Therapeutic effect of CXCR3-expressing regulatory T cells on liver, lung and intestinal damages in a murine acute GVHD model.

Adoptive transfer of CD4+CD25+ regulatory T cells has been shown to have therapeutic effects in experimental graft-vs-host disease (GVHD) models. Chemokines play an important role in the recruitment of alloreactive donor T cells into target organs during GVHD. In this study, we investigated the effectiveness of targeted delivery of CD4+CD25+ regulatory T cells via a transfected chemokine receptor on reduction of organ damage during acute GVHD. High levels of expression of Th1-associated chemokines (CXCL9, CXCL10 and CXCL11) and their receptor CXCR3 were observed in the liver, lung and intestine of GVHD-induced recipient mice. Recipient mice that had undergone transfer of CD4+CD25+Foxp3+ CXCR3-transfected T cells (CXCR3-Treg cells) showed significant amelioration of GVHD changes in the liver, lung and intestine in comparison with recipient mice that had received CD4+CD25+Foxp3+ T cells (Treg cells) or naturally occurring CD4+CD25+ regulatory T cells. This was due to more pronounced migration of CXCR3-Treg cells and their localization for a longer time in Th1-associated chemokine-expressing organs, resulting in stronger suppressive activity. We succeeded in preparing chemokine receptor-expressing Treg cells and demonstrated their ability to ameliorate disease progression upon accumulation in target organs. This method may provide a new therapeutic approach for organ damage in acute GVHD.

Association of platelet response to cilostazol with clinical outcome and CYP genotype in patients with cerebral infarction.

INTRODUCTION: Cilostazol, an anti-platelet drug that inhibits phosphodiesterase 3, is beneficial for patients with atherothrombosis. In contrast to other anti-platelet drugs such as aspirin and thienopyridines, little information is available on the relationship between platelet responses to cilostazol and clinical outcomes. MATERIALS AND METHODS: We conducted a prospective study on patients with cerebral infarction who were treated with cilostazol. The platelet response to cilostazol was assessed with our new assay for the phosphorylation of vasodilator-stimulated phosphoprotein (VASP) subsequent to the pharmacological action of cilostazol. Patients were followed up for 2 years and the relationship between VASP assay results and the recurrence of thrombotic events was examined. We also investigated the effects of CYP3A5 and CYP2C19 genotypes involved in the metabolism of cilostazol on the platelet response to cilostazol. RESULTS: Among the 142 patients enrolled, 130 completed the 2-year follow-up and the recurrence of thrombotic events was noted in 8 (6.2%). VASP phosphorylation levels were significantly lower in patients with than in those without recurrence. The combined genotype of CYP3A5*1/*3 and CYP2C19*1/*1 was associated with a low level of VASP phosphorylation, while either genotype was not. A multivariate analysis showed that high residual platelet reactivity during the cilostazol treatment, which was defined by a low response of platelet VASP phosphorylation to cilostazol, was an independent risk factor for the recurrence of thrombotic events. CONCLUSION: A low platelet response to cilostazol determined by a new platelet assay was associated with the recurrence of thrombotic events in patients with cerebral infarction.

Hoek's formula, a cystatin C-based prediction formula for determining the glomerular filtration rate, is the most effective method for original adjusting the dosage of vancomycin.

OBJECTIVE: Some formulas using the serum cystatin C level to estimate the GFR have recently been reported. However, there has been no report of a serum cystatin C-based formula for adjusting the dosage of the drugs cleared by the kidney. In this study, we compared the predictive performance of the serum vancomycin trough concentration predicted using serum cystatin C-based formulas. METHOD: The data were collected from 158 hospitalized patients. Five formulas have been published to predict the GFR using serum cystatin C. The cystatin C-based formulas were divided into two groups, formulas with or without anthropometric data. We predicted the serum vancomycin trough concentrations using VCM-TDM S_edition ver. 1.00 software. RESULTS: In formulas with anthropometric data, the mean absolute error (MAE) using Hoek's formula was 2.38, the MAE using Grubb's 1 formula was 4.13, the MAE using Sjöström's formula was 2.90, and the MAE using Cockcroft and Gault formula based on creatinine was 4.42. On the other hand, in formulas without an anthropometric data group, the MAE using Larsson's formula was 3.07, and the MAE using Grubb's 2 formula was 3.63. CONCLUSION: These results suggested that Hoek's formula is the most useful formula for determining the initial dosage settings for vancomycin.

