Mechanism of NK cell activation induced by coculture with dendritic cells derived from peripheral blood monocytes.

Dendritic cells (DCs) have been regarded as one of the effective antigen-presenting cells, but the relationship between DCs and lymphocytes, in particular natural killer (NK) cells, remains unclear. In this study, we evaluated how DCs interact with both lymphocytes and NK cells using a coculture system. The number of lymphocytes increased significantly when cocultured with DCs (1.8-fold increase). In particular, the proliferation of NK cells was prominent. Furthermore, the coculture of DCs with lymphocytes induced a marked increase in IL-12 and IFN-gamma secretion. When contact between the DCs and lymphocytes was prevented, the secretion of both IL-12 and IFN-gamma was markedly reduced. IFN-gamma production was completely blocked by an anti-IL-12 antibody, indicating that IFN-gamma secretion was dependent on IL-12 secretion. The stimulating effect of the DCs on the proliferation of the lymphocytes was partially suppressed by anti-IL-12 antibodies, and was completely attenuated when cellular contact was prevented. Furthermore, the NK cell proliferation induced by coculture with DCs was significantly blocked by the inhibition of the interaction of either CD40-CD40L or CD28-B7 molecule. The coculture with DCs enhanced NK activity by 40%, and this was partially suppressed by anti-IL-12 antibodies and was completely blocked by the inhibition of cell-to-cell contact. These results indicate that the activation of NK cells by DCs is partially mediated by IL-12 secretion, and that direct contact between DCs and NK cells play a major role in this response.

Prevention of murine acute graft-versus-host disease by staphylococcal enterotoxin B treatment.

Retroviral superantigens such as minor lymphocyte stimulating (Mls) antigen play an important role in the pathogenesis of acute graft-versus-host disease (GVHD). However, it remains unclear how exogenous bacterial superantigens modulate acute GVHD. In this study, we tested the effects of staphylococcal enterotoxin B (SEB) on the development of acute GVHD in a model involving the systemic transfer of parental C57Bl/6 spleen cells into BDF1 mice. SEB treatment suppressed the expansion of donor-derived T cells and blocked the decrease in the number of host cells. Impaired haematopoiesis was actually rescued by treatment with SEB. In SEB-treated mice, both spontaneous proliferation and IL-2 production in T cells were suppressed on day 2 after parental cell infusion. On day 21, the number of donor-derived CD4+ Vbeta8+ T cells markedly decreased in the spleen of SEB-treated mice. Donor-derived CD4+ T cells failed to proliferate in response to host alloantigens, and both donor- and host-derived T cells were unable to produce IL-2 in response to concanavalin A stimulation, suggesting that SEB treatment induced a general immunosuppressive state. Our results indicate that SEB treatment prevents the development of acute GVHD by leading to unresponsiveness of donor-derived T cells against host alloantigens in a Vbeta-restricted and unrestricted manner.

[ALL complicated by obstructive jaundice due to choledocholithiasis after unrelated bone marrow transplantation].

A 22-year-old man with acute lymphocytic leukemia underwent allogeneic bone marrow transplantation (BMT) from an unrelated donor in October 1996. In April 1997, he suddenly developed severe abdominal pain with nausea and vomiting. The diagnosis was obstructive jaundice associated with gallstones in the gallbladder and common bile duct. The patient underwent laparoscopic cholecystectomy and endoscopic removal of the stones in the common bile duct. The major component of the gallstones was bilirubinate calcium. Although the pathogenesis of gallstones after BMT remains unclear, several factors including impaired contractility of the gallbladder, hemolysis, changes in bile composition, and biliary tract infection may play important roles.

Interleukin 6 receptor expression by human cord blood- or peripheral blood-derived primitive haematopoietic progenitors implies acquisition of different functional properties.

