The widespread use of potent marrow suppressing drugs in malignant diseases has gained increasing importance in the functional assessment of neutrophil system. A peripheral neutrophil count of 500 cells/cumm is considered to be a good indicator of risk of bacterial infection. However, despite extreme neutropenia, infection is rare in chronic benign neutropenia patients. It may be presumed that adequate marrow reserve delivers mature neutrophil during bacterial infection in these patients. Currently available methods such as determination of blood neutrophil concentration, differential counts, estimation of bone marrow cellularity provide a relatively crude estimates of the functional capacity of the neutrophil system. The use of endotoxin, etiocholanolone and steroids have been introduced to determine the marrow neutrophil reserve with greater accuracy. In this study, intravenous hydrocortisone 100 mg was used to evaluate marrow neutrophilic reserve in normal and aplastic anemia subjects. The results showed significant statistical differences (P less than 0.001) between controls and aplastic subjects in 2, 3, 4 and 5th hour groups after steroid injection. The sensitivity and specificity occurring 3 and 4 hours after steroid injection were 78.6% and 100%, respectively. The normal marrow neutrophilic reserve showed more than 1198/cumm of increment neutrophilic counts 4 hours after steroid injection. No complication was found. The present finding suggests that steroid may be useful as an accurate evaluator of marrow neutrophilic reserve.

Several primary factors related to establishment of mouse embryo pluripotent stem cell lines (ES cell lines) have been tested comparatively. The results of 825 embryos indicated: (1) Delayed blastocysts were more conducive than normal ones to proliferation of inner cell mass (ICM). (2) Culture medium DMEM with 4 500 mg/l glucose was more advantageous than DMEM with 1000 mg/l glucose to the attachment of explanted blastocysts and to the proliferation of ICM. (3) DMEM without sodium pyruvate was beneficial to appearance of ES cell colonies and to their growth. (4) Feeder layer prepared from the primary culture of mouse embryos had very good effects to the growth and culture of ES cells.

We report a case of Cockayne syndrome. A 6-year-old boy presented with a progeroid face, dwarfism, psychomotor retardation, skin photosensitivity and retinal pigmented degeneration. Neurological study disclosed slowed nerve conduction velocities and a brain CT showed calcification in the basal ganglia. Auditory brain stem evoked potential showed prolonged interpeak latency of wave I to wave V. Laboratory evaluation revealed mild liver dysfunction and peripheral eosinophilia. Fibroblast cultures from the patient and his family were exposed to ultraviolet (UV) light of 254 nm, ranging from 1 to 10 J/m2. Under 1 J/m2 irradiation, the surviving fraction of the fibroblasts from the patient, his mother, and a control subject were 40%, 50%, 90% respectively. If the fibroblasts of these subjects were exposed to 2 J/m2 and 3 J/m2 irradiation, the surviving fraction changed to 10%, 22%, 80% and 1.5%, 9%, 68%, respectively. However, fibroblasts from his sister and father showed the same surviving fraction as the control. The study showed that fibroblasts from the patient and his mother were extremely sensitive to UV light irradiation. We also study the concentration of the pyrimidine dimer of DNA in the patient and the control subject. Pyrimidine dimer showed no difference between the patient and the normal subject before and after 24-hour UV irradiation. These results suggest that the sensitivity to UV of Cockayne fibroblasts may be due to a ligase deficiency or to a replicon initiation disturbance in Cockayne cells.

