Disassociation of verbal learning and hippocampal volume in type 2 diabetes and major depression.

OBJECTIVE: The purpose of this study was to examine the relationship between verbal learning and memory performance and hippocampal volume in subjects with co-morbid type 2 diabetes and major depression compared with healthy control subjects and subjects with type 2 diabetes alone. METHODS: Twenty four subjects with type 2 diabetes and 20 subjects with type 2 diabetes and major depression were recruited from endocrinology clinics and were compared with 32 healthy control subjects recruited from the community. Subjects were scanned on a 1.5 T GE scanner, and hippocampal volumes were measured using Freesurfer. The California Verbal Learning Test assessed learning and memory. Significant predictors of verbal learning performance (e.g., age, gender, education, blood pressure, stroke risk, hemoglobin A1c, and hippocampal volume) were determined using a stepwise linear regression. RESULTS: Subjects with diabetes and depression had significantly worse performance on verbal list learning compared with healthy control subjects. Hippocampal volume was a strong predictor of performance in healthy control subjects, and age and hippocampal volume were strong predictors in subjects with type 2 diabetes alone. Age alone was a significant predictor of verbal learning performance in subjects with diabetes and depression. CONCLUSIONS: The relationship between hippocampal volume and performance on the California Verbal Learning Test is decoupled in subjects with type 2 diabetes and major depression and this decoupling may contribute to poor verbal learning and memory performance in this study population.

Effect of pH and hydration on the normal and lateral interaction forces between alumina surfaces.

Interaction forces between alumina surfaces were measured using an AFM-colloid probe method at different pHs. For an alpha-alumina-sapphire system at acidic pH, the force curve exhibited a well-defined repulsive barrier and an attractive minimum. At basic pH, the interactive force was repulsive at all separations with no primary minimum. Lateral force measurements under the same conditions showed that frictional forces were nearly an order of magnitude smaller at basic pH than those observed at acidic pH. This behavior was attributed to the hydration of the alumina surface. Normal and lateral force measurements with the strongly hydrated rho-alumina surfaces supported these findings.

Hyperfunction of neutrophils in a patient with BCR/ABL negative chronic myeloid leukemia: a case report with in vitro studies.

BACKGROUND: Among patients diagnosed with chronic myeloid leukemia (CML), a small percentage lack a BCR/ABL fusion gene, a landmark of CML. Their clinical features are distinct from patients with BCR/ABL positive CML, although to the authors' knowledge the pathogenesis to date has been unknown. METHODS: A 50-year-old female patient with BCR/ABL negative CML and multiple complications of Graves disease, Sweet syndrome, and a fatal pulmonary alveolar proteinosis (PAP) is described in the current study. To show a clonal origin of her myeloid cells, hypoxanthine phosphoribosyltransferase (HPRT) assay was applied. Because the patient developed a progressive and fatal neutrophilia, a screening of cell functions in neutrophilic lineage, including in vitro colony assay of her bone marrow cells and production of superoxide and interleukin-8 (IL-8) by blood neutrophils was performed. RESULTS: Southern blot analysis based on the polymorphism of the HPRT gene was compatible with monoclonality of her neutrophils. The patient had an increased amount of bone marrow granulocyte-macrophage progenitor cells, which formed colonies in response to a very low dose (0.1 ng/mL) of granulocyte-colony stimulating factor. In vitro production of superoxide and IL-8, which is an inducer of positive chemotaxis of neutrophils, by her peripheral neutrophils was markedly augmented. Her bronchoalveolar lavage fluid also contained a significant amount of IL-8 as well as an unusual infiltration of neutrophils. CONCLUSIONS: In the patient in the current study, hyperfunction of the neutrophils might have contributed to the onset of PAP as well as Sweet syndrome and to the pathogenesis of BCR/ABL negative CML.

Progress reports on immune gene therapy for stage IV renal cell cancer using lethally irradiated granulocyte-macrophage colony-stimulating factor-transduced autologous renal cancer cells.