The significance of interleukin 6 receptor (IL-6R) expression by cord blood (CB)- and peripheral blood (PB)-derived primitive haematopoietic progenitors was investigated. IL-6R was preferentially expressed by PB-derived myeloid progenitors. Most PB-derived erythroid bursts (BFU-E) and mixed colony-forming cells (CFU-Mix) did not express this receptor. However, CB-derived primitive progenitor cells possessed multipotentiality, irrespective of IL-6R expression. Interestingly, the long-term culture-initiating cell (LTC-IC) population was enriched in PB-derived CD34+ IL-6R+ cells, but the extended LTC-IC (ELTC-IC) population, which represents a less mature class of haematopoietic progenitors, seemed to be equally distributed in the IL-6R+ and IL-6R- cell populations. In contrast, the number of LTC-ICs and ELTC-ICs was similar in CB-derived CD34+ IL-6R+ or IL-6R- cells. It is noteworthy that the number of LTC-ICs and ELTC-ICs in CB-derived CD34+ cells was markedly higher than that in PB-derived CD34+ cells regardless of IL-6R expression. Telomerase activity was consistently lower in PB-derived CD34+ IL-6R- cells than in CD34+ IL-6R+ cells. In contrast, telomerase activity was similar in CB-derived CD34+ IL-6R+ or IL-6R- cells. The pattern of telomerase induction upon cytokine stimulation differed between CB- and PB-derived CD34+ IL-6R+ or IL-6R- cells. However, overall telomerase activity per dish was well correlated with the proliferative potential of both cell populations, suggesting that induction of telomerase plays an important role in the escape from replicative senescence of primitive haematopoietic progenitors. Collectively, these results suggest that CB-derived primitive progenitors are less mature than PB-derived progenitors and that the expression of IL-6R by primitive haematopoietic progenitors may have different implications for PB- and CB-derived CD34+ cells.

Adsorption and pharmacokinetics of cyclosporin A in relation to mode of infusion in bone marrow transplant patients.

Two main factors that affect the pharmacokinetics of cyclosporin A (CsA) during 24-h durable intravenous (DIV) administration have been reported, namely physiological changes after bone marrow transplantation, and blood sampling through indwelling lines. In addition, it has been found that infusion sets made of polyvinyl chloride (PVC) markedly adsorb CsA. We conducted in vitro adsorption studies of CsA on infusion sets, and the administration routes that are used in the treatment of patients with bone marrow transplantation. We also examined the effects of administration route on CsA pharmacokinetics in clinical practice. The in vitro adsorption study using 30-mm segments of lumen from commercially available infusion sets showed that the degree of CsA adsorption per area of lumen made of PVC was significantly higher than that in those made of polyethylene (PE) or polybutadiene (PB), which showed no adsorption of CsA. Due to its adsorption, use of infusion sets made of PVC resulted in about a 40-50% loss of CsA dose, which affected the pharmacokinetic parameters during 24-h DIV, while those made of PE and PB did not. The use of non-PVC infusion sets should allow for accurate monitoring of CsA results, and provide cost benefit in the treatment of bone marrow transplantation.

Stromal cell-derived factor-1 (SDF-1) acts together with thrombopoietin to enhance the development of megakaryocytic progenitor cells (CFU-MK).

Stromal cell-derived factor-1 (SDF-1) is a CXC chemokine that acts as a stimulator of pre-B lymphocyte cell growth and as a chemoattractant for T cells, monocytes, and hematopoietic stem cells. More recent studies also suggest that megakaryocytes migrate in response to SDF-1. Because genetic elimination of SDF-1 or its receptor lead to marrow aplasia, we investigated the effect of SDF-1 on megakaryocyte progenitors (colony-forming units-megakaryocyte [CFU-MK]). We report that SDF-1 augments the growth of CFU-MK from whole murine bone marrow cells when combined with thrombopoietin (TPO). The addition of SDF-1 to interleukin-3 (IL-3) or stem cell factor (SCF) had no effect. Specific antagonists for CXCR4 (the sole receptor for SDF-1), T22, and 1-9 (P2G) SDF-1 reduced megakaryocyte colony growth induced by TPO alone, suggesting that many culture systems contain endogenous levels of the chemokine that contributes to the TPO effect. To examine whether SDF-1 has direct effects on CFU-MK, we developed a new protocol to purify megakaryocyte progenitors. CFU-MK were highly enriched in CD41(high) c-kit(high) cells generated from lineage-depleted TPO-primed marrow cells. Because the growth-promoting effects of SDF-1 were also observed when highly purified populations of CFU-MK were tested in serum-free cultures, these results suggest that SDF-1 directly promotes the proliferation of megakaryocytic progenitors in the presence of TPO, and in this way contributes to the favorable effects of the bone marrow microenvironment on megakaryocyte development.

Trisomy 10 as the sole abnormality in biphenotypic leukemia.

We observed a case of acute biphenotypic leukemia with trisomy 10 as the sole abnormality. The patient was an adult male diagnosed with ALL(L2), Cell marker studies showed positivity for CD3, CD7, CD13 and CD33, so the phenotypic diagnosis was determined to be biphenotypic leukemia. No case of biphenotypic leukemia with trisomy 10 has been previously reported, until now.

Sodium butyrate blocks interferon-gamma (IFN-gamma)-induced biosynthesis of MHC class III gene products (complement C4 and factor B) in human fetal intestinal epithelial cells.