In vitro induced differentiation of mouse embryonic stem cells (ES-5 cells), derived from 5-day 129 mouse blastocyst was studied with retinoic acid (RA) and dibutyryl cyclic adenosine monophosphate (dB-cAMP). RA only or RA with dBcAMP together can both induce monolayer ES-5 cells to differentiate into cells of two types: neuron-like cells and fibroblast-like cells. After treated with 10(-6)mol/L RA for 6 days, the differentiated cells were about 80% of all cells, among which most cells were fibroblast-like cells and others were neuron-like cells. While after 6 days of treatment with 10(-6)mol/L RA and 1 mmol/L dBcAMP, the ratio of differentiated cells can be up to 90-95%, and most cells (about 90-95% of differentiated cells) are neuron-like cells. Immunocytochemical analysis of phenotypic markers, especially GFAP and laminin, showed that the neuron-like cells were glia cells. DBcAMP affected the direction and efficiency of induction by RA. The induced differentiation by RA on attached aggregated ES-5 cells was studied as well. In this case, more cell types appeared, such as epitheloid cells, fibroblast-like cells and spindle shaped cells and so on. The exact nature of these differentiated cells was not identified. After attached culture for about 15 days, rhythmically contracting cardiac-like muscle cells were most attractive among those several differentiated cell types. The change of phenotypic markers during induced differentiation of ES-5 cells in monolayer and aggregated state was summarized in table 1. Transforming growth factor-beta 1 (TGF-beta 1) was also examined in undifferentiated and differentiated cells. Untreated ES-5 cells showed positive immunofluorescent reaction to TGF-beta 1 and various differentiated cells showed different reactions. Glia cells and cardiac-like cells displayed a much stronger TGF-beta 1 reaction. These results indicate that the exact role played by TGF-beta 1 during induced differentiation needs further investigation. The different effect of RA on monolayer and aggregated ES cells and the possible significance of cell to cell interaction in the latter case are discussed.

The results showed that marrow stromal cells include fibroblasts, reticular cells, macrophages and adipocytes. The capability of the adherent layer derived from marrow cells of 2 mouse femurs to support hematopoietic stem cells was stronger than those of layers derived from 0.5 or 1 mouse femurs. The radiosensitivity of bone marrow stromal cells was lower than that of hematopoietic stem cells. The radioprotective effect of AET and PLP (polysaccharide of Lobaria pulmonaria Hoffm) on the bone marrow stromal cells and their capability to support hematopoietic stem cells was clearly demonstrated.

SOKT1 -RTA immunotoxin, which consisted of an anti-pan-T cell monoclonal antibody SOKT1 and ricin toxin A-chain, decreased the peripheral blood lymphocyte transformation in response to PHA and to alloantigen by 92.9% and 84.7%, respectively. The generation of allocytotoxic T cell was also dramatically inhibited by this immunotoxin. After treatment of human bone marrow mononuclear cells with the immunotoxin, erythroid proliferation in CFU-E test preserved 62.9%. Based on these findings, we concluded that SOKT1-RTA immunotoxin can effectively inactivate human T lymphocytes in vitro without serious toxic effect on the hematopoietic progenitor cells. It may be an effective agent in aGVHD prophylaxis.

Using the double agar layer method of human tumor clonogenic assay, the anticancer effect of different combinations of anticancer drugs and interferons was tested on 3 lung cancer cell lines, PC-13, PC-14, and Calu-1. The anticancer drugs and the concentrations used in this study were cisplatin (1.0 microgram/mL), adriamycin (1.0 microgram/mL), mitomycin C (0.2 microgram/mL), VP-16 (5.0 micrograms/mL) and 5-FU (5.0 micrograms/mL). Three kinds of interferon, alpha, beta and gamma in 5,000 units/mL, were tested in combination or in sequence with other anticancer drugs on lung cancer cell lines. The results demonstrate an enhanced anticancer effect on PC-14 only with sequential or simultaneous combination of VP-16 with alpha, beta and gamma interferons; and on Calu-1, only with sequential use of adriamycin and beta-interferon. Our results indicate that there is no unique way of combining anticancer drugs and interferons which can obtain an enhanced anticancer effect on all lung cancer cell lines. The best combination of interferon and anticancer drugs seems to be influenced by the biological characteristics of the cancer cells.