There is no effective treatment for patients with stage IV renal cell cancer (RCC), although the introduction of new therapy is imminent. Cancer gene therapy is currently considered to be one of the most promising therapeutic modalities in the field of cancer treatment. Based on the results of animal studies, vaccination using autologous granulocyte-macrophage colony-stimulating factor-transduced renal cancer cells appears promising. Before initiating a clinical study using an ex vivo gene-transduced autologous cell vaccine-based immunogene therapy for RCC in Japan, in 1992 we initially planned a Japanese version of a clinical protocol in collaboration with a US group. In 1993, the original protocol was refined. We performed five preclinical qualification studies using RCC nephrectomy specimens from patients in 1997, and the results showed that preparation of RCC cells for autologous vaccines at the Clinical Cell Technology Facility, Research Hospital of the Institute of Medical Science, University of Tokyo, was feasible. Subsequently in August 1998, the Ministry of Health and Welfare and the Ministry of Education, Science, Culture, and Sport approved our clinical protocol. We have recruited two patients with stage IV RCC to our study so far. Here we report the background to the initiation of cancer gene therapy in Japan.

TCR-Vbeta repertoire analysis with RT-PCR was useful for the early detection of pulmonary relapsed T-cell lymphoma after autologous peripheral blood stem cell transplantation.

Pulmonary recurrence of malignant lymphoma is a rare event after stem cell transplantation. We report here a 45-year-old male who was successfully diagnosed with relapsed pulmonary T-cell lymphoma using an RT-PCR method. Clonal expansion of T cells expressing identical TCR V-D-J junction size (Vbeta5-Jbeta1.5) was demonstrated in lymphocyte groups obtained from both bronchoalveolar lavage fluid at relapse, and paraffin embedded lymph node samples resected when he was first diagnosed with angioimmunoblastic T-cell lymphoma. This method provided evidence to diagnose relapsed pulmonary angioimmunoblastic T-cell lymphoma in its early phase.

Identification of a melanoma antigen, PRAME, as a BCR/ABL-inducible gene.

In order to elucidate molecular events in BCR/ABL-induced transformation, we adopted a polymerase chain reaction (PCR)-based technique of differential display and compared mRNA expression in human factor-dependent cells, TF-1, with that in factor-independent cells, ID-1, which were established from TF-1 cells by transfection of BCR/ABL. Cloning and sequencing of a gene which was upregulated in ID-1 cells revealed that the gene was identical to a melanoma antigen, PRAME. Our present study demonstrated that PRAME was markedly expressed in primary leukemic cells with chronic myeloid leukemia (CML) in blastic crisis and Philadelphia (Ph)+-acute lymphoblastic leukemia (ALL), in which BCR/ABL played an important role as a pathogenic gene. Moreover, comparison of PRAME expression among CD34+ cells with CML in blastic, accelerated, and chronic phases revealed a higher expression in CML in advanced phases. Thus PRAME was considered to be a good candidate for a marker of Ph+-leukemic blast cells as well as a new target antigen of leukemic blast cells that cytotoxic T cells can recognize.

Pharmacokinetic studies of intravenous glycosylated recombinant human granulocyte colony-stimulating factor in various hematological disorders: inverse correlation between the half-life and bone marrow myeloid cell pool.

The pharmacokinetics of an intravenous bolus dose of glycosylated recombinant human G-CSF (rhG-CSF) was examined in 15 patients with various hematological disorders and 3 normal volunteers. The elimination half-life of rhG-CSF varied with the disorder. The half-life of an initial dose of rhG-CSF (2 micrograms/weight kg) was significantly prolonged in patients with aplastic anemia (2.7 +/- 0.3 h, n = 3) and myelodysplastic syndrome-refractory anemia (2.0 +/- 0.3 h, n = 3) when compared with those in normal controls (0.9 +/- 0.5 h, n = 3). In contrast, in patients with acute myelogenous leukemia which was overt leukemia from myelodysplastic syndrome-refractory anemia with excess of blasts in transformation, the half-life was shortened after chemotherapy (0.2 +/- 0.1 h, n = 3). The half-life of rhG-CSF in 2 patients with acute lymphoblastic leukemia in complete remission was prolonged (2.0 and 2.7 h) at the time of marrow-suppression after chemotherapy and then shortened (0.5, 1.0 h, respectively) in the recovery phase. The half-life of rhG-CSF was very weakly, inversely correlated with absolute neutrophil count in blood (n = 24, r2 = 0.32, P < 0.01), and was inversely correlated with the absolute count of bone-marrow myeloid cells (nucleated cell count in bone-marrow aspirates x the percentage of myeloid cells/100) of patients with aplastic anemia and myelodysplastic syndrome-refractory anemia (n = 12, r2 = 0.63, P = 0.002). These results suggest that the half-life of intravenously administered rhG-CSF (2 micrograms/kg) reflects the size of the myeloid cell compartment in vivo, and support the hypothesis that receptor-mediated consumption mainly accounts for the clearance of exogenous G-CSF.

