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It has been shown that some types of tumour cells produce activated transforming growth factor beta-1 (TGF-beta 1). However, the mechanism for the activation of TGF-beta 1 derived from tumour cells has not been fully elucidated. The present study was undertaken to characterise an activator of latent TGF-beta 1 secreted from a human gastric cancer cell line, KATO-III. Western blot analyses using antibodies for TGF-beta 1, latency associated peptide (LAP) and latent TGF-beta 1-binding protein (LTBP) revealed that, in the cell lysate of KATO-III, TGF-beta 1 protein was expressed as a small latent complex of TGF-beta 1 and LAP. This was also confirmed by a gel chromatographic analysis of the cell lysate obtained from KATO-III. A 2.5 kb transcript of TGF-beta 1 mRNA was detected in KATO-III cells by Northern blot analysis. A gel chromatographic analysis of the conditioned medium from KATO-III cells revealed, in addition to the active form of TGF-beta 1, a factor which activated latent TGF-beta 1 from NRK-49F cells at fractions near a molecular size of 65,000. This factor was inactivated by heat (100 degrees C), acidification, trypsin and serine protease inhibitors. TGF-beta 1 activity in KATO-III cell lysate was not detected in the untreated state, but potent TGF-beta 1 activity was detected after acid treatment. These results suggest that KATO-III releases not only a latent TGF-beta 1 complex but also a type of serine protease, different from plasmin, plasminogen activator, cathepsin D, endoglycosidase F or sialidase, which activates the latent TGF-beta 1 complex as effectively as acid treatment. Images Figure 1

To explore the mechanism of increased collagen deposition in scirrhous carcinoma of the stomach, an attempt was made to define the role of transforming growth factor beta 1 (TGF-beta 1), secreted from tumour cells, as a possible humoral factor which functions in a paracrine manner to stimulate the production of collagen in regional fibroblasts. Immunohistochemical staining revealed that tumour cells in scirrhous carcinomas were generally stained more intensively than those in other types of carcinomas. On Northern blot analysis the tumour cells established from scirrhous carcinoma (KATO-III, OCUM-1 and HSC-39) exhibited relatively strong signals compared with those from non-scirrhous carcinoma (MKN-28 and MKN-45). In the culture media of scirrhous carcinoma cells, the active form of TGF-beta 1 was detected, while in those of the non-scirrhous carcinoma cells the latent form was demonstrated by both colony and radioreceptor assays. The culture medium from KATO-III showed strong stimulating activity of collagen synthesis in fibroblasts, and this activity was partially neutralised by an anti-TGF-beta 1 antibody. These results suggest that tumour cells in scirrhous carcinoma produce more active-form TGF-beta 1 than does non-scirrhous carcinoma and thus is partially responsible for the observed enhanced collagen deposition in the region. Images Figure 1 Figure 2

We mobilized peripheral blood stem cells (PBSC) following CHOP plus rituximab (CHOP-R) therapy, and compared with the findings following CHOP therapy without rituximab. All patients were given G-CSF starting from day 11 after CHOP therapy. Patients in the CHOP-R group (n=8) were given rituximab on day 12. Target CD34(+) cells number was collected in a single leukapheresis on day 14, from all the eight patients in the CHOP-R group. PBSC mobilization kinetics, CD34(+) cells yield and colony-forming ability in the graft collection, toxicity during mobilization, and engraftment after transplantation of CHOP-R group were not significantly different from those in the CHOP group (n=8). In all patients given CHOP-R therapy, CD20(+) cells and immunoglobulin heavy chain (IgH) rearrangement in the graft collection were undetectable by flow-cytometric analysis and Southern blot analysis, respectively, but with PCR analysis two of eight grafts were positive for IgH rearrangement. While further studies are needed to evaluate the efficacy of purging and the outcome of patients undergoing autologous transplantation, CHOP-R therapy can be safely and effectively used in the mobilization phase of PBSC collection, without excess clinical toxicity or deleterious effect on PBSC mobilization kinetics or engraftment time.

We present a rare case of diffuse large B-cell lymphoma transformed from immunoglobulin (Ig) A-secreting marginal zone B-cell lymphoma. A 62-year-old woman was admitted to our hospital for examination of a disseminated pulmonary shadow. Gradual swelling of bilateral axilla and right inguinal lymph nodes were noted after admission. Histological examination of the lymph node biopsy specimen revealed the appearance of marginal zone B-cell lymphoma. The surface Ig of lymphoma cells was IgA-kappa, which coincided with the class of monoclonal Ig found in the patient's serum. The lymph node swelling and pulmonary shadow subsided, and the serum IgA level was normalized by 3 courses of systemic chemotherapy. However, after 4 courses of treatment, new tumor lesions at the right chest wall and left arm progressively became apparent. The biopsy specimen of the tumor showed a feature of diffuse large B-cell lymphoma. Despite intensive chemotherapy, the patient died of spreading tumor burden into the central nervous system.

