Interferon-gamma-induced gene expression in CD34 cells: identification of pathologic cytokine-specific signature profiles.

Hematopoietic effects of interferon-gamma (IFN-gamma) may be responsible for certain aspects of the pathology seen in bone marrow failure syndromes, including aplastic anemia (AA), paroxysmal nocturnal hemoglobinuria (PNH), and some forms of myelodysplasia (MDS). Overexpression of and hematopoietic inhibition by IFN-gamma has been observed in all of these conditions. In vitro, IFN-gamma exhibits strong inhibitory effects on hematopoietic progenitor and stem cells. Previously, we have studied the transcriptome of CD34 cells derived from patients with bone marrow failure syndromes and identified characteristic molecular signatures common to some of these conditions. In this report, we have investigated genome-wide expression patterns after exposure of CD34 and bone marrow stroma cells derived from normal bone marrow to IFN-gamma in vitro and have detected profound changes in the transcription profile. Some of these changes were concordant in both stroma and CD34 cells, whereas others were specific to CD34 cells. In general, our results were in agreement with the previously described function of IFN-gamma in CD34 cells involving activation of apoptotic pathways and immune response genes. Comparison between the IFN-gamma transcriptome in normal CD34 cells and changes previously detected in CD34 cells from AA and PNH patients reveals the presence of many similarities that may reflect molecular signature of in vivo IFN-gamma exposure.

Autologous gamete cryopreservation before hemopoietic stem cell transplantation.

BACKGROUND: Infertility after hemopoietic stem cell transplantation (HST) is a serious problem for young patients. Autologous gamete collection before HST may be a promising strategy to overcome infertility. MATERIAL/METHODS: From October 1988 to December 2003, six male and nine female patients with hematological malignancies had autologous gametes collected before HST. The data on autologous gamete collection were analyzed. RESULTS: Sperm could be collected from three patients. However, in two of the three, the numbers and motility of the sperm were severely depleted because they received chemotherapy for one and 11 cycles, respectively. Normal sperm was only collected from one patient with myelodysplastic syndrome who had no history of receiving chemotherapy. One or more oocytes could be collected in five of nine female patients, although the five received multiple cycles of chemotherapy. The successful oocyte collection was associated with an ovulation stimulant. CONCLUSIONS: Autologous oocye collection before HST may be possible, even if patients receive multiple cycles of chemotherapy. In contrast, autologous sperm collection before HST may be difficult after patients receive chemotherapy. Successful pregnancy using autologous gametes after HST remains extremely difficult, especially in female patients; however, it is important to give information on infertility and autologous gamete collection to patients scheduled for HST.

Severe hepatitis and complete molecular response caused by imatinib mesylate: possible association of its serum concentration with clinical outcomes.

A 40-year-old female with chronic myelogeneous leukemia (CML) in the chronic phase was treated with imatinib mesylate (STI571) because of interferon resistance. She achieved complete cytogenetic response but not complete molecular response 3 months after STI571 administration. Six months later, she developed severe liver damage without evidence of actively infectious hepatitis A, B, C, G, E, TT virus, Epstein-Barr virus or cytomegalovirus. A significant serum level of STI571 (107 ng/ml) was detected, although she had not taken the drug for 6 days. Liver biopsy demonstrated massive hepatic necrosis, consistent with drug-induced hepatitis. She achieved complete molecular response, although she did not take STI571 for 47 days after the development of hepatitis. These results suggest that both hepatitis and molecular response were associated with the serum STI571 concentration.

Distinctive gene expression profiles of CD34 cells from patients with myelodysplastic syndrome characterized by specific chromosomal abnormalities.

Aneuploidy, especially monosomy 7 and trisomy 8, is a frequent cytogenetic abnormality in the myelodysplastic syndromes (MDSs). Patients with monosomy 7 and trisomy 8 have distinctly different clinical courses, responses to therapy, and survival probabilities. To determine disease-specific molecular characteristics, we analyzed the gene expression pattern in purified CD34 hematopoietic progenitor cells obtained from MDS patients with monosomy 7 and trisomy 8 using Affymetrix GeneChips. Two methods were employed: standard hybridization and a small-sample RNA amplification protocol for the limited amounts of RNA available from individual cases; results were comparable between these 2 techniques. Microarray data were confirmed by gene amplification and flow cytometry using individual patient samples. Genes related to hematopoietic progenitor cell proliferation and blood cell function were dysregulated in CD34 cells of both monosomy 7 and trisomy 8 MDS. In trisomy 8, up-regulated genes were primarily involved in immune and inflammatory responses, and down-regulated genes have been implicated in apoptosis inhibition. CD34 cells in monosomy 7 showed up-regulation of genes inducing leukemia transformation and tumorigenesis and apoptosis and down-regulation of genes controlling cell growth and differentiation. These results imply distinct molecular mechanisms for monosomy 7 and trisomy 8 MDS and implicate specific pathogenic pathways.

Regulation of cellular gene expression in endothelial cells by sin nombre and prospect hill viruses.