Autologous bone marrow transplantation for patients with advanced chronic myelogenous leukemia.

We report on seven chronic myelogenous leukemia (CML) patients who received autologous bone marrow transplantation (ABMT) using bone marrow (BM) cells while at the chronic phase (CP) under the various treatments. Of the seven patients, four progressed to accelerated phase (AP) in 83-248 weeks after onset and three patients entered blastic crisis (BC) in 84-171 weeks after onset. All patients received high-dose chemoradiotherapy followed by infusion with 11.3 +/- 12.1 x 10(7) (average +/- S.D.) of bone marrow mononuclear cells (BM-MNCs)/kg IFN-alpha was resumed shortly after platelet recovery. Of the four patients in AP, one developed a recurrence of blastoma in 7 weeks, one progressed to second AP in 138 weeks after ABMT and two patients have survived the second CP for 159 and 330 weeks since ABMT, respectively. One of them achieved the complete disappearance of Ph1-positive metaphases for 33 weeks after ABMT. Of patients who received AMBT in BC, three relapsed within 8 weeks and died in 9, 17 and 58 weeks after ABMT, respectively. Hematological recovery was delayed in four patients. Therefore, we retrospectively re-evaluated the number of BM-MNCs collected through 50 procedures from 40 patients with CML-CP. The total MNCs obtained from 30 collections under IFN-alpha treatment was 27.4 +/- 30.9 x 10(8) cells (average +/- S.D.), being significantly lower than that obtained from 20 collections in pre-treatment state or with single chemotherapy other than IFN-alpha treatment (81.8 +/- 68.2 x 10(8) cells) (P < 0.005). The total number of MNCs correlated to white blood cell (WBC) count at BM collection (P < 0.01), which was also lower in the IFN-alpha(+) group than in the IFN-alpha(-) group (7.2 +/- 5.7 and 25.6 +/- 32.3 x 10(9)/l; P < 0.005). Our findings suggested that ABMT with the use of a sufficient number of progenitor cells might be helpful to CML patients in early AP and reach in extended periods of second CP. In addition, we suggest that BM collection is required before the start of IFN-alpha therapy because the total number of BM-MNCs correlated to the WBC count, which might be lower in IFN-alpha treatment.

Production of interleukin 1beta by human hematopoietic progenitor cells.

The production of interleukin 1beta (IL-1beta) by human hematopoietic stem/progenitor cells was studied to explore the concept that these cells are not merely responders to stimuli from their microenvironment, but can themselves produce a powerful biomodulator. Cells with a CD34+ CD45RA(lo) CD71(lo) phenotype were purified from human umbilical cord blood and cultured one per well in serum-free medium with a mixture of cytokines. Cells that had divided over 2-5 d to form doublets were identified and the daughter cells were studied individually. 91% (460/506) of daughter cells had clonogenic potential. Analysis of these individual daughter cells by reverse transcription-polymerase chain reaction showed that 29% of them (14/48) were positive for IL-1beta mRNA. One of the cells that was strongly positive for IL-1beta mRNA had a sibling that generated 366,000 cells of multiple lineages after 14 d. IL-1beta converting enzyme mRNA, which is necessary to produce IL-1beta, was also detected by reverse transcription-polymerase chain reaction at the single-cell level. Moreover, enzyme immunoassay for mature secreted IL-1beta in culture supernatants demonstrated the production of IL-1beta protein by these cells. This was confirmed by fluorescent immunostaining of the cells for human IL-1beta which showed a significant portion of positive cells. Taken together, the results demonstrate the capacity of early hematopoietic cells to synthesize IL-1beta. The capacity of human hematopoietic stem/progenitor cells to produce IL-1beta may be involved in regulation of their proliferation and differentiation under certain circumstances and dysregulation of this process may be modified in leukemogenesis.