Efficient engraftment of primary adult T-cell leukemia cells in newborn NOD/SCID/beta2-microglobulin(null) mice.

Adult T-cell leukemia (ATL) develops via multiple oncogenic steps in human T-cell leukemia virus type I (HTLV-I) carriers. To better understand pathogenesis of ATL, we developed a novel xenogeneic engraftment model in which primary ATL cells are intravenously transplanted into neonatal nonobese diabetic (NOD)/severe-combined immunodeficiency (SCID)/beta2-microglobulin(null) (NOD/SCID/beta2m(null)) mice. Acute-type ATL cells engrafted in the peripheral blood and in the lymph nodes of recipients at a high efficiency. Engrafted ATL cells were dually positive for human CD4 and CD25, and displayed patterns of HTLV-I integration identical to those of donors by Southern blot analysis. These cells infiltrated into recipients' liver, and formed nodular lesions, recapitulating the clinical feature of each patient. In contrast, in smoldering-type ATL cases, multiple clones of ATL cells engrafted efficiently in NOD/SCID/beta2m(null) mice. When smoldering-type ATL cells were retransplanted into secondary NOD/SCID/beta2m(null) recipients, single HTLV-I-infected clones became predominant, suggesting that clones with dominant proliferative activity can be competitively selected in this xenogeneic system. Taken together, the NOD/SCID/beta2m(null) newborn system is useful to understand kinetics, metastasis, and disease progression of ATL in vivo.

Inhibition of X-ray or chemical carcinogen-induced neoplastic transformation of C3H10T1/2 fibroblasts by lipopolysaccharides.

Oncogenic transformation of mouse 10T 1/2 fibroblasts induced upon exposure to X-ray or N-methyl-N'-nitro-N-nitrosoguanidine was suppressed if lipopolysaccharide (LPS) was present in the culture medium. The suppressive effect of LPS was exerted within 24 h after irradiation. Suppression was dependent on the concentration of LPS added and LPS (2 micrograms/ml) derived from Salmonella minnesota R595 reduced the number of transformed type III foci per dish from 0.39 to 0.15. Indomethacin (1 to 30 microM) further enhanced the effect of LPS in a dose-dependent manner.

Dominant negative isoform of the Ikaros gene in patients with adult B-cell acute lymphoblastic leukemia.

Gene targeting studies in mice have shown that the transcription factor Ikaros plays an essential role in lymphoid development and as a tumor suppressor in T cells, whereas the related gene Aiolos functions as a tumor suppressor in B cells. We analyzed the expression levels of the Ikaros gene family, Ikaros and Aiolos, in human bone marrow samples from patients with adult acute lymphoblastic leukemia [ALL (n = 46; B-cell ALL = 41; T-cell ALL = 5)]. Overexpression of the dominant negative isoform of Ikaros gene Ik-6 was observed in 14 of 41 B-cell ALL patients by reverse transcription-PCR, and the results were confirmed by sequencing analysis and immunoblotting. None of the other dominant negative isoforms of the Ikaros gene were detected by reverse transcription-PCR analysis. Southern blotting analysis with PstI digestion revealed that those patients with the dominant negative isoform Ik-6 might have small mutations in the Ikaros locus. We did not detect any overexpression of dominant negative isoforms of Aiolos in adult ALL patients. These results suggest that Ikaros plays a key role in human B-cell malignancies through the dominant negative isoform Ik-6.

