Kinetics of pDCs, mDCs, γδT cells and regulatory T cells in association with graft versus host disease after hematopoietic stem cell transplantation.

INTRODUCTION: Although the roles of each low-frequency immunocompetent cells such as dendritic cells (DCs), γδT cells, and Treg cells in induction of acute or chronic graft versus host disease (GVHD) have been discussed in several reports, there are few papers dealing with an evaluation of these immunocompetent cells together and simultaneously in patients with hematopoietic stem cell transplantation (HSCT) and explored the kinetics of these cells in association with GVHD. METHODS: In the present study, we assessed the number of plasmacytoid DCs (pDCs), myeloid DCs (mDCs), γδT cells and Treg cells serially in patients who received allogeneic HSCT and analyzed the relationship of these cells with acute or chronic GVHD (cGVHD) by using flow cytometry. RESULTS: The percentages and numbers of pDCs, mDC1s and γδT cells were significantly lowered in the patients with acute GVHD (aGVHD) compared with those with no GVHD. On the contrary, the percentages and numbers of Treg cells were significantly elevated in the patients with aGVHD compared with those with no GVHD. As to the association with cGVHD, Treg cells were elevated in the patients with cGVHD, compared with those with no GVHD. CONCLUSION: The present study revealed an association of pDCs, mDCs, γδT cells and Treg cells with induction or treatment of GVHD.

Combination use of immune complexes and a Ca2(+) channel blocker azelnidipine enhances interleukin-12 p40 secretion without T helper type 17 cytokine secretion in human monocyte-derived dendritic cells.

Immune complexes (ICs) improve the capacity of priming specific CD8(+) cytotoxic T cell responses of dendritic cells (DCs). ICs induce phosphorylation of mitogen-activated protein kinases (MAPK) and calcium influx, although the precise regulating mechanism still remains unclear. In the present study, we investigated the effect of a Ca2(+) channel blocker on the phosphorylation of p38 MAPK and extracellular signal-regulated kinase (ERK) in immature monocyte-derived DCs stimulated with lipopolysaccharide (LPS) or LPS-ICs, and the production of interleukin (IL)-12 family members (p40, p70, IL-23), T helper type 17 (Th17) cytokines (IL-6 and IL-23), tumour necrosis factor (TNF)-alpha and IL-10 were also investigated. In comparison with LPS stimulation, LPS-ICs stimulation enhanced p38 MAPK phosphorylation significantly, which was associated with an increase in IL-12 p40 monomer/homodimer secretion. LPS-ICs also enhanced TNF-alpha and IL-6 secretion, but suppressed IL-23 secretion. The use of azelnidipine (Aze), a long-acting L-type Ca2(+) channel blocker with a high lipid solubility, suppressed p38 MAPK phosphorylation stimulated with LPS or LPS-ICs, but surprisingly enhanced IL-12 p40 monomer/homodimer secretion stimulated with LPS-ICs. This IL-12 p40 secretion-enhancing effect was not accompanied by IL-10 or IL-23 production, but was associated with ERK phosphorylation. The use of Aze did not affect IL-12 p70 production. These results suggest that the use of Aze enhances ICs-mediated IL-12 p40 secretion without additional IL-23 secretion. Therefore, the use of Aze and ICs could be a new therapeutic approach to immunomolecular therapy, as it does not cause Th17 differentiation which induces autoimmunity or reduces anti-tumour immunity.

Cutaneous lymphocyte antigen-positive T cells may predict the development of acute GVHD: alterations and differences of CLA+ T- and NK-cell fractions.

Acute GVHD (aGVHD) is a serious complication after allogeneic SCT (allo-SCT). However, an adequate immunological index is not yet available for assessing its severity. We analyzed the fraction of cutaneous lymphocyte antigen (CLA)+ cells in peripheral blood T and natural killer (NK) cells in 33 patients and evaluated its association with aGVHD. The CLA+ T-cell fraction often increased 3-7 days before the onset of aGVHD, and the maximum percentage of CLA+ T cells in grades II-IV aGVHD cases was significantly higher than that in grade 0 or I aGVHD (P<0.01). When the cutoff value of the maximum CLA+ T-cell percentage was set at 20%, any higher percentage was a significant risk for the development of severe aGVHD (P<0.0001). The maximum CLA+ T-cell percentage was significantly correlated with a high body temperature, low percutaneous oxygen saturation, and fibrinogen/fibrin degradation product D-dimer level. The post-allo-SCT CLA+ T cells exhibited a high ability to produce IL-2 and IFN-gamma, and may be the effectors and immunological markers for aGVHD. The CLA+ NK-cell-fraction steadily increased 2-4 weeks after allo-SCT but was not influenced by aGVHD. The CLA+ T-cell percentage may predict the development of severe aGVHD in clinical settings.