Expression of interleukin-1 beta gene in candidate human hematopoietic stem cells.

Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect interleukin-1 beta (IL-1 beta) mRNA in candidate human hematopoietic stem cells. The cells, obtained from adult bone marrow (BM) or umbilical cord blood, had a CD34+ CD45RAlo CD71lo phenotype and were further fractionated into CD38+ and CD38- or Thy-1+ and Thy-1- subpopulations. The purity of these fractions was always more than 99%. IL-1 beta and CD34 mRNA were detected in pools of 30 BM-derived CD34+ CD45RAlo CD71lo cells. To further exclude any contribution by contaminating cells, individual cells were analyzed for CD34 and IL-1 beta mRNA. Positive results were obtained with 2 of 5 individual BM-derived CD34+ CD45RAlo CD71lo CD38+ cells isolated by micromanipulation after overnight culture in serum-free medium without any exogenous cytokines, and 1 of 10 individual CD34+ CD45RAlo CD71lo CD38- cells isolated immediately after sorting. Moreover, of 10 pools of three BM-derived CD34+ CD45RAlo CD71lo cells cultured overnight in the presence of a mixture of various cytokines (Steel factor, IL-3, IL-6, macrophage colony-stimulating factor [M-CSF], erythropoietin, and IL-3/granulocyte-macrophage colony-stimulating factor [GM-CSF] fusion protein), 5 were positive for IL-1 beta mRNA. This result was compatible with more than 20% (95% confidence limit 0.06-0.61) of the BM cells with the CD34+ CD45RAlo CD71lo phenotype expressing IL-1 beta mRNA. IL-1 beta expression was also consistently observed from day 0 to day 9 in liquid cultures of cord-blood-derived CD34+ CD45RAlo CD71lo Thy-1+ or Thy-1- cells. The cultures contained the same combination of cytokines and resulted in an expansion of cell numbers of up to 400-fold. GM-CSF mRNA was not detected in the equivalent of 75 cells at any day, even though it could be detected with high sensitivity in control stromal cells. Because IL-1 beta is a powerful and pleiotropic biomodulator of cytokines and adhesion molecules, our observations suggest that at least some primitive hematopoietic cells do not merely respond passively to signals from their environment, but may themselves regulate the paracrine production of cytokines from neighboring stromal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Production of human granulocyte colony stimulating factor by various kinds of stromal cells in vitro detected by enzyme immunoassay and in situ hybridization.

Production of human granulocyte colony stimulating factor (G-CSF) by stromal cells was studied in vitro. Induction of G-CSF by interleukin 1 (IL-1) and lipopolysaccharide (LPS) was compared using enzyme immunoassay in various kinds of stromal cells. Primary human bone marrow stromal cells, a human bone marrow-derived stromal cell line (KM-102), and peripheral blood monocytes secreted small amounts of G-CSF without stimulation, while vascular endothelial cells and skin fibroblasts secreted G-CSF only when induced by IL-1 or LPS. The production of G-CSF by monocytes was stimulated predominantly by LPS, whereas that by KM-102 cells, endothelial cells, and fibroblasts was induced by IL-1 but much less so by LPS. IL-1 and LPS stimulated similar levels of G-CSF production by primary bone marrow stromal cells which consisted of various types of cells. In situ hybridization for G-CSF mRNA showed that only a small proportion of primary bone marrow stromal cells expressed a large amount of G-CSF mRNA upon stimulation. The positive cells were round or oval in shape, while most of the spindle-shaped stromal cells were negative for specific grains. Although further characterization of positive cells is needed, the results suggest that bone marrow stromal cells are heterogeneous in terms of their capacity for G-CSF production.

[Pharmacokinetics of hematopoietic growth factors and granulopoiesis. The pathophysiology of human granulocyte colony-stimulating factor].

