A sporadic case of spinocerebellar degeneration with prominent involvement of the motor system has recently been encountered. A 54-year-old man without family history noticed speech disturbance at the age of 46 and weakness in his right hand the following year. The muscle weakness and atrophy were slowly progressive and made walk impossible at the age of 50, when his dysphagia increased. At the age of 54, he was admitted to our hospital when neurological findings revealed marked amyotrophy of general skeletal muscle and tongue with fasciculation. Deep tendon reflexes were decreased. Cerebellar ataxia was impossible to evaluate because of profound muscle weakness. And sensory disturbance was suspected in the distal portion of the lower extremities. CT scan revealed progressive atrophy of the brain stem and cerebellum. The patient died at the age of 54 due to CO2 narcosis. The clinical course was 8 years. A summary of the pathological findings was as follows: 1) Marked neuronal loss of the anterior horn of the spinal cord and motor cranial nerve nuclei except for oculomotor nuclei, with mild degeneration of pyramidal tract below lumbosacral level. 2) Degeneration of cerebellipetal system, spinocerebellar tract, Clarke's column and the middle root zone and cerebellifugal system, dentate nucleus, superior cerebellar peduncles, and red nucleus. 3) Mild degeneration of pontine nuclei, inferior olivary nuclei, pontine transverse fibers, the middle and inferior cerebellar peduncles, cerebellar white matter and Purkinje cells as in OPCA.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitrosourea compounds have been widely used in the chemotherapy of malignant brain tumors, because of their blood-brain barrier permeability. However, drug resistance to nitrosoureas has been recently a major concern. Using an in vitro colony formation assay, intrinsic and acquired resistances to an anticancer nitrosourea, 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU), were analyzed in rat 9L and C6 glioma cells. 9L and C6 cells were treated with varying doses of ACNU for 2 hours. Ten days after, the cells were fixed and stained with crystal violet. Colonies consisting more than 50 cells were counted. The survival fraction following treatment is the ratios of colony efficiency of treated cells to the colony efficiency of untreated control cells. The dose-response curve for ACNU indicated the existence of a shoulder (Dq, quasithreshold dose) at doses and an exponential cell-killing at higher doses with D0(37% survival dose). Based on dose-response curves corresponding to multitarget single-hit model, 9L cells showed 7.4 microM, 2.9 microM, and 14 microM at Dq, D0, and SD10 (10% survival dose) values, respectively, whereas C6 cells showed respective values of 6.4 microM, 30 microM, and 75 microM. 9L cells had significantly less intrinsic resistance to ACNU than C6 cells at the p less than 0.005 level by a covariance analysis of the curves. As with changes of drug susceptibility after ACNU treatment, both parent cells were treated every other day (1, 5, and 10 repeated times) with various doses up to approximately 1% survival dose of the parent cells.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated a new chemosensitivity test, MTT-hybrid assay, which was a hybrid of MTT colorimetric assay and double-layered soft agar colony assay, using human bone and soft tissue tumor cells. MTT formazan crystals produced by viable cells in the soft agar medium were solubilized by SDS at 60 degrees C. The absorbance (560 nm) is directly proportional to the cell number over a wide range. The absorbance increased in proportion to colonial growth of osteosarcoma cells, while it decreased in a human diploid cell strain in a few days. Drug sensitivity of tumor cells is supposed to be assessed without contaminating normal cells by MTT-hybrid assay in primary tumor samples. Good correlation of IC50 was observed between MTT-hybrid assay and colony assay. The MTT-hybrid assay shows potential value as a rapid predictive test for chemotherapeutic agents in an individual patient.