Thirteen cases (10.5%) of squamous cell carcinoma of the lung with neuroendocrine differentiation were identified ultrastructurally in 124 cases of resected pulmonary carcinoma. In addition to the features of epidermoid differentiation, i.e. presence of tonofilaments and desmosones, a small amount of cancer cells containing neurosecretory granules were found in all the 13 cases. Ultrastructures indicated that both epidermoid and neuroendocrine differentiations were present simultaneously in individual cancer cell. Immunohistochemical staining for NSE was performed in 12 cases. Positive reaction was obtained in all but one (91.7%). NSE positive cancer cells distributed focally or in single cell scattered in the cancer cell nests. The results suggest that biphasic differentiation of pulmonary squamous cell carcinoma diagnosed by light microscopy may not originate from different cells of different derms, but may be derived from a common stem cell of the bronchial epithelium which possesses the ability of multipotential differentiation.

63 patients with aplastic anemia were studied by using in vitro assay for committed erythroid and granulomonocytic progenitors from the bone marrow. The T cell-mediated effects of suppression of normal hematopoiesis were observed by PBMNC of the patients when cocultured with normal bone marrow. The stimulated effects of androgen on BFU-E and CFU-E with methyl testosterone were also studied. The results showed that the PBMNC of 44.4% of the 63 patients suppressed normal hematopoiesis. 41.3% of the patients responded to methyl testosterone (MT) and 14.3% of the patients had very obvious decrease or absence of BFU-E, CFU-E and CFU-GM without evidence of immunological effects or response to androgen. According to these findings, it may be useful for clinicians to choose better therapeutic regimens for aplastic anemia. Such as BMT for the patients with hematopoietic stem cells deficiency; immuno-suppression treatment or splenectomy may be of benefit for those who suffered from immune mediated aplastic anemia (IMAA) and androstenones chosen for those sensitive to MT in vitro. There were 15 patients with IMAA treated with immuno-suppressive agents and 19 patients sensitive to MT treated with androgens. All of them had satisfactory results.

McAbPD4 could inhibit the proliferation of transformed cells Rat3-3 with an inhibition rate of 72.3% in vitro. Moreover, McAbPD4 decreased the Rat3-3's colony forming ability on soft agar and tumorigenicity in nude mice. By Flow Cytometry, it was indicated that the inhibitory effect was due to a blockade between G1 and S phase. It was shown by Western Blotting that the McAbPD4-defined antigen was a 40 KD protein, named p40, which was located on the membrane of target cells MGC803 and transformed cells Rat3-3 by immunofluorescence assay. The antigen p40 was highly expressed on Ha-, Ki-, Nb- and Src oncogene transfected cells, but was undetectable or at the best only faintly on cells transformed with mos or raf. The antisense oligonucleotide AS-1 of Ha-ras distinctively inhibited the proliferation of Rat3-3, the level of Ha-ras oncogene product p21 and p40 were decreased by 47.8%, 36.5%, and 52.0%, respectively. The inhibitory effect of AS-1 to GCM3T3 was more marked, each with 54.2%, 47.9% and 65.9%. It is suggested that p40 not only is a tumor-associated antigen, but also a transformation-related cell surface protein, which is closely associated with and might be regulated by the activation of ras or Src oncogene and its product. For the inhibitory effect of McAbPD4 to Rat3-3 cells, it is deduced that p40 might be a growth factor receptor mediating autocrine growth of Ha-ras transformed cells.

