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.

We produced lethally irradiated retrovirally GM-CSF-transduced autologous renal tumor cell vaccines (GVAX) from six Japanese patients with stage IV renal cell cancer (RCC). Four patients received GVAX ranging from 1.4 x 10(8) to 3.7 x 10(8) cells on 6-17 occasions. Throughout a total of 48 vaccinations, there were no severe adverse events. After vaccination, DTH skin tests became positive to autologous RCC (auto-RCC) in all patients. The vaccination sites showed significant infiltration by CD4(+) T cells, eosinophils, and HLA-DR-positive cells. The kinetic analyses of cellular immune responses using peripheral blood lymphocytes revealed an enhanced proliferative response against auto-RCC in four patients, and cytotoxicity against auto-RCC was augmented in three patients. T cell receptor beta-chain analysis revealed oligoclonal expansion of T cells in the peripheral blood, skin biopsy specimens from DTH sites, and tumors. Western blot analysis demonstrated the induction of a humoral immune response against auto-RCC. Two of the four patients are currently alive 58 and 40 months after the initial vaccination with low-dose interleukin-2. Our results suggest that GVAX substantially enhanced the antitumor cellular and humoral immune responses, which might have contributed to the relatively long survival times of our patients in the present study.

In order to elucidate molecular events in BCR/ABLinduced 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.z 2000 Federation of European Biochemical Societies.

The production of interleukin 1 ␤ (IL-1 ␤ ) 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 ϩ 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% (

Abstract. 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 endothelid 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 strom d 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

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.

No abstract

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).