For the study of immunological phenomena peculiar to liver transplantation, it would be important to know well hematolymphoid system in the liver. In this paper, we report the restoring mechanism of hemopoiesis in the murine regenerating liver. 1) We found hemopoietic stem cells (HSC) and wheat germ agglutinin positive cells (WAG-PC) in both fractions of peripheral blood lymphocytes (PBL) and intrahepatic lymphocytes (IHL) from untreated C3H/He mice. The numbers of HSC and WGA-PC in each fraction gradually increased after partial hepatectomy. This change was remarkable in the population of IHL, associated more strongly with the liver. Colonies, which were generated from IHL in a fibrin clot culture system, are mainly composed of granulocytes, macrophages and mast cells. 2) We found colony-stimulating activity not only in the culture supernatant of normal nonparenchymal liver cells but in that of normal parenchymal liver cells. Furthermore, we clarified that one of colony-stimulating factors in both culture supernatants is GM-CSF. This result suggests parenchymal liver cells also release cytokines such as GM-CSF. These facts show that hematolymphoid system in the liver is a complicated network, composed of parenchymal liver cells, nonparenchymal liver cells, hemopoietic stem cells, and other cell populations.

Interleukin-6 (IL-6) is a pleiotropic cytokine regulating immune response, production of acute phase reactants in hepatocytes, growth of hematopoietic stem cells and other cellular functions in many cell lineages. The increased production of IL-6 is often seen in infections diseases, chronic inflammatory diseases, and certain tumors which accompany polyclonal B cell activation and increased level of CRP. Recent progress in the study of the basic aspects on IL-6 will be discussed, which includes the regulation mechanisms of IL-6 gene, the structure of IL-6 receptor complex (IL-6, 80 KDa IL-6 receptor and signal transducing gp130) and IL-6 signal transduction pathways.

The in vitro assay of colony forming unit-erythroid (CFU-E) colony is a direct and most reliable method to evaluate a biological activity of erythropoietin (EPO). In general, two kinds of cells are employed in this assay system as an effector cell, e.g. bone marrow cell and fetal mouse liver cell, and the latter cell has more sensitivity for EPO. In this paper, a fundamental methodological process in vitro CFU-E colony formation using a fetal mouse liver cell was described in detail and sensitivity for EPO as well as reproducibility of formed colony count were checked up. In addition, an inhibitory effect was indicated in plasma, even though sampled from a healthy volunteer and it was confirmed to be eliminated sufficiently by simple pretreatment with acidification combined with boiling.

We have intended to improve gene-transfer technique into hematopoietic stem cells for somatic gene therapy. 1) We have developed a new packaging cell line, ampGPE for retroviral production. LTR-less gag, pol or env genes from Moloney murine leukemia virus were separately inserted into BMGNeo vector. Packaging cell lines containing 20-50 copies of these two kinds of plasmid were obtained. Retrovirus stock for gene-transfer have been produced at a high titer (10(5)-10(6) cfu/ml) and without replication-competent viruses by using ampGPE. 2) Retrovirus-transduced murine CFU-GM have been found to selectively proliferate (5-10 fold/week) in liquid capture with recombinant murine IL-3, human IL-6 and G418 to consequently obtain enough amount of, highly concentrated (70-100%), and gene-transferred murine CFU-GM for gene-delivery system.

Gene therapy, which is treatment of diseases by introducing normal genes into the body, is becoming feasible as the result of advances in genetic engineering. The hematopoietic stem cells have been considered as the appropriate target for gene transfer in many genetic diseases for which allogeneic bone marrow transplantation has been employed successfully. However, there are still many problems to be solved. In particular, expression from retrovirally transduced genes in bone marrow cells has been transient and unstable. On the other hand, an alternative approach to somatic cell gene therapy using nonhematopoietic cells, including skin fibroblasts, endothelial cells, keratinocytes, and lymphocytes, has been shown to possess several advantages. This kind of approach is usually applied to supplementation therapy in not only hereditary disorders but also various acquired diseases, such as cancer or infectious diseases. Recently, clinical application of gene transfer into lymphocytes to treat cancer and immunodeficiency have been approved at NIH (USA). The trial could represent the start of a new era in molecular medicine.