This paper, used an improved methylcellulose-agar double layer method in vitro to culture megakaryocyte progenitors (CFU-meg) of 15 normal bone marrow donors and 20 idiopathic thrombocytopenic purpura (ITP) patients bone marrow cells in order to observe the action of Shen-Xue-Ling (SXL). The results showed that the colony efficiency of CFU-meg of ITP group (13.75 +/- 6.93/10(5) MNC) was obviously lower than that of the control group (21.22 +/- 9.06/10(3) MNC, P less than 0.01). The serum of the patients whose PAIgG was markedly higher than normal value inhibited growth of CFU.meg derived from normal bone marrow cells (P less than 0.01). SXL could increase the colony efficiency of CFU-meg for ITP patients at several doses. The average colonies were 15.77 +/- 6.31/10(5) MNC (at 100 mg/ml, P greater than 0.05), 18.72 +/- 5.54/10(5) MNC (at 200 mg/ml, P less than 0.05), 21.60 +/- 7.35/10(5) MNC (at 300 mg/ml, P less than 0.01) and 21.84 +/- 7.15/10(5) MNC (at 500 mg/ml, P less than 0.01) as compared with the group of ITP patients. Incubated with patient's serum (1%) and SXL (at 300 mg/ml), the number of CFU-meg extracted from normal bone marrow cells was significantly higher than that from patients (P less than 0.01) and was close to normal group (P greater than 0.05). The results suggested that SXL might inhibit the antiplatelet antibodies, facilitate reproduction, division and maturity of CFU-meg.

Having been passed for 160 generations, a cell line designated as H 22-F 25/L was established from a murine tumor lymphatic metastatic model H 22-F 25 which had been set up in our college. The cell line was in suspension culture with a rapid proliferation and stable growth. The peak time of cell division and proliferation was 48 and 96 hours after culture. In a week, the cell number was increased by 25 times. H 22-F25/L still keeps the features of a poorly differentiated cancer. Its tumor inducing rate (in vivo) was 100% in 615 mice. Lymph node metastasis rate was 50% and pulmonary metastasis rate 10%. H 22-F 25/L is a population of heterogenetic tumor cells including 2 stem cell lines (the model number of chromosomes being 43 in 40% tumor cells and 86 in 32%) and some side lines. The common marker chromosomes M1, M2, M3 and M4 were present in all stem and side lines.

Bleomycin A6 (A6), a single component of bleomycin complex, is highly active against human liver cancer cells in vitro and xenografts in nude mice. A6 was conjugated to monoclonal antibody H111 directed against human hepatoma BEL - 7402 cells, using Dextran T40 as an intermediate. The conjugate consisted of a coupling molar ratio of 1:264 for H111 and A6, and retained 6.3% of A6 activity. As determined by clonogenic assay with hepatoma BEL - 7402 cells exposed to the agents for 1 h, the IC90 values for H111 - A6 conjugate, free A6 and M3 - A6 conjugate (an irrelevant conjugate) were 0.17 mu mol/L, 17 mu mol/L and 7 mu mol/L respectively. The cytotoxicity of Hill - A6 conjugate to target cells was markedly blocked by unconjugated H111 but not by irrelevant monoclonal antibody M3. The H111 - A6 conjugate exhibited 78% inhibition on the growth of hepatoma BEL - 7402 xenografts in nude mice, whereas the equivalent doses of free A6, M3 - A6 conjugate and H111 plus A6 mixture showed approximately 30% inhibition. Histopathological examination showed no toxic changes in the liver, lung, kidney and bone marrow in the H111 - A6 conjugate--treated animals. These results suggest that the conjugate of monoclonal antibody and bleomycin A6 exhibits specific cytotoxicity to target liver cancer cells and the conjugate is highly effective against liver cancer xenografts in nude mice with more marked tumor inhibition than free A6 at comparable dose levels.

Six cases of acute leukemia patients were treated with APHSCT and the clinical results were satisfactory. The preparatory marrow ablative chemoradiotherapy regimen before APHSCT was same as that for autologous bone marrow transplantation (ABMT). The number of mononuclear cells infused to restore the hematopoietic function ranged from 1.65 to 2.92 x 10(8)/kg and these cells yielded 0.17 to 2.26 x 10(4)/kg CFU-GM. The hematopoietic and immunological function of patients returned to normal level 25 to 38 days after APHSCT; the recovery was faster than that of ABMT and FLT. One patient each died of recurrence of leukemia and fungi septicemia 90 and 70 days after transplantation respectively, whereas the other four patients have survived for 9, 8, 4, and 2 months respectively.