Leukemia-associated fusion proteins, dek-can and bcr-abl, represent immunogenic HLA-DR-restricted epitopes recognized by fusion peptide-specific CD4+ T lymphocytes.

Although CD4(+) helper T lymphocytes have been demonstrated to play an important role in antitumor immune response, only a few epitopes of tumor-associated antigens recognized by HLA class II-restricted CD4(+) T lymphocytes have been identified. In the present study, we addressed the question of whether leukemia-associated fusion proteins are recognized by CD4(+) T lymphocytes. Immature dendritic cells (DCs) were loaded with necrotic or apoptotic leukemia cells with t(6;9) or t(9;22) and then cocultured with the dek-can fusion peptide-specific or the bcr-abl fusion peptide-specific CD4(+) T lymphocyte clone. The dek-can peptide-specific and bcr-abl peptide-specific CD4(+) T lymphocyte clones produced interferon-gamma (IFN-gamma) when they were cocultured with HLA-DR-matched but not with mismatched DCs which had been loaded with apoptotic as well as necrotic leukemia cells with t(6;9) and t(9;22), respectively. IFN-gamma production by CD4(+)T lymphocyte clones in response to stimulation with DCs loaded with leukemia cells was inhibited by the anti-HLA-DR monoclonal antibody. These data indicate that the acute myelogenous leukemia-associated fusion protein, dek-can, and chronic myelogenous leukemia-associated fusion protein, bcr-abl, are both processed and presented by DCs to the fusion peptide-specific CD4(+) T lymphocytes.

Identification of novel MUNC13-4 mutations in familial haemophagocytic lymphohistiocytosis and functional analysis of MUNC13-4-deficient cytotoxic T lymphocytes.

BACKGROUND: Familial haemophagocytic lymphohistiocytosis (FHL) has an autosomal recessive mode of inheritance and consists of at least three subtypes. FHL2 subtype with perforin (PRF1) mutation accounts for 30% of all FHL cases, while FHL with MUNC13-4 mutation was recently identified and designated as FHL3 subtype. OBJECTIVE: To examine MUNC13-4 mutations and the cytotoxic function of MUNC13-4 deficient T lymphocytes in Japanese FHL patients METHODS: Mutations of MUNC13-4 and the cytotoxicity of MUNC13-4-deficient cytotoxic T lymphocytes (CTL) were analysed in 16 Japanese families with non-FHL2 subtype. RESULTS: Five new mutations of the MUNC13-4 gene were identified in six families. The mutations were in the introns 4, 9, and 18, and exons 8 and 19. Two families had homozygous mutations, while the remaining four had compound heterozygous mutations. Cytotoxicity of MUNC13-4 deficient CTL was low compared with control CTL, but was still present. Clinically, the onset of disease tended to occur late; moreover, natural killer cell activity was not deficient in some FHL3 patients. CONCLUSIONS: MUNC13-4 mutations play a role in the development of FHL3 through a defective cytotoxic pathway.

Antileukemia multifunctionality of CD4(+) T cells genetically engineered by HLA class I-restricted and WT1-specific T-cell receptor gene transfer.

To develop gene-modified T-cell-based antileukemia adoptive immunotherapy, concomitant administration of CD4(+) and CD8(+) T cells that have been gene modified using identical HLA class I-restricted leukemia antigen-specific T-cell receptor (TCR) gene transfer has not yet been fully investigated. Here, using CD4(+) and CD8(+) T cells that had been gene modified with a retroviral vector expressing HLA-A*24:02-restricted and Wilms' tumor 1 (WT1)-specific TCR-α/ß genes and siRNAs for endogenous TCRs (WT1-siTCR/CD4(+) T cells and WT1-siTCR/CD8(+) T cells), we examined the utility of this strategy. WT1-siTCR/CD4(+) T cells sufficiently recognized leukemia cells in an HLA class I-restricted manner and provided target-specific Th1 help for WT1-siTCR/CD8(+) T cells. By using a xenografted mouse model, we found that WT1-siTCR/CD4(+) T cells migrated to leukemia sites and subsequently attracted WT1-siTCR/CD8(+) T cells via chemotaxis. Therapy-oriented experiments revealed effective enhancement of leukemia suppression mediated by concomitant administration of WT1-siTCR/CD4(+) T cells and WT1-siTCR/CD8(+) T cells. Importantly, this augmented efficacy in the presence of WT1-siTCR/CD4(+) T cells was correlated with longer survival and enhanced formation of memory T cells by WT1-siTCR/CD8(+) T cells. Collectively, our experimental findings strongly suggest that this strategy would be clinically advantageous for the treatment of human leukemia.