Induction of recipient cell-specific donor T-cell anergy by UV-C-irradiated recipient immature monocyte-derived dendritic cells.

The induction of donor T-cell anergy to recipient cells for reducing GVHD could be one way of expanding donor candidates for HLA-mismatched hematopoietic SCT. The present study was designed to clarify whether recipient cell-specific T-cell anergy could be induced by priming donor lymphocytes with recipient monocyte-derived DCs (mo-DCs) irradiated with ultraviolet-C (UV-C). By irradiation of mo-DCs with UV-C, the expression of DC-associated surface phenotypes such as CD83, CD80, CD86 and CD40 was reduced and the antigen-presenting ability of UV-C-irradiated mo-DCs was clearly decreased. By co-culturing normal donor 1 lymphocytes with UV-C-irradiated donor 2 immature mo-DCs, the response of the lymphocytes to donor 2 mature mo-DCs was markedly reduced as compared with that of the lymphocytes prestimulated with non-irradiated donor 2 immature mo-DCs or UV-C-irradiated mo-DCs derived from a different individual donor 3. The present study demonstrated that recipient cell-specific T-cell anergy could be induced by priming donor lymphocytes with UV-C-irradiated recipient immature mo-DCs in hematopoietic SCT. These data suggest the applicability of donor graft cells, which have been prestimulated with UV-C-irradiated recipient immature mo-DCs, for expanding donor candidates in HLA-mismatched hematopoietic SCT.

High-dose cytosine arabinoside and etoposide with total body irradiation as a preparatory regimen for allogeneic hematopoietic stem-cell transplantation in patients with acute lymphoblastic leukemia.

One approach to improving the outcome of allogeneic hematopoietic stem-cell transplantation for acute lymphoblastic leukemia (ALL) is to intensify the pretransplant conditioning regimen without increasing toxicity. We used an intensified conditioning regimen consisting of high-dose cytosine arabinoside (3 g/m(2) twice daily i.v. for 3 consecutive days, total six doses), high-dose etoposide (1 g/m(2) once daily i.v. during the first 2 days) and total body irradiation (TBI) (HDACE-TBI) in ALL patients. We retrospectively analyzed 21 patients treated with HDACE-TBI, of whom 18 were in complete remission (CR) and three were in non-CR at transplantation. Although gastrointestinal toxicities were common, critical regimen-related toxicities were not seen in any patients. One patient demonstrated veno-occlusive disease, which could be controlled conservatively. The disease-free survival rate of 18 patients in CR at transplantation was 61%. These results demonstrate that the HDACE-TBI combination regimen is a feasible alternative to other preparatory regimens and does not increase the regimen-related toxicity.

High-dose chemotherapy and autologous peripheral blood stem cell transplantation for treatment of unspecified peripheral T-cell lymphoma presented with hepatosplenomegaly and hypercytokinemia syndrome: report of three cases.

We report here three cases of peripheral T-cell lymphoma unspecified (PTCL-US), which presented with bone marrow infiltration and hepatosplenomegaly and were successfully treated with high-dose chemotherapy (HDCT) and autologous peripheral blood stem cell transplantation (auto-PBSCT). The patients were all characterized by cytokine-induced symptoms such as fever, anasarca, cytopenia, poor general condition, and disseminated intravascular coagulation syndrome. Laboratory data showed extremely high levels of soluble interleukin-2 receptor, beta(2)-microglobulin, and ferritin. All three patients were negative for anti-adult T-cell leukemia antibody. In one patient, hemophagocytosis was revealed by a histological examination of the bone marrow. The International Prognostic Index was high for all three patients, and they all achieved complete remission after the intensive chemotherapy for remission induction. During complete remission, they were treated with HDCT [modified interleukin-converting enzyme regimen] followed by auto-PBSCT. The recovery of hematopoiesis after auto-PBSCT was prompt and sustained engraftment was obtained. No serious adverse effects other than myelosuppression were noted. One patient died due to cerebrovascular disease without relapse 18 months after auto-PBSCT. The other two patients are still alive and have not suffered from relapse. Our observations suggest that auto-PBSCT following HDCT may be an effective and safe therapeutic modality for high-risk PTCL-US patients characterized by hepatosplenomegaly and cytokine-induced syndrome.

