Keisuke Sakurada

The important balance between cytokines derived from type 1 and type 2 helper T cells in the control of graft-versus-host disease

We have investigated cytokine mRNA expression in the peripheral blood mononuclear cells of 20 patients who received allogeneic hematopoietic stem cell transplants to assess the cytokine network after transplantation. IL-4 mRNA expression decreased in five of five (100%) patients with ⩾grade III (severe) acute GVHD and increased in 10 of 22 (45%) patients without severe GVHD. In contrast, IL-12 mRNA expression increased in two of two (100%) patients with severe GVHD, but increased in only six of 18 (33%) patients without severe GVHD. Furthermore, IL-10 and/or IL-13 mRNA expression increased in 19 of 22 (86%) patients without severe GVHD, but increased in only one of three (33%) patients with severe GVHD. In patients with allogeneic PBSCT who had severe acute GVHD, the cytokine mRNA expression in patients with allogeneic PBSCT, who had no severe GVHD, showed a similar pattern to that in patients with allogeneic BMT. IL-4 mRNA expression increased in three of five (60%) patients and IL-10 and/or IL-13 mRNA expression increased in five of five (100%) patients. In contrast, IL-12 mRNA expression increased in only one of three (33%) patients. Serum IL-4 concentration in allogeneic PBSCT patients in the early engraftment phase was relatively high, while serum IL-12 concentration was low. These findings suggest that severe GVHD may be related to the cytokine imbalance between type 1 helper T (Th1) cells and type 2 helper T (Th2) cells.

Cytokine gene expression in peripheral blood mononuclear cells during graft-versus-host disease after allogeneic bone marrow transplantation

Summary. Cytokine gene expression in peripheral blood mononuclear cells during the development of graft‐versus‐host disease (GVHD) in patients who underwent allogeneic bone marrow transplantation (allo BMT) was analysed using a semiquantitative reverse‐transcriptase polymerase chain reaction (RT‐PCR). The expression of interleukin (IL)‐lβ, IL‐6, and tumour necrosis factor (TNF)‐α mRNA was increased during the development of GVHD and the degree of this increment depended on the severity of the disease. IL‐2 expression was not detected at all and interferon‐γ expression was not much changed during GVHD. In patients with hepatic veno‐occlusive disease (VOD), another transplantation‐related complication, the expression of IL‐1β and TNF‐a mRNA was increased but IL‐6 mRNA expression showed little increase. These findings suggest that IL‐lβ, IL‐6 and TNF‐α produced by peripheral blood mononuclear cells play an important role in the development of GVHD. Furthermore, liver dysfunction due to GVHD or VOD may be distinguishable by this type of cytokine analysis. Analysis of cytokine mRNA expression in peripheral blood mononuclear cells after allogeneic bone marrow transplantation may provide important information concerning the immune response and the cytokine network system in marrow transplant patients.

Possible roles of an adult T-cell leukemia (ATL)-derived factor/thioredoxin in the drug resistance of ATL to adriamycin

Chemotherapy for adult T-cell leukemia (ATL) has been reported to fail to induce complete remission because of drug resistance in most patients. We have examined the expression of an ATL-derived factor (ADF)/thioredoxin in relation to resistance to adriamycin (ADM) in various T-cell leukemia cell lines including ATL cell lines. Immunoblot analysis demonstrated that ATL cell lines expressed ADF/thioredoxin at levels 2.8 to 12 times those of other T-cell acute lymphocytic leukemia (T-ALL) cell lines, and that ATL cell lines were 2 to 15 times more resistant to ADM than other T-ALL cell lines. Therefore, we established ADM-resistant cell lines from three different ATL cell lines, and examined the correlation between ADM resistance and expression of ADF/thioredoxin. ADM-resistant ATL cell lines were also found to be resistant to other drugs such as cisplatin and etoposide, and they expressed ADF/thioredoxin at levels 5 to 10 times those of parent ATL cell lines. Diamide and sodium selenite, which have been reported to inhibit ADF/thioredoxin, restored the sensitivity to ADM in ATL and ADM-resistant ATL cell lines. The MDR-1 gene product, a membrane P-glycoprotein (Pgp), was not expressed on ATL cell lines or ADM-resistant ATL cell lines. Topoisomerase II and glutathione peroxidase activities in T-cell leukemia cell lines were not correlated with ADM resistance. These results suggest that ADF/thioredoxin may play an important role in the drug resistance of ATL cells to ADM.