One human body is composed of 6 x 10(13) cells, and eyes are also composed of many cells of different functions. The cellular functions and intercellular interaction are regulated by many regulators including cytokines and growth factors to maintain the homeostasis. The transforming growth factor-beta (TGF-beta) superfamily, a large family of multifunctional factors, regulates various cellular functions, including cellular proliferation, migration, differentiation, apoptosis and extracellular matrix production. The TGF-beta superfamily contains about 30 multifunctional factors, and is divided into several families according to the sequence homology. The TGF-beta family, the activin family, and bone morphogenic proteins belong to the TGF-beta superfamily. TGF-beta superfamily members transduce signals through type I and type II serine/threonine type transmembrane receptors. The signals are transduced from receptors through nuclei by Smad family members, which are phosphorylated by the activated type I receptors and translocate from cytoplasm into nuclei. TGF-beta family members and the TGF-beta superfamily receptor family are expressed in ocular tissues including the cornea, ciliary epithelium, lens epithelium, retina, and blood vessels. This observation suggests the importance of the TGF-beta superfamily in eyes. Smad family members (Smad 1, Smad 2, Smad 3 and Smad 4) are expressed in the cultured retinal pigmant epithelial cell line (D407), in which TGF-beta and activin A stimulate the translocation of Smad 2, but not Smad 1 into nuclei, whereas bone morphogenetic protein (BMP) stimulates that of Smad 1, but not Smad 2. TGF-beta superfamily members play important roles in the pathogenesis of retinal neovascularization and in the wound healing process of corneal tissue. TGF-beta inhibits the endothelial functions, but, stimulates angiogenesis in vivo. TGF-beta is involved in the formation of abnormal connective tissue in corneal wound healing. In these processes, many cytokines and growth factors are involved, interacting with each other and forming networks. It is mandatory to clarify the networks to investigate molecular pathogenesis and new therapeutic agents.

A 84-year-old man was admitted with diabetes mellitus, hypertension and chronic renal failure in September 1994. In October 1995, his renal function gradually worsened with hyperkalemia. Sodium polystyrene sulfonate (Kayexalate) was administered orally for the treatment of hyperkalemia. However, after 7 days from the start of Kayexalate therapy, thrombocytopenia progressed gradually, and 12 days later the initial platelet count of 20.7 x 10(4)/microliter decreased to 8.6 x 10(4)/microliter. This thrombocytopenia rapidly improved after cessation of Kayexalate administration. In December 1995, readministration of Kayexalate for the treatment of hyperkalemia induced thrombocytopenia again. Bone marrow aspiration biopsy revealed normal counts of nucleated cells and megakaryocytes with no increase in blasts. No other disorders which cause thrombocytopenia were seen in this patient. The complication of thrombocytopenia associated with Kayexalate has not been reported. This is the first reported case of thrombocytopenia caused by Kayexalate administration.

Human peripheral blood progenitor cells (PBPC) are currently used as a source of hematopoietic reconstitution by autologous transplantation after myeloabrative chemotherapy for malignancies. PBPC would also be useful for allogeneic transplantation since the collection of PBPC is much safer than that of bone marrow stem cells (BM). For allogeneic transplantation, it is imperative to confirm that PBPC contains self-renewable stem cells that can sustain a long lasting hematopoiesis. In the present study, we examined the reconstitution of human hematopoiesis in severe combined immunodeficiency (SCID) mice by transplanting peripheral blood CD34+ cells in which the neo gene was transduced as a marker. In 2 of 4 mice receiving PB-CD34+ cell transplantation, the neo gene appeared as early as 4 weeks and lasted as long as 24 weeks in all DNA preparations of bone marrow, peripheral blood and spleen cells from the SCID mice, while in 2 of 4 mice receiving BM-CD34+ cell transplantation, although the neo gene also lasted as late as 24 weeks, it did not appear as early as in the mice receiving PB-CD34+ cell transplantation. A similar observation was noted in clinical trials, i.e. the white blood cell and platelet recovered earlier by transplantation of PBPC than of BM. In mice who had the neo gene, we were also able to demonstrate by FACS the presence of human lineage specific antigen in the cells as late as 24 weeks after transplantation with PB-CD34+ cells, and the presence of human IgG in the sera 10 weeks after transplantation. These findings indicate that PB-CD34+ cells contain long-term repopulating stem cells which undergo differentiation in SCID mice.