We have studied the production of human granulocyte colony-stimulating factor (hG-CSF). Enzyme immunoassay showed that hG-CSF was produced by primary bone marrow stromal cells, peripheral blood monocytes, fibroblasts, and endothelial cells in vitro. These cells produced variable levels of hG-CSF depending on the type of inducers. Interestingly, in situ hybridization showed that only a small proportion of bone marrow stromal cells and blood monocytes expressed a large amount of hG-CSF mRNA. Secondly, we have estimated serum hG-CSF level and clearance of exogenous hG-CSF in patients with various hematological disorders. Endogenous hG-CSF was undetectable (< 30 pg/ml) in sera of normal volunteers. On the other hand, the serum hG-CSF level was elevated in infection, malignancy, and neutropenia, suggesting the presence of reactive, ectopic, and unknown mechanisms for hG-CSF production, respectively. The half-life of recombinant hG-CSF was prolonged in disorders with reduced myeloid cell mass, especially in aplastic anemia, whereas it was shortened in myeloid leukemia, and in recovery phase after chemotherapy. This finding suggests the possibility of receptor-mediated consumption of hG-CSF in vivo. These in vitro and in vivo studies on hG-CSF would be of value for understanding the pathophysiological roles of hG-CSF.

Effect of interferon-alpha in patients with chronic myelogenous leukemia in the accelerated phase: cytogenetic and molecular studies.

Human interferon-alpha (IFN-alpha) has been shown to be effective in the treatment of Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia (CML) in the benign stable phase. The present study indicates that IFN-alpha may have a suppressive effect on Ph1-positive clones not only in the early stable phase but also in the accelerated phase with additional chromosomal abnormalities in some patients. In this study, in addition to 5 benign-phase patients, 3 patients with CML in the accelerated phase who had additional chromosomal abnormalities were treated with IFN-alpha. The presence of the Ph1-positive clone was estimated by chromosomal analysis and by Southern analysis at the DNA level using a 3' breakpoint cluster region (bcr) probe. Hematological remission and the suppression of proliferation of Ph1-positive clone to various extents were achieved by IFN-alpha treatment in 2 benign-phase patients and 3 patients with additional chromosomal abnormalities. Interestingly, in one of the latter three patients, Ph1-positive clones with or without additional chromosomal abnormalities were completely suppressed judging from chromosomal analysis and from the disappearance of bcr gene rearrangements.

Conceptus uptake of the 106RuNO-nitro complex in relation to gestational stages.

106RuNO-nitro complex was administered intravenously to pregnant rats to study uptake of the radioruthenium complex by the conceptus in relation to gestational stages. Each conceptus was sampled periodically with respect to its placenta, fetal membrane, fetal fluid and fetus. Perceptible radioactivity in the fetus was detected only in the later stage of gestation and its relative concentration, defined as the ratio radioactivity per unit weight in the body tissue at sacrifice to that in the whole-body at dosing, was very low compared with other tissues. The average number of fetuses in one litter was 13 and the transfer rate of nitro complex into the fetuses 24 hr after injection to rats on the 20th day of gestation was about 1% of initial maternal dose. The relative concentration in the placenta and fetal membrane was much higher than in the fetus and decreased with time after injection. These results indicate that the placenta and fetal membrane play significant roles as barriers to the transfer of 106RuNO-nitro complex into the fetus.

Implantation of fibroblasts transfected with human granulocyte colony-stimulating factor cDNA into mice as a model of cytokine-supplement gene therapy.

A fibroblast-mediated gene delivery method was used for the endogenous expression of human granulocyte colony-stimulating factor (G-CSF) as a model for cytokine supplement therapy. Human G-CSF cDNA was inserted into the plasmid expression vector BMGNeo, which contains a partial sequence of bovine papilloma virus and a selectable marker gene. The recombinant plasmid (BMGNeo-GCSF) was transfected into NIH/3T3 fibroblasts by the calcium phosphate coprecipitation method, and the stably transformed cells were isolated by G418 selection. An appropriate clone producing a large amount of G-CSF was selected by enzyme immunoassay of the culture supernatants. Southern blot analysis suggested that the BMGNeo-GCSF plasmid replicated mainly as an episome, and Northern blot analysis demonstrated the high expression of human G-CSF mRNA in the cells. After the implantation of the G-CSF-producing fibroblasts into nude mice, prominent neutrophilia, about 30-fold the level of normal control, was observed within seven days. Moreover, the number of hematopoietic progenitor cells in spleen remarkably increased for all cell lineages in these mice. To regulate the in vivo expression of G-CSF, we designed a subcutaneous diffusion chamber apparatus that contains the G-CSF-producing fibroblasts. The leukocytosis (neutrophilia) induced in C3H mice after embedding the device quickly disappeared after ethanol treatment of the chamber. Furthermore, reinjection of the G-CSF-producing fibroblasts into the chamber caused a second neutrophilia.