Lymphomatous polyp of mantle cell type in the duodenum complicated by gastric cancer: a case of trisomy 3 and t(11;14)(q13;q32).

We experienced a rare case of a lymphomatous polyp of mantle cell type forming a polypoid mass lesion in the duodenum bulbous together with advanced gastric cancer. A total gastrectomy was performed, and the specimen revealed atypical small- to medium-sized lymphoid cells with indented nuclei, which infiltrated the Peyer's patch and formed a nodular mass in the lamina propria and submucosa of the duodenum. The lymphoma cells also infiltrated the lymphoid follicle of the gastric mucosa, spleen, and regional lymph node with a typical mantle zone pattern. Flow cytometric analysis of the single cells of the lymph node and immunohistochemistry of a paraffin-embedded specimen revealed that the lymphoma cells expressed surface CD5, CD19, CD20, and nuclear cyclin D1. Chromosomal analysis of this single cell suspension revealed that these lymphoma cells have trisomy 3 in conjunction with t(11;14)(q13;q32), which is frequently seen in mucosa-associated lymphoid tissue lymphomas (MALToma) in the stomach and is also reported in mantle cell lymphoma as a secondary genetic alteration. Our report suggests that trisomy 3 may be a common chromosomal abnormality in lymphomatous polyps of mantle cell type.

Erythroid involvement in CD36 deficiency.

OBJECTIVE: The CD36 molecule is expressed in platelets, monocytes, erythroblasts, and other different tissues. The two types of platelet CD36 deficiency, types I and II, are associated with the absence and presence of CD36 on monocytes, respectively. To clarify the involvement of the erythroid lineage in CD36 deficiency, we investigated the phenotype and RNA expression of CD36. MATERIALS AND METHODS: CD36 expression was examined in 296 patients with several cardiovascular diseases in our outpatient clinic. There were 12 patients with type I deficiency and 16 with type II CD36 deficiency. A bone marrow sample was examined in five type I and four type II patients. Expression of CD36 mRNA was examined in burst-forming unit-erythroid (BFU-E). The sequences of reverse transcriptase polymerase chain reaction (RT-PCR) products of the CD36 mRNA from monocytes were examined. RESULTS: As expected, CD36 was deficient in erythroblasts from all five patients with type I deficiency. CD36 was present in erythroblasts from three of the four with type II deficiency, suggesting that their abnormality is restricted to platelets (type IIa). CD36 was unexpectedly absent from erythroblasts of a single type II patient (type IIb). CD36-specific mRNA was identified in BFU-E from each of two normals, six type I, and six type II patients, including type IIb. The sequences of RT-PCR products of the CD36 mRNA in a patient with type IIa and another with type IIb showed homozygous wild alleles. CONCLUSION: The findings provide evidence for further heterogeneity among CD36-deficient individuals and the existence of a basic principle mechanism of type II, such as glycosylation abnormality.

Pathogenetic analysis of three cases with a bleeding disorder characterized by defective platelet aggregation induced by Ca2+ ionophores.

We report three cases of platelet dysfunction characterized by defective Ca2+ ionophore-induced platelet aggregation without impaired production of thromboxane A2 (TXA2). The patients had mild to moderate bleeding tendencies, and their platelet aggregation and secretion induced by ADP, collagen, arachidonic acid, stable TXA2 (STA2) and Ca2+ ionophore A23187 was defective or much reduced. However, ristocetin- or thrombin-induced platelet aggregation was normal. The analysis of second messenger formation showed that inositol 1,4,5-triphosphate formation or Ca2+ mobilization induced by thrombin, STA2 or A23187 was normal. Furthermore, the phosphorylation of 47 kDa protein (pleckstrin) and 20 kDa protein (myosin light chain, MLC) in response to those agonists was normal. These findings suggest that the defective site in the patients' platelets lies in the process distal to or independent of protein kinase C activation, Ca2+ mobilization and MLC phosphorylation.