A 69-year-old woman was admitted with apoplexy after operation of mitral valve stenosis and gastrectomy due to a gastric ulcer. In June 1994, her condition gradually worsened after acute pneumoniae in her right lung. Intravenous hyperalimentation with cimetidine administration was started to improve her undernourishment, because she had a history of gastric ulcer. However, after 10 days from the start of cimetidine therapy, anemia progressed rapidly. Biochemical examinations revealed that the serum indirect bilirubin and LDH levels were elevated and no serum haptoglobin was detected. These results indicated the development of hemolytic anemia, but at that time we could not clarify the reason. In October 1994, thrombocytopenia gradually progressed, and we halted the administration of cimetidine to ranitidine. Both hemolytic anemia and thrombocytopenia was dramatically improved after cessation of cimetidine administration. We then changed the drug from cimetidine, however the same phenomena have appeared again. The patient was in stable condition, after cessation of H2-blockers administration. The complication of hemolytic anemia and thrombocytopenia associated with H2-blocker administration in Japan.

In a search for the optimal method to harvest peripheral blood progenitor cells (PBPC) without myeloablative chemotherapy, we administered recombinant human granulocyte colony-stimulating factor (G-CSF) to adult, healthy volunteers and investigated the mobilization rate of the PBPC. The consecutive subcutaneous administration of G-CSF in a dose of 2 micrograms/kg/d for 5 days significantly increased colony-forming units-granulocyte/macrophage (CFU-GM) up to 2340 +/- 980 per mL whole blood with a 30 +/- 21-fold mobilization of PBPC, range 11- to 76-fold. Burst-forming units-erythroid (BFU-E) and mixed erythroid progenitors (CFU-Mix) were also increased, and the mobilization rate was 8.4 +/- 4.5-fold and 7.6 +/- 4.7-fold, respectively. A time course study of PBPC after final G-CSF injection was done for 0 to 30 hours on days 1, 3, and 5. After a single administration of G-CSF (day 1), there was no increment of PBPC during the subsequent 30 hours, but the white blood cell count (WBC) markedly increased. Three days after the injection of G-CSF, no increase of CFU-GM was seen during the first 2 hours, then a significant, time-dependent increase occurred for up to 30 hours. Five days after the injection of G-CSF, there was no increase of CFU-GM during the first 2 hours, and the significant increase at 24 hours was maintained for up to 30 hours. BFU-E and CFU-Mix showed the same pattern of expansion as seen with CFU-GM. Thus, subcutaneous administration of 2 micrograms/kg/d G-CSF for 5 days will lead to a successful PBPC harvest for transplantation, and the most suitable period for this is 24 to 30 hours after the final G-CSF administration.

In the present paper, we investigated the pathophysiological implication of TGF-beta from megakaryocytes or megakaryoblasts in the development of myelofibrosis. In the bone marrow of myelofibrosis, proliferation of megakaryocytes is often noticed. We therefore investigated the TGF-beta expression in the bone marrow megakaryocytes from 12 chronic myeloproliferative disorder patients with myelofibrosis by immunohistochemical analysis. About all the specimen showed strong positivity for TGF-beta. In order to examine whether megakaryoblasts produce TGF-beta, we then measured TGF-beta activity in the conditioned medium (CM) of megakaryoblasts from a patient with acute megakaryoblastic leukemia who had profound myelofibrosis. The CM showed strong collagen synthesis stimulating activity which was nullified by addition of anti TGF-beta antibody. Since TGF-beta exists as latent form in platelets, TGF-beta was considered to be altered from active to latent form during megakaryocytes differentiation. In this context, MEG-01, a megakaryoblastic cell line which produces active TGF-beta was underwent differentiation to produce platelet-like bleb with TPA treatment. During the differentiation, MEG-01 showed the decrease of active TGF-beta production and increase of latent TGF-beta together with the production of LTBP. These results suggest that megakaryoblasts produce active TGF-beta and may may cause myelofibrosis, while more differentiated megakaryocytes produce latent TGF-beta. Mechanism by which megakaryoblast escape from negative autocrine of active TGF-beta was also investigated. MEG-01 was found to express mutated p53 which is considered to be responsible for impaired signal transduction of TGF-beta.