A new bioassay for human granulocyte colony-stimulating factor (hG-CSF) using murine myeloblastic NFS-60 cells as targets and estimation of its levels in sera from normal healthy persons and patients with infectious and hematological disorders.

[3H]thymidine uptake by NFS-60 cells in microcultures was found to increase in a linear fashion with the increasing doses of purified recombinant human granulocyte colony-stimulating factor (rhG-CSF). Such increases were found neither with rhG-CSF samples pretreated with rabbit anti-rhG-CSF serum nor with other human colony-stimulating factors such as granulocyte-macrophage colony-stimulating factor (hGM-CSF) or macrophage colony-stimulating factor (hM-CSF). Based on these findings, sera from normal persons and patients with severe infections or various hematological disorders were tested after dialysis using this system in order to determine whether G-CSF levels in sera can be estimated or not. In ten normal persons, five patients with acute myelogenous leukemia (AML M1, M2, and M3), five with myelodysplastic syndrome, and four with chronic myelogenous leukemia, no increases in [3H]thymidine uptake were found within the dose range of 0.4 microliters to 50 microliters. In contrast, linear dose responses parallel to a G-CSF standard curve were observed in one patient with a severe bacterial infection, four with aplastic anemia, two with acute myelomonocytic leukemia (AMMoL) (M4), and two with idiopathic neutropenia tested. From the standard curve, the probable levels of G-CSF were calculated as follows: approximately 200 pg/ml with infection, 130-220 pg/ml with aplastic anemia, 150 and 200 pg/ml with AMMoL, and 1120 and 1200 pg/ml with idiopathic neutropenia. The activities of sera were reduced by the anti-rhG-CSF serum pretreatment in the same way as documented in the case of rhG-CSF. Furthermore, the level in a patient with a severe infection became undetectable soon after elimination of the infection and blood neutrophil counts had returned to normal. These findings indicate that the microbioassay system will be useful for measuring circulating G-CSF levels which would fluctuate in accord with requirements for stimulating neutrophil production or with abnormal production of hG-CSF.

Serum granulocyte colony-stimulating factor levels in healthy volunteers and patients with various disorders as estimated by enzyme immunoassay.

In order to better understand the patho-physiologic role of granulocyte colony-stimulating factor (G-CSF), we estimated its serum levels in healthy persons and patients with various disorders, using a newly developed enzyme immunoassay (Motojima et al). In 49 of 56 normal healthy persons (88%), the levels were beneath the sensitivity of the assay (less than 30 pg/mL), while in the remaining seven healthy persons, the levels ranged from 33 to 163 pg/mL. On the other hand, nine of 11 patients (82%) with idiopathic aplastic anemia (AA), one patient with Fanconi's anemia, six of 12 patients (50%) with myelodysplastic syndrome (MDS), five of 12 patients (42%) with acute leukemia without any blast cells in the blood (M4: one, M5: one, L1: one, and L2: two), six of 18 patients (33%) with chronic myeloid leukemia (CML), one of two patients with chronic lymphoid leukemia (CLL), two of four patients with lung cancer, one patient with cyclic neutropenia, two of seven patients with malignant lymphoma, and four patients with acute infection had G-CSF levels ranging from 46 pg/mL to greater than 2,000 pg/mL. Interestingly, a reverse correlation between blood neutrophil count and serum G-CSF level was clearly demonstrated for aplastic anemia (r = -.8169, P less than .01). Moreover, it was found that the G-CSF level rose during the neutropenic phase of cyclic neutropenia and after chemotherapy or bone marrow transplantation (BMT) in three patients with leukemia; also high G-CSF levels were positively correlated to blood neutrophil counts in some cases of infectious disorders and lung cancer. The cellular sources and the mechanisms for production and secretion of circulating G-CSF were not investigated in this study, but the data presented here strongly indicate that G-CSF plays an important role as a circulating neutrophilopoietin.