Human peripheral blood mononuclear cells (PBMC) obtained from healthy volunteers gave rise to three types of colonies that could be distinguished by their unique morphological characteristics; 5.1 +/- 0.9 (mean +/- SE) loose colonies of small cells, 2.1 +/- 0.9 packed colonies of larger cells, and 1.6 +/- 0.7 mixed colonies, were formed when PBMC (2 x 10(5) cells/dish) from 10 healthy volunteers were cultured in the medium containing methylcellulose. Cytochemical analysis with Astra blue-eosin dual staining revealed that all types of colonies consisted of various proportions of basophils, eosinophils, and hybrid (eosinophilic and basophilic) granulocytes which contained both of granules. These hybrid granulocytes were also identified by the ultrastructural features of two kinds of the granules. Relationships between cell numbers added to culture and formed colony numbers indicated colony of the cells to form the colonies. The colonies formed from untreated patients with chronic myelogenous leukemia (CML) during chronic phase were sevenfold of those from healthy volunteers. The colonies formed from treated patients with CML were normal in number. The number was 40 times greater in culture from a patient with basophilic crisis and a patient with myeloid crisis than normal, whereas that from a patient with lymphoid crisis were within normal limit. The number of the colonies from PBMC of patients with eosinophilia were in normal range, whereas those from bone marrow were six times or more than those from PBMC. These findings suggest that PBMC contains common basophil-eosinophil progenitors, and the culture used in this study in considered to be useful in the examination of basophil and eosinophil production from PBMC. Further studies using more purified cell population and other sources of colony stimulating factors such as interleukin-3 (IL-3), IL-4, IL-5, and granulocyte-macrophage colony stimulating factor should be carried out in order to clarify the significance of hybrid granulocytes in basophil and eosinophil proliferation and differentiation.

Morphologic features of the autopsied specimen of 22 cases with supratentorial gliomas treated by surgery, radiation and/or chemotherapy were analysed, and the characteristics of recurrence of gliomas were searched for. The cases consisted of anaplastic 12 astrocytoma and 10 glioblastoma. The results were as follows: 1) Characteristic CT findings before death were regrowth of the tumor mass or the occurrence of a new enhanced lesion in 21 out of 22 cases. The enhanced lesion showing regrowth of the tumor located in the same site as the previous tumor mass in 21 cases. The new enhanced lesion resulting from a trans-or subependymal tumor spread, was seen in the ventricular wall, and these findings were a characteristic feature of the recurrence of gliomas. 2) Modes of extension of the tumor were subdivided into 3 types. One was the expansive or infiltrative type caused by regrowth of the residual tumor. In the second pattern, a spread of tumor cells occurred along the myelinated fiber tracts to the brain stem (60%), or to the contralateral cerebral hemisphere through the corpus callosum (50%). The third mode of tumor propagation was cerebrospinal fluid seeding with intraventricular or subarachnoid tumor regrowth (45%). 3) Characteristic histological findings shown in the original tumor bed were those of increased cellularity with endothelial proliferation, widespread necrosis with occlusion of the blood vessels, occurrence of the gemistocytic astrocytes and large bizarre cells. Thickening of wall of the blood vessels due to effect by radiation was followed by occlusion of the blood vessels. Large necrosis in the tumor tissue was caused by those process and others. Necrotic area was mainly circumscribed and corresponded to the territory of the vessels. One of the specific findings in the morphological changes of the tumor cells was giant cell formation which were monstrous cell, giant cell (12 cases out of 22), and gemistocytic cell (in all cases). These specific cells were supposed to the degenerative changes of the tumor cells exposed while withstanding such adverse conditions as hypoxia, radiation and chemotherapy. 4) Infiltration distant from the primary lesion which were defined only by microscopical examination was demonstrated as both through myelinated fiber tracts in 8 cases and through perivascular spaces in 2 cases. Reinvasion of the tumor cells from the subarachnoid spaces to the brain parenchyma was along the Virchow-Robins spaces of the penetrating blood vessels in the latter cases.(ABSTRACT TRUNCATED AT 400 WORDS)

In order to investigate the role of the human spleen on hematopoiesis, hematopoietic stem cells and stimulates were evaluated in fetal and adult spleens. BFU-E and CFU-C were existed in 20 weeks and 23 weeks fetal spleens (BFU-E 145 +/- 45/10(5) mononuclear cells, CFU-C 55 +/- 6/10(5) mononuclear cells). In adult spleen, a few stem cells were recognized, which may be contaminated from peripheral blood in sinus of the spleen. We tested conditioned media from adult spleen cells for the stimulative activity on the in vitro growth of BFU-E and CFU-C from bone marrow mononuclear cells. Spleen conditioned medium stimulated proliferation of these precursor cells. It seemed that PHA-stimulated spleen conditioned medium augmented BFU-E, whereas CFU-C growth was suppressed. Adult and fetal spleens were studied immunohistochemically using anti-G-CSF, GM- CSF and erythropoietin antibodies. The cells with G-CSF and GM-CSF were shown in fetal spleens. In adult spleens, however, only GM-CSF was detected.