Diverse effects of tumor necrosis factor-alpha on three subclones from human myelomonocytic leukemia cell line ME-1 exhibiting different differentiation stages.

The effects of tumor necrosis factor-alpha (TNF-alpha) were examined in three subclone cells from human myelomonocytic leukemia cell line ME-1. These three subclone cells exhibit different differentiation stages of the myelomonocytic lineage. TNF-alpha exerted a growth-suppressive effect on the least mature subclone cells, ME-F2 cells. On the other hand, TNF-alpha induced the most mature ME-F1 cells and intermediate ME-F3 cells to differentiate along the monocytic pathway. TNF-alpha also enhanced interferon-gamma (IFN-gamma)-induced complement C2 production by ME-F1 and ME-F3 cells but did not affect production by differentiated ME-F1 and ME-F3 cells. These results suggest that the diversity of the effects of TNF on subclone cells from ME-1 depends on the stage of cell differentiation.

Characterization of self-glutamic acid decarboxylase 65-reactive CD4+ T-cell clones established from Japanese patients with insulin-dependent diabetes mellitus.

To investigate autoimmunity to glutamic acid decarboxylase (GAD) 65 in Japanese patients with insulin-dependent diabetes mellitus (IDDM, type I diabetes), we established seven CD4+ T-cell clones, by stimulating peripheral blood mononuclear cells (PBMC) of six IDDM patients, using a mixture of overlapping human GAD65 peptides. No GAD65 autoreactive T-cell clones were evidenced in four healthy controls. Specificities of T-cell clones were as follows: (a) two clones specific to GAD65 p111-131 (residue 111 to 131) + DR53 (DRB4*0103); (b) one clone specific to GAD65 p413-433 + DR1 (DRB1*0101); (c) two clones specific to GAD65 p200-217 + either DR9 (DRB1*0901) or DR8 (DRB1*0802); and (d) two clones specific to GAD65 p368-388 + DP2 (DPA1*01 or 0201-DPB1*0201). Two DR53-restricted and one DR1-restricted T-cell clones, responded to a recombinant human GAD65 protein, and showed cytotoxicity against B lymphoblastoid cell lines pre-pulsed with the peptides. Six T-cell clones exhibited the Th1-like phenotype. Interestingly, two DR53-restricted T-cell clones killed a Fas-deficient B lymphoblastoid cell line, thereby indicating that cytotoxicity was not completely dependent on a Fas-Fas ligand interaction. Thus, the T-cell epitopes were mapped in a limited portion of GAD65 protein, with a tendency to be restricted by disease-associated HLA-DR, but not DQ molecules.

T-cell large granular lymphocytic leukemia occurring after autologous peripheral blood stem cell transplantation.

A 61-year-old man with angioimmunoblastic lymphoma in first complete remission underwent autologous peripheral blood stem cell transplantation. At 1 month post transplant, asymptomatic large granular lymphocytosis developed. The surface marker profile of the cells was CD3+CD8+CD56-CD57+. The disease course was chronic and indolent. The patient remains in complete remission from angioimmunoblastic lymphoma more than 6 months post transplant with persistent large granular lymphocytosis (lymphocyte count, 5-15 x 10(9)/l). Although post transplantation T-cell lymphoproliferative disorders have mostly occurred in allogeneic transplantation recipients and presented as aggressive lymphomas/leukemias, we suggest that chronic indolent T-cell large granular lymphocytic leukemia can occur after autologous stem cell transplantation.