Motor neurons innervating the vertical yoke muscles in cats were investigated using fluorescent dyes by the double labeling method. In 4 cats, the right superior rectus muscle (SR) and left inferior oblique muscle (IO) were injected with 3% fast blue (FB) and 5% diamidino yellow dihydrochloride (DY), respectively, for observing motoneurons innervating yoke muscles involving upward gaze. In 4 other cats, the left inferior rectus muscle (IR) and right superior oblique muscle (SO) were injected with 3% FB and 5% DY, respectively, to observe motoneurons innervating yoke muscles involving downward gaze. After SR and SO injections labeled cells were found in the bilateral oculomotor nucleus and the bilateral trochlear nucleus, respectively, predominantly on the contralateral side. Following IR and IO injections labeled cells were found only in the ipsilateral oculomotor nucleus. No double labeled cell was observed in any labeled neurons, which indicates that each of the yoke muscles was innervated by independent individual motoneurons. According to the present observation and past reports on rats, cats and monkeys, motoneurons innervating SR and SO have developed from lower mammals such as the rat into predominantly contralateral innervation in the monkey, from a phylogenetic point of view. On the other hand, compared to contralateral innervation in lower mammals, motoneurons innervating IR and IO have developed into ipsilateral innervation in the monkey.

Implantation of genetically manipulated fibroblasts is now coming considered to be one of the important methods for gene therapy. Before the clinical application of this method, we still need to resolve several problems encountered. We have recently developed a model system for the fibroblast-mediated cytokine supplementation gene therapy. BMGNeo (bovine papilloma virus-derived plasmid) (gifted from Dr. Karasuyama) was used for expression of hG-CSF cDNA or hIFN-alpha cDNA (gifted from Dr. Nagata). The two plasmid DNAs (BMGNeoG-CSF and BMGNeoIFN) were individually transfected into NIH/3T3 fibroblasts by the calcium phosphate coprecipitation method. Cell clones producing a large amount of G-CSF or IFN-alpha were selected by the enzyme immunoassay methods and were called G-CSF3T3 or IFN3T3 respectively. Nude mice implanted with G-CSF3T3 highly produced G-CSF in vivo. Remarkable increases in both blood neutrophils and spleen hematopoietic stem cells/progenitor cells (CFU-S, BFU-E, CFU-E, CFU-GM and CFU-MK) were observed. To regulate the production of G-CSF by G-CSF3T3 in vivo, we developed a diffusion chamber system as the cells can be treated easily. We could control the peripheral neutrophil count in nude mice. In the same manner, IFN3T3 was implanted in nude mice bearing a CML cell line, KU812. KU812 tumor growth was significantly suppressed by implantation of IFN3T3 into the chamber. The fibroblast-mediated cytokine supplementation gene therapy might be useful for the treatment of patients requiring for continuous dosing of cytokines.

The 9-year-old boy was admitted to Shizuoka Children's Hospital because of cervical lymphoadenopathy. Complete blood count showed normal RBC and platelet counts. WBC was 2700/microliters with no tumor cells. Bone marrow aspirate showed normocellularity with 34% tumor cells. Lymph node biopsy from his right neck was performed and the patient was diagnosed as non-Hodgkin's lymphoma (lymphoblastic type). Surface marker analysis disclosed that the tumor cells were positive for CD5, CD7, CD19, CD38, CD71, and Ia antigen. Chromosomal analysis of the cervical lymph node revealed 46, XY, t(7;14) (p15;q32). Molecular investigation with appropriate probe showed germ-line configurations of IgH gene, TcR beta gene, and TcR gamma gene, and one rearranged band of TcR delta gene. Monoclonality of tumor cells was demonstrated from chromosomal analysis and molecular study. CD7 and CD19 are not lineage specific antigens because CD7 is expressed on immature AML cells and CD19 is expressed on T ALL cells or AML cells. Moreover, TcR delta rearrangement is considered to occur at early phase of hematolymphoid cells. Based on these data, tumor cells of this patient is considered to originate from immature lymphoid cell, so-called lymphoid stem cell.