Spontaneous complete remission of four months' duration was observed in a 51-year-old male with hypoplastic leukemia. Cytogenetical analysis revealed that leukemia cells of this patient were tetraploid. The diameter of leukemia cells involving myeloid cells ranged from 30 to 50 mu. The remission was apparently associated with repeated blood transfusions and severe infection. Complete remission was confirmed by normal morphology and karyotype of the bone marrow cells, although in vitro marrow stem cell growth did not return to normal. Thus, normal hematopoiesis may not have recovered when the diagnosis of spontaneous remission was made.

PHA-leukocytes conditioned medium (PHA-LCM) prepared from the patients with chronic renal failure and normal control was tested for BPA by progenitor cell assay with methylcellulose culture method. The BPA of uremic patients, expressed as a percentage of standard CM, was significantly lower than that of normal subjects (96 +/- 9%). However, there was no correlation between BPA and the severity of anemia. The number of circulating BFU-E per milliliter of blood was significantly lower in uremic patients (71 +/- 77) than in normal controls (131 +/- 96), and the number correlated with the severity of anemia. From these results, the maturation process of erythroid series in uremic patients appeared to be impaired at a stage between pluripotent stem cell and BFU-E, and might be secondary to inefficient production of BPA by lymphocytes.

We studied the effect of interleukin-2, and alpha and gamma-interferon on the growth of B cell colonies in three patients with hairy cell leukemia (HCL). Cells in the colonies from HCL were morphologically similar to the circulating cells, but HCL colonies were morphologically different from B-CLL colonies. HCL colonies had several satellite cells. Spell out (PHA) was essential for the growth of HCL. HCL responded to PHA-MTCM, the supernatant derived from 24 hr incubation of PHA, silica, monocytes and T cells. HCL cells didn't respond to IL-2 or alpha-interferon. However, gamma-interferon significantly enhanced HCL cell proliferation.

We tested purified recombinant hemopoietic factors for their effects on the proliferation and differentiation of murine myelomonocytic leukemia cells (WEHI-3B-Y1) in serum-free agar culture. We found that purified recombinant human G colony-stimulating factor (CSF) markedly increased the colony number of WEHI-3B-Y1 cells and differentiation-inducing activity. However, at low colony densities [( 100/dish), G-CSF did not induce the differentiation of WEHI-3B-Y1 cells. We conclude that G-CSF does not induce the differentiation of WEHI-3B-Y1 cells directly, but induce the differentiation as a result of the secondary autoinduction of differentiation. Interleukin 3 (IL-3) slightly enhanced but erythropoietin (Epo) did not alter the colony number of WEHI-3B-Y1 cells. GM-CSF or M-CSF decreased the colony number of WEHI-3B-Y1 cells. Such purified recombinant human IL-3, Epo, GM-CSF and M-CSF did not induce morphologically the distinct differentiation of WEHI-3B-Y1 cells.

We investigated the inhibitory effect of autologous sera on erythroid colony formation (CFU-E) of bone marrow cells from patients with chronic renal disease and the clinical effect of recombinant erythropoietin. Colonies formed in cultures using autologous serum (AS) decreased in 15 among 30 cases as compared with those using fetal calf serum (FCS). This inhibitory effect of autologous sera was diminished by treatment with activated charcoal in all these cases. The degree of hemoglobin increase after administration of recombinant erythropoietin appeared to correlate with the intensity of inhibitory activity of AS. These data indicate the clinical significance of the inhibitor(s) of erythropoiesis in uremic sera and suggest that the clinical effects of erythropoietin in this disease are further improved if the inhibitor(s) can be effectively removed.