Human intestinal epithelial cells have been established as local sites for complement biosynthesis. In this study, we investigated the effects of IFN-gamma and sodium butyrate on biosynthesis of MHC class III gene products (complement C4 and factor B) in the human fetal intestinal epithelial cell line INT-407. IFN-gamma induced a dose- and time-dependent increase in C4 and factor B secretion. However, sodium butyrate dose-dependently inhibited IFN-gamma-induced C4 and factor B secretion. These effects were also observed at the mRNA level. Immunoblotting indicated that IFN-gamma induced a rapid activation of Stat1alpha, and fluorescence immunohistochemistry detected a translocation of Stat1alpha into the nucleus within 1 h. However, the translocation of Stat1alpha was not affected by the addition of sodium butyrate. Nuclear run-on assay indicated that IFN-gamma induced a weak increase in the transcription rate of factor B gene, and sodium butyrate did not affect this response. IFN-gamma and sodium butyrate induced a counter-regulatory effect on C4 and factor B secretion: IFN-gamma acted as a potent inducer, but sodium butyrate potently abrogated these responses. These are mainly regulated through the post-transcriptional mechanism.

[Phase II clinical study of SH L 573 (fludarabine phosphate) in patients with chronic lymphocytic leukemia].

We conducted a multicenter phase II clinical study of fludarabine phosphate, a new purine nucleotide analogue, in patients with chronic lymphocytic leukemia (CLL). Fludarabine phosphate was administered at a dose of 20 mg/m2/day intravenously for 5 days every 4 weeks as one course. Six courses as a maximum were repeated. The response rate was 38.5% (95% confidence intervals: 20.2% to 59.4%), with 1 complete remission and 9 partial remissions out of 26 treated patients. Major drug-related adverse reactions were fever, nausea, weakness, and paresthesia of the fingers; as a grade-3 reaction, varicella was also reported. Neutropenia and thrombocytopenia were observed as manifestations of hematologic toxicity. Clinical laboratory test results revealed abnormalities in hepatic function, including increased GPT, but none of these was rated grade 3 or 4.

[Effective pentostatin-based treatment of adult T cell leukemia in a patient with severe arthritis].

We report a 48-year-old woman with adult T-cell leukemia who had refractory arthralgia, intense headaches, and fever. Leukemic cell infiltration of the cerebrospinal fluid was detected but no other acute signs were observed. Abnormal lymphocytes with lobulated nuclei were found in the synovial fluid, and a histologic examination revealed proliferation into the synovium. Because combination chemotherapy did not elicit a favorable response, the patient was treated with a pentostatin bolus injection. The articular symptoms disappeared and complete remission was obtained. Six months later, she experienced arthralgia again together with a gradual increase of abnormal lymphocytes in peripheral blood. Sixteen months later, the patient was given pentostatin and achieved a complete remission again. She is still free from relapse without further therapy after 36 months, and her articular symptoms have not returned either. There were no adverse effects due to pentostatin. The patient's serum IL-6 level was elevated, suggesting that IL-6 may play a role in arthropathy.

Haematopoietic action of flt3 ligand on cord blood-derived CD34-positive cells expressing different levels of flt3 or c-kit tyrosine kinase receptor: comparison with stem cell factor.

We compared the effect of human flt3 ligand (FL) and stem cell factor (SCF) on cord blood (CB)-derived CD34+ cells expressing different levels of flt3 or c-kit tyrosine kinase (TK) receptor in clonal cell culture. The c-kit receptor was expressed by 58.5+/-16.7% of CB CD34+ cells (n=19), in which c-kit(high), c-kit(low) and c-kit cell populations could be identified. In contrast, the flt3 receptor (FR) was weakly expressed on 58.6+/-8.3% (n=9) of CB CD34+ cells. FL+erythropoietin (Epo) failed to support erythroid burst (BFU-E) formation by any subpopulation of CD34+ cells. However, SCF + Epo supported BFU-E and erythrocyte-containing mixed (CFU-mix) colony formation from all subpopulations. Interestingly, FL markedly augmented CFU-mix colony formation supported by interleukin (IL)-3 + Epo when CD34+c-kit(low) or CD34+FR+ cells were used as the target. On the other hand, SCF significantly enhanced CFU-mix colony formation supported by IL-3 + Epo when CD34+c-kit(high) or low and CD34+FR+ cells were used. The replating potential of CFU-mix supported by IL-3 + Epo+ FL was greater when CD34+c-kit(low) or CD34+FR+ cells were used. When the CD34+c-kit(low) cells were used, the number of lineages expressed in secondary cultures of CFU-mix colonies derived from primary cultures containing IL-3 + Epo + FL or SCF was significantly larger than when the primary cultures contained IL-3 + Epo. Furthermore, the number of long-term culture-initiating cells found in CD34+FR+ cells was larger than that in FR cells. CB-derived CD34+c-kit(low) cells represent a less mature population than c-kit(high) cells, as reported previously. Therefore, these results indicate that both FL and SCF can act on primitive multipotential progenitors. However, it is still uncertain whether CB-derived CD34+FR+ cells are less mature than CD34+FR- cells.