A female with asymptomatic primary biliary cirrhosis associated with pernicious anemia.

We experienced a female case with asymptomatic primary biliary cirrhosis that was associated with pernicious anemia after 16 years from the onset. She was 52 years old when she first visited a clinic in 1981 for liver dysfunction treatment. Antimitochondrial antibody was negative and antipyruvate dehydrogenase complex antibody was positive in a low titer in its immunoglobulin (Ig)M type. Histological examination of her liver revealed a presence of definite chronic non-suppurative destructive cholangitis with numerous epithelioid cell granuloma. She had been given 600 mg of the oral daily dose of ursodeoxycholic acid since 1992. Macrocytic anemia incidiously appeared in September 1999. An immunological examination detected negative antiparietal cell antibodies and positive anti-intrinsic factor antibodies. Her bone marrow smear showed numerous megaloblasts and serum vitamin B12 in her blood was low at 99 pg/mL. Severe reversed atrophic-type gastritis (type A gastritis) was demonstrated by the use of dye-endoscopy with Congo red. Her macrocytic anemia dramatically improved after intramuscular administration of vitamin B12. In conclusion, attention should be given to the association of pernicious anemia during the follow up of primary biliary cirrhosis.

Pathogenesis of a bleeding disorder characterized by platelet unresponsiveness to thromboxane A2.

A platelet disorder characterized by the absence of thromboxane A2 (TXA2)-induced platelet aggregation is a new clinical entity of platelet dysfunction. The platelets of three patients had the ability to bind exogenous TXA2, but synthetic TXA2 mimetic-induced postreceptor biochemical events, such as IP3 formation, Ca2+ mobilization, phosphatidic acid formation, and GTPase activities, were selectively defective, suggesting impaired coupling between the TXA2 receptor and phospholipase C activation. Gene analysis of the TXA2 receptor showed a substitution of Leu for Arg60 in the first cytoplasmic loop in all patients, and this mutation seemed to be responsible for this platelet disorder.

Thrombopoietin activates the growth of megakaryoblasts in patients with chronic myeloproliferative disorders and myelodysplastic syndrome.

The effects of thrombopoietin (TPO) on cell proliferation and differentiation, and the relation between these effects and the expression of c-mpl on leukemia cells were studied in seven acute myelogeneous leukemia cell lines and seven myelogeneous blast cell preparations from patients with chronic myeloproliferative disorders (CMPDs) and myelodysplastic syndrome (MDS). Among the leukemia cells, five preparations of megakaryoblastic leukemia cells from patients and one megakaryoblastic cell line, CMK 11.5, proliferated in response to TPO in vitro. CMK 11.5 and the blastic cells from one patient diagnosed with MDS with myelofibrosis differentiated with increasing expression of CD41a in response to TPO. However, TPO had no effect on the cells lacking megakaryocytic characteristics. Some patients with CMPD and MDS develop acute transformation with blasts demonstrating megakaryocytic features, and some of these cells show growth in response to TPO. Therefore, in vivo administration of TPO should be considered carefully for patients with CMPD or MDS, since TPO may induce leukemic cell proliferation.

Cell kinetic study of normal human bone marrow hematopoiesis and acute leukemia using 7AAD/PY.

We have used the 7AAD/PY method to analyze the cell cycle status of normal human bone marrow hematopoiesis, and found that the cell kinetics differed. There were cells with relatively low levels of RNA in the S-phase (Type I) and a high level in the S-phase (Type 11). T-cells, B-cells, nucleated red cells and CD34+/CD19+ early B-cells in bone marrow were Type I, whereas myelomonocytic subset and CD34+/CD33-dim+ common myeloid cells were Type II. AC133+/CD38-dim cells, which were thought to be lineage-marker negative hematopoietic stem cells, had intermediate amounts of RNA in the S-phase between Type I and II (Type 0). Seventy-four cases of acute leukemia were also analyzed. Most of the T- and B-ALL cases were found to be Type I, most of the ANLL cases were Type II, and there were 10 cases that were Type 0. These findings yielded fundamental information about normal hematopoiesis and acute leukemia.