Inhibition by gossypol of phospholipid-sensitive Ca2+-dependent protein kinase from pig testis

Gossypol, a polyphenolic binaphthalene-dialdehyde extracted from cotton plants which possesses male antifertility action in mammals, is a potent inhibitor of phospholipid-sensitive Ca2+-dependent protein kinase from pig testis. Gossypol inhibited Ca2+-dependent activity of the enzyme without affecting its basal activity. The IC50 value (concentration causing 50% inhibition) was 31 microM when lysine-rich histone was used as substrate. Kinetic analysis indicated that the compound inhibited the enzyme non-competitively with respect to ATP (Ki = 31 microM) or lysine-rich histone (Ki = 30 microM), and competitively with respect to phosphatidylserine (Ki = 2.1 microM). With Ca2+, irrespective of the presence or absence of 1,3-diolein, the compound lowered Vmax and increased the apparent Ka for Ca2+. The compound also inhibited phosphorylation by the enzyme of high-mobility-group 1 protein (one of the endogenous substrates in the testis for the enzyme located in nucleosome), with an IC50 value of 88 microM. These results suggested that a phospholipid-sensitive Ca2+-dependent protein phosphorylation system in the testis is involved in the regulation of spermatogenesis.

Establishment of a novel granulocytic sarcoma cell line which can adhere to dermal fibroblasts from a patient with granulocytic sarcoma in dermal tissues and myelofibrosis

Summary A novel human myeloid cell line, designated HSM‐1, has been established from the pleural effusion of a patient with granulocytic sarcoma (GS) who had been followed as having primary myeiofibrosis for 10 years. When he was diagnosed as having granulocytic sarcoma in dermal tissues, no evidence of malignant transformation into leukaemia was found in both the peripheral blood and bone marrow. The established cell line was positive for myeloperoxidase, Sudan black B. Naphthol AS‐D chloroacetate esterase. Surface marker analysis revealed that HSM‐1 expressed CD4. CD13, CD11a, CDllb, Leu8. CD49b. CD49d, CD49e, CD29 and HLA‐DR.

Th2 cytokines (IL-4, IL-10 and IL-13) and IL-12 mRNA expression by concanavalin A-stimulated peripheral blood mononuclear cells during chronic graft-versus-host disease

To the Editor: We have reported previously that the response of IFN-)I mRNA expression to the stimulation of concanavalin A (ConA) in peripheral blood mononuclear cells (PBMC) in patients who had extensive chronic graft-versus-host disease (cGVHD) after allogeneic bone marrow transplantation (allo BMT) was not increased compared with those without extensive cGVHD. A similar low response of IL-2 and IL-5 mRNA expression to ConA was observed in such patients with extensive cGVHD (1). Based on these findings, we have investigated the response of IL-4, IL-10 and IL-13 (Th2 cytokines) as well as IL12 (Th2-suppressing cytokine) mRNA expression in PBMC to the stimulation of ConA in patients receiving allo-BMT, since IFN-)I and IL-2 are produced mainly by Thl cells and IL-5 is produced mainly by Th2 cells. Three patients with extensive cGVHD, 5 patients with limited cGVHD, 4 patients without cGVHD, 2 patients in acute phase (3 months after allo-BMT), 1 patient each after autoand syngeneic BMT and 2 normal individuals were analysed in the present study. PBMC were obtained from heparinized fresh blood samples and cultured at 106/ml in RPMI-1640 medium containing 10% fetal calf serum and 5 x M 2-mercaptoethanol. Replicate cultures then received either no stimulation or stimulation by addition of Con A (Pharmacia Fine Chemicals, Uppsala, Sweden) at a final concentration of 5 pg/ml for 12 h. The cells were harvested and the total RNA was extracted and then cytokine gene expression was analysed by semiquantitative RT-PCR as reported previously (Table 1) ( 1-5). Each 5 pg of total RNA was reverse-transcribed with 600 U of murine Moloney leukemia virus reverse transcriptase (BRL, Grand Island, NY, USA) and 150 pmol of random hexamer. An aliquot (1/20th) of the resulting cDNA was used for the semiquantitative polymerase chain reaction (PCR). The following primers were synthesized using a 380B DNA synthesizer (Applied Biosystems):