A 62 year old male patient was previously admitted to another hospital in February 1989 because of palpitation. Peripheral blood examination revealed pancytopenia. Although the number of nucleated cells in the bone marrow aspirate was in the normal range. It contained 2.2% of blastic cells and dysplastic cells. He was diagnosed to be myelodysplastic syndrome with refractory anemia. He subsequently received repeated blood transfusions and other symptomatic treatments as an outpatient until 1990. When he was admitted to our hospital because of severe pancytopenia. The numbers of WBC, RBC, and platelets were 2,000/microliters, 134 x 10(4)/microliters, 2.9 x 10(4)/microliters respectively. Bone marrow aspiration resulted in dry tap, and biopsy at the iliac bone showed remarkable fibrosis with marked decrease of normal hematopoietic cells. Chromosome analysis revealed multiple aberrations such as 47XY, +8, 13q-, 14p+, 48XY, +8, +9, 13q+. The patient was treated with BHAC-AMP combination chemotherapy. After 3 cycles of the therapy, pancytopenia was improved and chromosomal aberration disappeared. This case was considered to be an acute myelofibrosis developed from myelodysplastic syndrome and worth to reporting with a review of literature, because drastic combination chemotherapy was extremely effective.

Specific DNA probes for genes encoding immunoglobulins (Ig) and the T cell receptor (TCR) are useful diagnostic tools in lymphoproliferative disorders. Gene rearrangement analysis was carried out in 2 cases of conjunctival lymphoid lesions. A 36-year-old man (case 1) had a 1-year history of left conjunctival tumor. A biopsy was performed and histopathological findings showed diffuse proliferation of small lymphocytes, but monoclonality was not revealed by immunophenotypic analysis. A right conjunctival lesion developed and five months later a biopsy of the left conjunctiva was performed again. A frozen sample was analyzed and immunoglobulin heavy chain gene rearrangement was found. A 53-year-old woman (case 2) had a 6-month history of bilateral conjunctival tumor. The first biopsy did not reveal monoclonality immunophenotypically. A second biopsy with a frozen specimen was analyzed and immunoglobulin heavy chain gene rearrangement was found. We diagnosed these two cases as B-cell lymphoma. We discuss the clinical value of gene rearrangement analysis as a diagnostic method for lymphoproliferative disorders.

A 63-year-old male with refractory anemia with excess of blasts (RAEB) and hypoplastic marrow was treated with K-18 (240 mg/day P.O.). On admission, peripheral blood revealed pancytopenia. Bone marrow specimen revealed severe hypocellularity with 18.9% of the blast cells. Ten months later, the blast cells in the bone marrow decreased to 3.8%, and complete remission (CR) was obtained. CR was eight weeks. Duration of response (CR + PR) continued for about eight months. K-18 is an antitumor agent with minimal side effects, and seems to be effective for RAEB with hypoplastic marrow.

Attempts were made to clarify the mechanism of platelet aggregation and to characterize the platelet aggregating material employing established human cancer cell lines. Eleven out of the nineteen human cancer cell lines investigated showed platelet aggregating activity. The existence of divalent cation was required for the platelet aggregation induced by HMV-1 tumor cells. The platelet aggregations induced by tumor cells (HMV-1, PC-10, 3LL) were not suppressed by specific thrombin inhibitor (MD-805). The platelet aggregating activities of tumor cells (HMV-1, M 7609) were diminished by treatment with trypsin but not with collagenase or neuraminidase. Aggregating activity was preserved with a preparation of membrane from these tumor cells, although it was abolished by heating(100 degrees C 15 min) or sonication. By SDS PAGE (autoradiography), membrane proteins with MW of 20,000 daltons which specifically bound to platelets were commonly found in cells with platelet aggregating activity (HMV-1, M 7609), but were absent in platelet non-aggregating cells (HGC-25). It is therefore concluded that platelet aggregation induced by human tumor cells does not require the coexistence of thrombin, but is evoked by direct interaction of platelets with aggregating proteins (MW 20,000 daltons) on the cell membrane.

To elucidate the correlation of platelet aggregating activity of tumor cells and their metastatic potentials, we established a highly and a low metastatic clone with a different platelet aggregating activity from murine fibrosarcoma (Meth A). The parental Meth A cell and its low metastatic clone (ML-01) showed a typical platelet aggregation pattern with a certain lag time, while a highly metastatic clone (MH-02) showed a biphasic aggregation with no lag time in which the first reversible aggregation was caused by ADP released from MH-02, since it was eliminated by apyrase treatment. The highly metastatic potential, however, was not solely due to the platelet aggregating activity because the administration of PGI2-analogue TEI-8153A did not completely inhibit their pulmonary metastasis. In fact, MH-02 attached more preferentially to type IV collagen or endothelial cell than ML-01 in vitro. These results suggest that MH-02 exerts its high metastatic property through the mechanisms involving multiple factors such as the increased platelet aggregating potential or enhanced adhesiveness.