Neoplastic transformation of plateau-phase mouse 10T1/2 cells following single and fractionated doses of X rays.

The induction of cell transformation and cell killing in plateau-phase 10T1/2 mouse cells by single and fractionated doses of X rays was investigated. The single dose-transformation curve was composed of a first phase (doubling dose = 0.2 Gy), a second phase (doubling dose = 1 Gy), and a third phase (no increase of transformation at doses more than 6 Gy). Two-dose fractionation experiments revealed that transformation damage was reduced when total doses of 0.93, 1.86, and 3.72 Gy were given with two equal fractions separated by intervals of 3 to 15 hr, as compared with the equivalent single doses. This not only represents repair of subtransformation damage but also is consistent with previous findings of repair of potential transformation damage. The fractionated dose-transformation curve obtained 3 hr after the first (conditioning) dose indicated that damaged cells had recovered, at least in part, from transformation damage, as shown by the reappearance of the first phase of transformation induction. No alteration of sensitivity to the second dose seemed to develop; however, the transformable fraction of the cell population was apparently increased by the conditioning dose. From the present results with single and fractionated doses, a resolution between repair of sub- and potential transformation damage did not seem possible. Plateau-phase cells were found to be useful for dose fractionation studies.

Radiation-induced neoplastic transformation of C3H10T1/2 cells is suppressed by ascorbic acid.

X-ray induced transformation of C3H10T1/2 cells was suppressed in a concentration-dependent manner by administration of ascorbic acid after irradiation (0.1-20 micrograms/ml for the first week) in the culture medium. The dose-response curve was shifted about 60% downward and was slightly steeper in the presence of ascorbic acid (5 micrograms/ml for the first week) than in its absence. The 1-week treatment procedure revealed that cells initiated by radiation remained susceptible to ascorbic acid until the time of morphological phenotype expression. The neoplastically transformed phenotype expressed after incubation for 8 weeks could no longer be suppressed by ascorbic acid even after culture transfer. Similarly, the neoplastically transformed phenotype suppressed for 8 weeks by ascorbic acid treatment was not subsequently expressed in the absence of ascorbic acid. On the basis of the oxygen-detoxifying nature of ascorbic acid, we postulated that expression of the neoplastically transformed phenotype is promoted by reactive oxygen species and peroxy radicals generated in cells during the whole assay period. The data may be useful as a guide for chemopreventive efforts against radiation carcinogenesis.

Expression of the Gfi-1 gene in HTLV-1-transformed T cells.

Human T-cell leukemia virus type I (HTLV-I) is the causative agent of adult T-cell leukemia/lymphoma (ATLL) and immortalizes human T cells interleukin-2 (IL-2)-dependently in vitro. Protracted culture of HTLV-I-infected T cells enables them to grow IL-2-independently. Although acquisition of IL-2-independent growth has been correlated with activation of signal transducers and activators of transcription (STATs), the precise mechanism of IL-2-independent growth is unknown. We found that expression of the Gfi-1 (growth factor independence-1) gene was elevated in most HTLV-I-transformed IL-2-independent cell lines but in few HTLV-I-infected IL-2-dependent cell lines. We also found elevated expression of Gfi-1 in fresh leukemic cells of ATLL patients. Although expression of Gfi-1 is correlated with activation of STAT3, induction of the dominant negative form of STAT3 in the HUT102 cell line does not alter the level of Gfi-1 expression. Furthermore, MT2 cells treated with Gfi-1 antisense oligonucleotide had reduced [3H]thymidine uptake compared with MT2 cells treated with Gfi-1 sense oligonucleotide. These findings indicate that Gfi-1 activation is involved in the IL-2-independent growth of HTLV-I-transformed T cells in vitro and in the development of ATLL in vivo, but is not induced by STAT activation.