Administration of Cepharanthin (ceph) resulted in a significant acceleration of the recovery from leukopenia in 5-Gy X-ray irradiated mice. Then we examined the function of ceph on the recovery of hematopoietic stem cells after irradiation. The number of GM-CFU (granulocyte-macrophage committed stem cells) in the femur and spleen increased significantly in ceph compared to phosphate buffer solution administered groups. However, the time course of the recovery of GM-CFU did not differ between the two groups. These enhancing effects of ceph on GM-CFU were not observed when it was added to a bone marrow culture system. Therefore, the accelerating effects of the recovery from leukopenia after irradiation appears to be an indirect function of ceph.

An improved method for the detection of thymidine kinase (TK) activity with the use of 125I-iododeoxyuridine as the substrate. Radioimmunoassay of prolifigen TK "Daiichi", was used for this assay. Eighty-seven serum samples were collected from 40 patients with acute nonlymphocytic leukemia in four institutions. The levels of TK were measured in the time of pretreatment, remission, and recurrence, and the relationship between the levels of TK and either LDH, marrow blasts, or circulating blasts was also examined. The levels of TK were significantly lower in the state of remission than in the pretreatment. However, the level of TK in remission was much elevated in the majority of cases than normal range (less than or equal to 5 units/l). On the contrary, LDH in remission was within normal limits in the majority of cases. The level of TK in the state of relapse was significantly higher than in the remission. The level of TK correlated in some extent with the percentage of marrow blasts (r = 0.508), and the level of TK in more than 5% of marrow blast was most beyond normal range. Correlation coefficient between the percentage of circulating blasts and TK (r = 0.577) was slightly higher than that between the percentage of marrow blasts and TK. Correlation coefficient between the level of TK and the number of marrow blasts was higher than that between LDH and number of marrow blasts. The levels of TK correlated well with serum LDH (r = 0.778), and was more sensitive than LDH. In conclusion, it was suggested that TK could be used as one of an useful and supplemental tumor marker in the follow-up of treatment of acute nonlymphocytic leukemia and to monitor the effect of therapy.

The chemical structure and immunobiological activities of the cell wall peptidoglycan isolated from Capnocytophaga species was investigated. Peptidoglycan was isolated from Capnocytophaga species strain SE2-2 by boiling in 4% sodium dodecyl sulfate and by digestion with pronase, trypsin and alpha-amylase. Analysis of amino acids and amino sugars of the peptidoglycan revealed that glucosamine, muramic acid, D-glutamic acid, alanine, and diaminopimelic acid (A2pm) were the principal components. Serine and glycine were not found. Dinitrophenylation method revealed that about half of A2pm residue had a free amino group, and analysis by hydrazinolysis showed that a small part of alanine and A2pm located at the C-terminal. The above results indicate that one of the amino groups of A2pm residue at one strand of the stem peptide subunit cross-linked to the carboxyl group of alanine of the neighboring strand. It was thus revealed that the peptidoglycan of Capnocytophaga species belonged to the Al gamma type of the classification by Schleifer and Kandler. Peptidoglycan isolated from Capnocytophaga species strain SE2-2 was found to be definitely adjuvant-active in induction of delayed type hypersensitivity against ovalbumin when administered to guinea pigs as water-in-oil emulsion and in stimulation of increase serum antibody levels. Regarding mitogenicity on splenocytes of BALB/c and BALB/c nu/nu mice, peptidoglycan from Capnocytophaga species was markedly enhanced the uptake [3H] thymidine in dose of 10 micrograms/10(5) cells, however thymocytes were not reactive. Stimulation effects on peritoneal macrophages from a guinea pig to incorporation of 14C-glucosamin was exhibited by addition of 100 micrograms of this peptidoglycan. These findings indicate that peptidoglycan of Capnocytophaga species might eventually be responsible for destruction of periodontal tissue by host mediated activities.

Harvest of peripheral blood stem cells (PBSC) for autografts is a safe, reliable procedure with low morbidity in children with cancer, and cryopreserved PBSC are useful in reducing cytopenia following marrow-ablative chemotherapy. The colony forming unit granulocyte/macrophage (CFU-GM) content of the thawed grafts is an important determinant of hematopoietic recovery after PBSC autografts. The possible value of high-dose chemotherapy without total body irradiation and PBSCT compared to intensified chemotherapy in the treatment of children with very high-risk lymphoid malignant disorders was reported.