcytosine (BH-AC) on the cell cycle of murine leukemic cells (L 1210 cells) were compared with those of 1-beta-D-arabinofuranosylcytosine (ara-C), known to be effective for acute leukemia. In a cytokinetic study, a combination of Feulgen microcytofluorometry and tritiated thymidine autoradiography (3H-TdR ARG) was used to measure DNA content and to determine DNA synthesis simultaneously in a single cell. Administration of 200 mg/kg of BH-AC significantly prolonged the survival time of mice bearing L 1210. In addition, the cells in the G1 + S1 phases increased with time, accounting for 94.4 per cent of all cells measured at 48 h after the administration, compared to 73.7 per cent before administration. On the other hand, following the administration of 86 mg/kg of ara-C (equivalent to 200 mg/kg of BH-AC), the percentage of cells in the S1 + S2 phases increased maximally to 75.9 per cent at 18 h. Cytokinetic studies further showed that BH-AC administration blocks DNA synthesis of the cells in the S-phase for a longer period than does ara-C. These results suggest that the prolonged inhibition by BH-AC on DNA synthesis allows cells to accumulate in the S-phase to a greater degree.

A method was developed for measuring the nuclear DNA content in single cells previously identified on a bone marrow smear stained by the Wright-Giemsa method. The smear was first photographed and the location of individual cells, identified by morphology, was recorded on a cell map. The smear was then bleached with 50% acid ethanol and absolute methanol, and re-stained by the Feulgen method in 0.05% pararosaniline Schiff's reagent (pH 2.3) at 7 degrees C for 10 min. Nuclear red fluorescence was observed and the intensity of this fluorescence was proportional to the amount of DNA after prior irradiation of smears with green light for 9 hr. The method is useful for measuring cell DNA content in heterogeneous cell populations when morphological cell identification is required.