Amplitude reduction of H-reflex during mental movement simulation in elite athletes.

In order to investigate the changes of motoneuron excitability during mental simulation of a voluntary movement (motor imagery; MI), the soleus H-reflex and several activities of autonomic effectors were recorded simultaneously when MI of speed skate sprint was performed. The subjects were seven elite speed skate athletes ranging in age from 18 to 24 years old. They were all skilled in MI, and could imagine full vivid skating movement internally as if they were really exercising. The subjects were awake, relaxed and blindfolded. At first, rest (5 min), positive relaxation (4 min) and concentration periods (2.5 min) were set up. Then, after the previous announcement for the start, MI was initiated by the sound of a signal gun which was recorded by a tape recorder. MIs were continued for each subject from about 36 s to 38 s, and these imaging times were very close to the actual personal best records of each subject. The autonomic effectors represented by skin conductance response (SCR), heart rate (HR), and respiration rate (RSR) became remarkably active during MI in all trials. SCR was increased by 51% on the average. Furthermore, HR and RSR were increased by 57% and 76% respectively, compared with those during resting period. These changes in the autonomic effectors were all significant and were those which were activated in actual movements. The amplitudes of H-reflex were almost all constant before MI initiation. However, the reduction of the H-reflex amplitude arose just after the start and lasted to the end of MI.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunotherapy for leukemia targeting the Wilms' tumor gene.

The Wilms' tumor (WT1) gene encodes a zinc finger transcription factor, which is preferentially expressed in acute leukemia cells and chronic myelogenous leukemia cells in blast crisis, but not in most normal cells. These findings strongly suggest that WT1 is a potential target of immunotherapy for human leukemia. We have established a CD8+ cytotoxic T lymphocyte (CTL) clone, designated TAK-1, which is specific for a WT1-derived 9-mer peptide consisting of HLA-A24-binding anchor motifs. TAK-1 lysed both HLA-A24-positive allogeneic cells and autologous cells that were loaded with a WT1-derived peptide. TAK-1 was cytotoxic to HLA-A24-positive leukemia cells, but not to HLA-A24-positive lymphoma cells that did not express WT1, to HLA-A24-negative leukemia cells, or to HLA-A24-positive normal cells. Treating leukemia cells with an antisense oligonucleotide complementary to WT1 reduced TAK-1-mediated cytotoxicity. TAK-1 did not inhibit colony formation of HLA-A24-positive normal bone marrow cells. Recently, other groups have also reported the establishment of HLA-A2-restricted anti-leukemic CTLs specific for WT1-derived peptide. In addition, a murine model of immunotherapy against WT1-expressing tumors has been reported. Recent studies have demonstrated that WT1 is also aberrantly expressed in various kinds of cancer cells. Taken together, these results suggest that immunotherapy targeting WT1 should be effective against both solid tumors and leukemia.

Human T-lymphotropic virus type I provirus and T-cell prolymphocytic leukemia.

T-cell prolymphocytic leukemia (T-PLL) is a rare type of post-thymic T-cell neoplasm, the etiology of which is unknown. Patients with T-PLL have been found to be seronegative for human T-lymphotropic virus type-I (HTLV-I), and their leukemia cells do not retain monoclonally integrated HTLV-I provirus. Recently, we have demonstrated the presence of defective HTLV-I provirus by polymerase chain reaction in the DNA extracted from peripheral blood cells or affected lymph nodes of T-PLL patients. Although there is a possibility, from our observation, that an alternative mechanism is operating in HTLV-associated leukemogenesis, it is still unknown whether and how HTLV-I can contribute to the leukemogenesis of T-PLL. In this review, we describe controversial issues and discuss a role of HTLV-I in the leukemogenesis of T-PLL.