Aggressive jejunal gamma deltaT-cell lymphoma derived from intraepithelial lymphocytes: an autopsy case report.

A 69-year-old man with massive ascites was referred to our hospital. Paracentesis revealed exudative ascites with many abnormal lymphocytes, which expressed T-natural killer (T-NK) cell surface markers and gamma deltaT-cell receptor (TCR). Although the ascites resolved for a short time with chemotherapy, gastrointestinal bleeding occurred and acute retention of ascites was observed. The patient died of panperitonitis. At autopsy, part of the jejunum revealed ulceration and marked mucosal thickening, and was perforated at the site of the severe ulceration. Histological examination showed massive infiltration of abnormal lymphocytes that were positive for CD45RO. Therefore, the cells responsible for the jejunal lymphoma and ascites were thought to be derived from gamma deltaIEL.

Acute lupus peritonitis successfully treated with steroid pulse therapy.

A 21-year-old man with systemic lupus erythematosus (SLE) who developed acute lupus peritonitis is described. Acute lupus peritonitis appeared during generalized lupus flare, with nausea, vomiting, frequent diarrhea, and abdominal tenderness with rebound and guarding. The patient was afebrile and had decreased bowel sounds. Abdominal ultrasonography and computed tomography revealed marked thickening of the gastric, duodenal, and jejunal walls, massive intraluminal fluid collection, and increasing ascites. Gastrointestinal endoscopy showed edematous mucosa with multiple erosions of the stomach and duodenum. The ascitic fluid was remarkable for low complement levels and elevated anti-DNA antibody. These manifestations of acute lupus peritonitis resolved after steroid pulse therapy with methylprednisolone. We should consider acute lupus peritonitis in a patient with SLE when abdominal symptoms are severe. Experience with our patient indicates that steroid pulse therapy is effective for this rare but severe manifestation of SLE.

A case of interstitial pneumonitis associated with natural alpha-interferon therapy for myelofibrosis.

A 55-year-old man with myelofibrosis was treated with natural alpha-interferon with a good hematologic response. Initially, he had anemia, leukocytosis, thrombocytosis and hepatospleomegaly. A bone marrow biopsy showed replacement with fibrosis with an increase in megakaryocytes. Natural alpha-interferon (alpha-IFN) was started at a dose of 3 x 10(6) units/day. The leukocyte and platelet counts gradually normalized, and the liver and spleen decreased in size. However, the patient complained of a dry cough and dyspnea on the 61st treatment day, when the accumulated dose of alpha-IFN treatment had reached 1.8 x 10(8) units. He subsequently developed acute respiratory failure (PaO2 < 60 mm Hg) with bilateral lung infiltrations, suggesting the occurrence of interstitial pneumonitis associated with alpha-interferon therapy. Immediately, the alpha-interferon was discontinued and high-dose methylprednisolone (1.5 g/day) was administered for 3 days. This treatment was followed by oral prednisone therapy. Steroid therapy brought about gradual improvement as suggested by a repeat radiograph. Since high levels of fibrogenic cytokines, such as PDGF and TGF-beta, have been reported in patients with myelofibrosis, it is necessary to pay attention to interstitial pneumonia as a complication in alpha-IFN therapy for myelofibrosis.

Severe anemia in a patient with isolated adrenocorticotropin deficiency.

A 64-year-old man was referred to our hospital for evaluation of progressive anemia. On admission, he had a severe normocytic normochromic anemia [hemoglobin 7.5 g/dl] requiring a blood transfusion. Endocrinological studies demonstrated an isolated ACTH deficiency. After receiving glucocorticoid replacement therapy, his anemia was rapidly corrected, his hematocrit and hemoglobin remained elevated for approximately 4 months. We present evidence that glucocorticoid plays an important role in the physiological regulation of human erythropoiesis.

Thalassemia incidentally found by marked erythrocytosis due to an ochre mutation at codon 35 in a Japanese man.