An 80-year-old male was admitted because of dizziness and palpitation. Laboratory investigation revealed pancytopenia. A bone marrow aspirate showed a markedly hypocellular marrow with 41.6% blast cells. Peroxidase activity was negative and PAS reaction was block positive in the blast cells. Surface markers of these cells were positive for HLA-DR antigen and partially positive for CD13 (MY7). Other markers, such as T, B or myeloid antigens were all negative. These blast cells were classified as L1 according to the FAB system but suggested essentially unclassifiable in cell differentiation. The patient was treated successfully with vincristine and prednisolone and induced into complete remission although repeated marrow examination findings revealed hypocellular. As for the classification of hypoplastic leukemia, lymphoid or primitive "stem cell" leukemia also should be considered as other categories of acute leukemias and be treated according to each case.

This study investigated the possibility of using electrically evoked auditory brainstem response (EABR) for predicting surviving spiral ganglion cell populations. EABR recordings were made from six kanamycin induced deaf cats by the round window monopolar stimulation (RW) and the scala tympani bipolar stimulation (ST). On completing the electrical stimulation, each animal was sacrificed and prepared for histological examination. The spiral ganglion cell populations were estimated by classifying them into four groups (0-25%, 25-50%, 50-75%, 75-100%) as a percentage of survival cells under magnification. EABR thresholds and input-output function of amplitude were compared with microscopic findings of surviving spiral ganglion cell populations. The results were as follows: 1) The rate of amplitude growth in response to increased stimulus intensity of electrically evoked potentials from ST was gradual, but that of RW was steep. 2) The vestibular potentials were elicited by RW, and at a lower stimulus intensity the RW waveform began to become distorted by the myogenic potentials. 3) EABR threshold was a poor predictor of surviving spiral ganglion cell populations. 4) There was a correlation between surviving spiral ganglion cell populations and the slope of the input-output functions of ST EABR. However, there was no correlation between that of RW EABR and the slope.

The radiosensitivity and radio-chemosensitivity of 3 series of human cancer cell lines were evaluated by human tumor clonogenic assay. The sources of cell lines were gingiva carcinoma (Ca9-22), uterus carcinoma (Hela) and gastric carcinoma (MKN-45). BLM and CDDP were used, and chemosensitivity of gingiva carcinoma tended to be higher than other cell lines. Radiosensitivity was same as MKN-45. Isobologram were employed for quantitation of the interaction between the irradiation and anti-cancer agents. In Ca9-22, the interaction of between gamma-rays, BLM and CDDP was supra-additive. Hela was also supra-additive, but in MKN-45, the interaction of between gamma-rays and BLM was sub-additive.

The effects of cepharanthine on the suppression of hemopoiesis by X-ray irradiation were studied. A whole body X-irradiation (3 Gy) induced decrease of leucocyte count, nucleated cell count of bone marrow, myeloid stem cell count (CFU-C), and spleen weight. Oral administration of cepharanthine (25 mg/kg BW or 50 mg/kg BW) tended to decrease these damage on hemopoiesis, and increased spleen weight on 5th day after irradiation. Histological examinations revealed that the administration of cepharanthine accelerated the hemopoietic recovery in the red pulp of spleen.