Cytokine Gene Expression after Allogeneic Bone Marrow Transplantation

Cytokines produced by T lymphocytes, monocytes/macrophages, and fibroblasts play a central role in the immune response and in the development of graft-versus-host disease (GVHD). Also, it has been reported that dysregulated production of cytokines maybe the primary mediator of clinical manifestation of acute GVHD. Regarding cytokine gene expression after human allogeneic bone marrow transplantation (allo BMT), we have demonstrated increased IL-1 beta, IL-6, and TNF-alpha mRNA expression in peripheral blood mononuclear cells during the development of acute and chronic GVHD and that the degree of the increase was dependent on the severity of the disease. Furthermore, overexpression of these cytokine mRNAs could be detected before the clinical manifestations of GVHD developed. In contrast, IL-2 mRNA expression was not detected in peripheral blood mononuclear cells in GVHD patients. On the other hand, we have reported that increased mRNA expression and protein product of IL-2 and IFN-gamma were evident in the mixed lymphocyte culture of the cases who developed severe lethal transplantation-related complications. Therefore, the detection of increased IL-2 and IFN-gamma gene expression in MLC appeared to be useful for predicting transplantation-related complications in BMT patients. Furthermore, we found increased IL-2 receptor alpha subunit mRNA expression in the peripheral blood mononuclear cells during GVHD. These findings may indicate the important role of inflammatory cytokines such as IL-1 beta, IL-6 and TNF-alpha in the development of the clinical manifestation of GVHD and also may be indicative of the important role of IL-2 and the IL-2 receptor in allo response perhaps mainly as an autocrine effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of mixed chimaerism and origin of bone marrow derived fibroblastoid cells after allogeneic bone marrow transplantation

Summary We assessed the origin of bone marrow derived fibroblastoid cells (BMF) in long‐term cultures of 13 samples obtained from nine patients after allo‐BMT by polymerase chain reaction (PCR) amplification of MCT118, one of the variable number of tandem repeats regions (VNTR). BMF showed a complete recipient pattern in nine samples obtained from seven patients; however, a recipient‐predominant mixed chimaeric pattern was detected in BMF from four patients. Also, two of the four patients died with bone marrow hypoplasia. These data suggest that mixed chimaeric pattern of BMF may be correlated with bone marrow hypoplasia.

All-Trans Retionic ACid in the Treatment of Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are a group of hematopoietic disorders characterized by uni- or multilineage maturation defects of the bone marrow. Controversial therapeutic results have been obtained using growth factors or differentiating agents such as 13-cis retinoic acid. In this pilot study we evaluated the effects of all-trans retinoic acid (ATRA) in 10 MDS patients (5 male, 5 female). Six patients had refractory anemia (RA), 1 had refractory anemia with excess of blasts (RAEB), and 3 had refractory anemia with excess of blasts in transformation (RAEB-t). All patients received the same dose of ATRA (45 mg/sqm/day) orally for 6 weeks. A rise in hemoglobin concentration > 1g/dl was observed in 3/10 patients, while 5/10 patients showed an increase in granulocyte count > 0.5 x 10(9)/l without concomitant increase in the percentage of blast cells in the bone marrow. A rise in the platelet count > 50 x 10(9)/l was observed in 1/10 patients. All the effects were transient and maximal responses were obtained by the fourth week of treatment. Thereafter, the peripheral blood counts started to drop again, reaching pre-therapy values by the end of the treatment. This phenomenon could be attributed either to the exhaustion of an ATRA-responding cell pool, the development of cellular resistance to ATRA or to a reduction of plasma ATRA levels after prolonged treatment. According to our results, it seems that ATRA might have therapeutic efficacy in MDS, particularly if its effect could be improved by combinations with other differentiating agents or growth factors.

Reversal effect of itraconazole on adriamycin and etoposide resistance in human leukemia cells

Itraconazole is a triazole antifungal agent that inhibits cell membrane serol biosynthesis. Currently, itraconazole is a potent candidate for in vivo use to revert multidrug resistance in acute leukemias, with the added benefit of its antifungal effect. As previously reported, itraconazole, as well as verapamil, reversed adriamycin-resistant K562 cells (K562/ADR) and HL60 cells (HL60/ADR) in dosages compatible to the plasma levels achieved by the therapeutic dosages used for the treatment of fungal infections. By RT-PCR analysis of mdrl, mdr3, and mrp mRNA, these adriamycin-resistant cells showed a higher expression of the transcript of these genes than those of the parent cells. By FACS analysis, both the adriamycin-resistant cells showed a higher expression of P-glycoprotein on their cell surfaces. These results suggested the involvement of itraconazole in the mdr gene and/or mrp gene product-associated resistance. Furthermore, itraconazole partially reversed etoposide resistance in both the K562 and K562/ADR cells. The present study suggests that itraconazole may reverse multidrug resistance, at least in part, via a classical MDR-associated mechanism.