We present a patient with beta-thalassemia minor diagnosed on the basis of the incidental findings of marked hypochromic and microcytic erythrocytosis. Hemoglobin analysis revealed increased hemoglobin (Hb) A2 levels and decreased beta/alpha synthesis ratio in both the propositus and his mother. Further molecular studies identified a single base substitution of TCA to TAA within codon 35 in heterozygous state, which creates a premature terminator resulting in a defect of effective beta globin synthesis. This is the first report of beta-thalassemia due to a nonsense mutation at codon 35 of beta-thalassemia gene in the Japanese population.

[A case of acute myelogenous leukemia accompanied with myelofibrosis and megakaryocyte-like giant bizarre blasts].

A 45-year-old man was admitted with high fever and leukocytosis in August 1993. The diagnosis of acute myelogenous leukemia (AML; M2) was made on the basis of morphological, cytochemical and immunological characteristics of the blasts in the bone marrow. The induction therapy with BHAC, daunorubicin, 6-MP was unsuccessful in achieving remission; the bone marrow biopsy specimen revealed the proliferation of the remaining leukemic cells and massive fibrosis accompanied with unusual megakaryocyte-like giant bizarre cells. These megakaryocyte-like giant cells were positive for myeloperoxidase and CD34, but not GPIIIa and factor VIII, indicating that those were derived from myelogenous stem cells. Following the low-dose Ara-C therapy, improvement of fibrosis and disappearance of these giant cells were observed in the bone marrow. After the reinduction therapy with high-dose Ara-C and MIT against markedly increased blasts, the patient died of systemic fungal infection. The presence of myelofibrosis and giant atypical blasts might allow resistance to therapy and poor prognosis.

Band 4.2 Shiga: 317 CGC-->TGC in compound heterozygotes with 142 GCT-->ACT results in band 4.2 deficiency and microspherocytosis.

A novel compound heterozygous mutation of 317 CGC-->TGC with 142 GCT-->ACT in human red cell band 4.2 deficiency is described. A proband and his son suffered from compensated haemolysis with nearly complete deficiency of red cell band 4.2. Their red cell morphology exhibited microspherocytosis resembling classic hereditary spherocytosis (HS). Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed band 4.2 to be nearly missing (< 1% of normal controls) with the presence of 74 kD and 72 kD isoforms in trace amounts. Other family members (daughters older and younger than the son) exhibited nearly normal amounts of 72kD as a wild form of band 4.2 on SDS-PAGE with the presence of the 74kD isoform in a trace amount. The proband and his son demonstrated two compound heterozygous mutations in trans: i.e. nucleotide (nt) 949 CGC-->TGC (codon 317 Arg-->Cys) in exon 7 and nt 424 GCT--ACT (codon 142 Ala-->Thr) in exon 3 of the band 4.2 gene. The two daughters demonstrated only the mutation of nt 949 CGC-->TGC in exon 7 in heterozygous states, but no 142 mutation. Therefore the proband and his son were compound heterozygotes of these two mutations in trans. It is interesting to note that the 74 kD isoform of band 4.2 protein existed in a trace amount in the two daughters in spite of the absence of the 142 Ala-->Thr mutation. In addition, even in the presence of the 142 mutation in one allele in the proband and his son, their red cell morphology demonstrated classic HS with microspherocytosis, although a homozygous state of the 142 mutation known as the Nippon type of band 4.2 deficiency exhibits ovalostomatocytosis.

Anaplastic large cell Ki-1 lymphoma with bone involvement: report of two cases.

Two cases of anaplastic large cell Ki-1 lymphoma involving bone as the most prominent and initial manifestation are reported. The first patient was a 20-year-old male who had back pain and incomplete paraparesis due to vertebral involvement. The second was a 14-year-old girl, whose first clinical signs were fever of unknown origin and sternal bone pain. Radiologically, skeletal lesions were lytic and destructive. Histopathologically, the tumour cells had pleomorphic bizarre nuclei and abundant basophilic cytoplasm. Immunohistochemically, Ki-1(CD30) reactivity was strongly positive in both cases. Tumour cells were also CD3, CD4, epithelial membrane antigen and interleukin-2 receptor positive in the first case, and CD10, HLA-DR positive in the second case. The former tumour was considered to be of T-cell lineage and the latter of lymphoid progenitor cell origin. Radiation and chemotherapy were temporarily effective. However, both patients died 14 and 7 months after diagnosis, respectively, due to systemic lymph node involvement. These observations suggest that the prognosis for Ki-1 lymphoma involving bone is poorer than indicated in previous reports.