With human tumor clonogenic assay, the direct antiproliferative activity of recombinant human leukocyte interferon alpha (IFN-alpha) was investigated on human renal cell carcinomas (RCCs), which consisted of a human RCC cell line (ACHN), two human RCC xenografts and fifteen primary RCCs. The combination effect of IFN-alpha with a cancer chemotherapeutic agent was studied, as well, with the assay system. IFN-alpha showed a dose-dependent antiproliferative activity against the human RCCs. The clonal growth of ACHN cell line was inhibited by less than 50% at the concentration of 1,000 IU/ml. Two xenografts had a different sensitivity to IFN-alpha, in which the percent colony formation was less than 20% in RCC-3 at the concentration of 100-100,000 IU/ml, while in RCC-4 more than 50% even at the high concentration of 10,000 IU/ml. In 15 primary tumors obtained at surgery, two types of response to IFN-alpha were demonstrated. One was the response in which the colony formation was inhibited in a dose-dependent manner as an increment of IFN-alpha concentration, and the other in which the colony formation was not sufficiently inhibited even at the high concentration of IFN-alpha. The dose-dependent inhibition of colony formation was demonstrated in 10 out of 15 specimens (66.7%). When the colony formation suppressed to less than 50% of control was considered to be sensitive to IFN-alpha, 6.7% of these 15 primary tumors were sensitive to IFN-alpha at 100 IU/ml, 20.0% at 1,000 IU/ml and 20.0% at 10,000 IU/ml. Combination effects of IFN-alpha and with each of four different cancer chemotherapeutic agents (vinblastine, adriamycin, methotrexate, 5-fluorouracil) were investigated on the ACHN cell line. Every combination type produced a subadditive or synergistic combination effect. In particular, the combination of IFN-alpha with vinblastine of more than 0.1 microgram/ml concentration yielded a combination effect of statistical significance (p less than 0.001). Even against premary tumors, the combination of IFN-alpha with vinblastine showed a synergistic effect in one out of every three tumors. These results suggested that the combination of IFN-alpha with a cancer chemotherapeutic agent would enhance the clinical effect of IFN-alpha alone in only a certain situation.

The pathogenesis of Cefmetazole (CMZ) induced agranulocytosis was investigated in the case of a 40-year-old man who developed agranulocytosis while he was under treatment with CMZ. Although concentration of CMZ in serum is not detected, patient's serum drawn at the time of diagnosis suppressed normal allogeneic marrow GM colonies in vitro. Normal IgG purified from serum had no effect on GM colony formation in vitro. However, patient's IgG purified from serum drawn at the time of diagnosis suppressed normal allogeneic marrow GM colony in vitro without CMZ. These studies suggest that CMZ-induced agranulocytosis is immunologically mediated through an IgG inhibitor which seems to exert its effect on GM colony formation.

The influence of bone marrow fibroblasts in healthy subjects and patients with aplastic anemia on normal erythroid colony formation was studied using the methylcellulose method. These fibroblasts were treated with methylprednisolone as well. Bone marrow fibroblasts of healthy subjects and patients with aplastic anemia, and the supernatant of their conditioned medium significantly inhibited normal erythroid colony formation. A significantly marked inhibition of normal erythroid colony formation was observed of bone marrow fibroblasts (or the conditioned medium) of the aplastic anemia, when compared with that of the bone marrow of healthy subject fibroblasts (or the conditioned medium). By treating both groups of the bone marrow fibroblasts with methylprednisolone the inhibition was slightly improved. From the above experimental results, it was suggested that the bone marrow fibroblasts and the conditioned medium inhibited normal erythroid colony formation through humoral factors secreted by the fibroblasts, and through contact between the fibroblasts and erythroid colony formation cells.

To prolong the survival of patients with chronic myeloid leukemia (CML), 19 patients were treated with busulfan to keep their leukocyte counts within normal range by controlling bone marrow hyperplasia. The duration of chronic phase in these patients was significantly longer than that in historical controls who were treated conventionally with busulfan. This prolongation was not ascribable to the difference in such prognostic factors between the two therapy groups as splenomegaly, leukocyte count and percentage of peripheral blasts. There was a significant difference again in the duration of chronic phase between the two therapy group even when restricted to each 11 patients with intermediate relative risk (0.7-1.5) according to Sokal et al. Four patients showed thrombocytopenia less than 5 x 10(4)/microliters, but all these patients recovered within 4 months and there was no further critical side effect except subcutaneous bleeding. This study suggests that maintenance of leukocyte count within normal range and suppression of granuloid hyperplasia in bone marrow with busulfan may prolong chronic phase of CML. Probability of clonal evolution may be decreased by reducing the total leukemic cell mass and suppressing cellular turnover of primitive CML stem cells. Another possibility is that prolongation of chronic phase might be dependent on the appearance of normal karyotype clone after long-term bone marrow suppression just like after intensive chemotherapy or alpha-interferon therapy.