[Clinical application of reticulated platelet analysis using the matic-modified thiazole orange method and flow cytometry].

Reticulated platelet (RP) analysis using thiazole orange (TO) and a flow cytometer is a convenient and promising method for estimating platelet kinetics in patients with thrombocytopenia. Among many different modifications of the TO method, a novel protocol reported by Matic in 1998 seems to be superior because it allows RP analysis using a sample of whole blood very quickly and accurately. Therefore, we used this method to analyze platelet kinetics in patients with idiopathic thrombocytopenic purpura (ITP) and hemophagocytic syndrome (HPS), as well as patients with hematopoietic malignancies (HM) after intensive chemotherapy. The proportion of RP was increased in the patients with ITP and HPS, and furthermore showed a negative linear correlation with the platelet count. The change in the proportion of RP occurred about one week before the change in the circulating platelet count in the patients with HM after chemotherapy. This modification of the TO method by Matic is expected to become a standard protocol for RP analysis.

Arg60 Leu mutation in the first cytoplasmic loop of the platelet thromboxane A2 receptor is not essential for mediating inhibitory coupling between the receptor and adenylyl cyclase.

Human platelet thromboxane A2 (TXA2) receptor (TXR) has been reported to functionally couple to the inhibitory GTP-binding protein for adenylyl cyclase (Gi). However, it still remains unclear which portions of the TXR structure are critical determinants in that coupling. We have previously reported several patients with platelet dysfunction, whose platelets showed impaired coupling between TXR and phospholipase C caused by an Arg60 to Leu mutation in the first cytoplasmic loop. To investigate whether this portion is essential for mediating inhibitory coupling between TXR and adenylyl cyclase, we analyzed the inhibition by the TXA2 analog of the PGE1 or forskolin-induced platelet cAMP increase in patients' platelets, and found that the inhibition occurred normally. This suggests that Arg60 in the first cytoplasmic loop of the TXR is not involved in TXR-Gi coupling.

Inhibition of ATRA-induced myeloid differentiation in acute promyelocytic leukemia by a new protein tyrosine phosphatase inhibitor, 3,4-dephostatin.

We previously reported that the expression and the activity of SHP-1, a non-transmembrane protein tyrosine phosphatase (PTPase) increased during myeloid differentiation of an acute promyelocytic leukemia cell line (HT93) induced by all-trans retinoic acid (ATRA). To examine whether inhibition of SHP-1 activity attenuates myeloid differentiation, we used a new PTPase inhibitor, 3,4-dephostatin, and studied its effect on myeloid differentiation. Suppressive effects on immunoprecipitated SHP-1 phosphatase activity and myeloid cell differentiation were detected. These results suggest that SHP-1 is a substrate for 3,4-dephostatin, and that SHP-1 PTPase activity is closely related to myeloid differentiation.

Mutations of the platelet thromboxane A2 (TXA2) receptor in patients characterized by the absence of TXA2-induced platelet aggregation despite normal TXA2 binding activity.

Previously, we reported five cases of platelet dysfunction characterized by the absence of thromboxane A2 (TXA2) - induced platelet aggregation despite normal TXA2 binding activity. In this platelet disorder, patients were divided into two groups; i.e. those whose platelets lacked or did not lack phospholipase C (PLC) activation (Group A and Group B, respectively) (Thromb Haemost 1996; 76: 1080). Furthermore, in one of the patients, we showed that a single amino acid substitution (Arg60 to Leu) in the first cytoplasmic loop of the TXA2 receptor (TXR) was responsible for this platelet disorder. However, mutational analysis of the TXR in the remaining patients has not been performed. Based on this background, we investigated the mutations of the TXR in these patients, and found that all of the patients have the same abnormality of the TXR (Arg60-->Leu), although the Group A patients were homozygous and the Group B patients were heterozygous for this mutation. This mutation is the only abnormality which has been found in this platelet disorder, and in patients heterozygous for this mutation, the mutant type TXR suppresses wild-type receptor-mediated platelet aggregation by a mechanism independent of PLC activation.