The antitumor activity of a new podophyllotoxin spin-labeled derivative, 4-(4''-(2'',2'',6'',6''-tetramethyl-1''-+piperidinyoxy)amino)-4'- demethylepipodophyllotoxin (GP-7) First synthesized by us, was studied in vitro. It was found that the proliferation of SGC-7901 cells was markedly inhibited by GP-7 depending the concentration and exposure time. At concentration of 0.04-100 mg/L, the inhibition rates were 15.5-92.6% ID50 was 0.42 mg/L. After exposure to GP-7 greater than 0.5 mg/L for 24, 48, 72 and 96 h, the inhibition rates of cells were 25.1, 49.0, 71.4 and 84.9% respectively. The dose-response curve of GP-7 on SGC-7901 cell was similar to that of etoposide (VP-16). The colony formation of SGC-7901 cell was also inhibited by GP-7 in a concentration dependent fashion with ID50 1.63 mg/L. At concentrations of 0.1-0.5 mg/L, the inhibitory effects were stronger than that of VP-16. GP-7 decreased the mitotic index (MI) of SGC-7901 cell and had no effect on microtubule assembly and disassembly in vitro, which suggested that GP-7 did not act on M phase.

As evaluated by clonogenic assay, bleomycin A6 was found to be highly active against established human cancer cell lines derived from colon cancer (HT-29) and cecum cancer (Hce-8693). These human cancer cells have been serially transplanted in nude mice. At a tolerable dosage level, bleomycin A6 exerted remarkable growth inhibition on human colon cancer HT-29 and cecum cancer Hce-8693 xenografts (approximately 90% inhibition). No histopathological changes were found in the organs of treated animals. Compared on the basis of equitoxic doses (1/9 LD50), bleomycin A6 exerted much stronger growth inhibition against colon cancer HT-29 xenografts in nude mice than 5-fluorouracil and mitomycin C, with inhibition rates of 82%, 12% and 53%, respectively. More extensive necrosis was found in tumors treated with bleomycin A6 than in those treated with mitomycin C or 5-fluorouracil. The ratio values of non-necrotic tumor tissue to whole tumor tissue for bleomycin A6, mitomycin C and 5-fluorouracil were 0.33, 0.65 and 0.57, respectively. These observations indicate that bleomycin A6 is a potent antitumor agent against colon cancer xenografts and may be useful in human colon cancer chemotherapy.

The distribution of 5-hydroxytryptamine (5-HT) immunostaining cells in the digestive tracts (hibernation and nonhibernation), the brain and other various tissues of Bufo bufo gargarizans was studied by peroxidase anti-peroxidase immunocytochemical method. In the brain, 5-HT immunostaining cells were localized in the raphen nuclear area of brain stem and in the ependyma cell area of the ventriclus tertius of diencephalon. These immunostaining cells were round or oval. The cells usually possess processes which were filled with immunoreactive substance. Some of the processes were contact with the processes of other cells. A few 5-HT positive reactive nerve fibers were observed in the brain stem and the diencephalon. The density of 5-HT immunostaining cells in the digestive tubes were the highest in the pylorus, fundus, cardia of gaster, and moderate in the esophagus and duodenum and the lowest in the large intestine and the small intestine. The density of 5-HT immunostaining cells in the digestive tubes were higher in nonhibernant toads than in hibernant toads. By the statistical method, the difference of the density between the two sorts of toads were notable (P less than 0.05). The 5-HT immunostaining cells were visualized to distribute between the epithelium cells of the mucosa or the epithelium cells of gland. These positive cells usually had one or more processes which contained 5-HT immunoreactive substance. Some were reached into lumen surface of the gland or intestine. Some were extended into lamina propria through the basal membrane. These results indicate that the 5-HT immunostaining cells in digestive tubes could release 5-HT by both endocrine and exocrine ways.