Mechanisms of hantavirus-induced vascular leakage remain unknown. This study was initiated to determine whether hantavirus-induced changes in endothelial cell gene expression may provide insight into disease mechanisms. Additionally, by using pathogenic Sin Nombre virus (SNV) and non-pathogenic Prospect Hill virus (PHV), we wanted to identify cellular responses that are likely to differentiate pathogenic from nonpathogenic hantaviruses. Using the Affymetrix DNA Array, we found that PHV and SNV did not significantly differ in the number of activated genes (18 versus 14 genes) in infected endothelial cells at 4 h PI. However, a smaller group of genes (36) were up-regulated by PHV compared to SNV (175) at 12 h PI. Only two genes were down-regulated in SNV-infected cells. Expression of the functionally diverse group of genes was altered at an early stage of infection (4 and 12 h PI). The genes affected include putative anti-viral factors, transcription factors, growth factors, chemokines, receptors, structural proteins, metabolism, and kinases. Although many genes were activated in cells infected with SNV and PHV, overall cellular transcriptional responses were more altered by pathogenic SNV compared to non-pathogenic PHV.

The relationship of aplastic anemia and PNH.

Bone marrow failure has been regarded as one of the triad of clinical manifestations of paroxysmal noctumal hemoglobinuria (PNH), and PNH in turn has been described as a late clonal disease evolving in patients recovering from aplastic anemia. Better understanding of the pathophysiology of both diseases and improved tests for cell surface glycosylphosphatidylinositol (GPI)-linked proteins has radically altered this view. Flow cytometry of granulocytes shows evidence of an expanded PNH clone in a large proportion of marrow failure patients at the time of presentation: in our large NIH series, about 1/3 of over 200 aplastic anemia cases and almost 20% of more than 100 myelodysplasia cases. Clonal PNH expansion (rather than bone marrow failure) is strongly linked to the histocompatability antigen HLA.-DR2 in all clinical varieties of the disease, suggesting an immune component to its pathophysiology. An extrinsic mechanism of clonal expansion is also more consistent with knock-out mouse models and culture experiments with primary cells and cell lines, which have failed to demonstrate an intrinsic proliferative advantage for PNH cells. DNA chip analysis of multiple paired normal and PIG-A mutant cell lines and lymphoblastoid cells do not show any consistent differences in levels of gene expression. In aplastic anemia/PNH there is surprisingly limited utilization of the V-beta chain of the T cell receptor, and patients' dominant T cell clones, which are functionally inhibitory of autologous hematopoiesis, use identical CDR3 regions for antigen binding. Phenotypically normal cells from PNH patients proliferate more poorly in culture than do the same patient's PNH cells, and the normal cells are damaged as a result of apoptosis and overexpress Fas. Differences in protein degradation might play a dual role in pathophysiology, as GPI-linked proteins lacking an anchor would be predicted to be processed by the proteasome machinery and displayed in a class I H.A. context, in contrast to the normal pathway of cell surface membrane recycling, lysosomal degradation, and presentation by class II HLA. The strong relationship between a chronic, organ-specific immune destructive process and the expansion of a single mutant stem cell clone remains frustratingly enigmatic but likely to be the result of interesting biologic processes, with mechanisms that potentially can be extended to the role of inflammation in producing premalignant syndromes.

Analysis of baseline and cisplatin-inducible gene expression in Fanconi anemia cells using oligonucleotide-based microarrays.

BACKGROUND: Patients with Fanconi anemia (FA) suffer from multiple defects, most notably of the hematological compartment (bone marrow failure), and susceptibility to cancer. Cells from FA patients show increased spontaneous chromosomal damage, which is aggravated by exposure to low concentrations of DNA cross-linking agents such as mitomycin C or cisplatin. Five of the identified FA proteins form a nuclear core complex. However, the molecular function of these proteins remains obscure. METHODS: Oligonucleotide microarrays were used to compare the expression of approximately 12,000 genes from FA cells with matched controls. Expression profiles were studied in lymphoblastoid cell lines derived from three different FA patients, one from the FA-A and two from the FA-C complementation groups. The isogenic control cell lines were obtained by either transfecting the cells with vectors expressing the complementing cDNAs or by using a spontaneous revertant cell line derived from the same patient. In addition, we analyzed expression profiles from two cell line couples at several time points after a 1-hour pulse treatment with a discriminating dose of cisplatin. RESULTS: Analysis of the expression profiles showed differences in expression of a number of genes, many of which have unknown function or are difficult to relate to the FA defect. However, from a selected number of proteins involved in cell cycle regulation, DNA repair and chromatin structure, Western blot analysis showed that p21waf1/Cip1 was significantly upregulated after low dose cisplatin treatment in FA cells specifically (as well as being expressed at elevated levels in untreated FA cells). CONCLUSIONS: The observed increase in expression of p21waf1/Cip1 after treatment of FA cells with crosslinkers suggests that the sustained elevated levels of p21waf1/Cip1 in untreated FA cells detected by Western blot analysis likely reflect increased spontaneous damage in these cells.

New human myelodysplastic cell line, TER-3: G-CSF specific downregulation of Ca2+/calmodulin-dependent protein kinase IV.

We have established a new hematopoietic cell line from a patient with myelodysplastic syndrome (MDS), which was refractory anemia with excess blasts (RAEB). This cell line, designated TER-3, depends on several cytokines for long-term survival and growth, and requires interleukin-3 (IL-3) for continuous growth. Cytochemical analysis revealed that TER-3 cells are weakly dianisidine positive and nonspecific esterase positive, but peroxidase negative. The surface marker profile shows that the TER-3 cells are strongly positive for myeloid, lymphoid, and megakaryocytic antigens such as CD15, CD19, and CD61, and negative for some common multilineage antigens such as CD13, CD33, and CD34. Thus, this cell line has a multilineage phenotype, suggesting that the transformation event occurred in multipotent stem cells. Dianisidine- and nonspecific esterase-positive TER-3 cells increase with granulocyte-colony stimulating factor (G-CSF) rather than with IL-3. These results suggest that the cell line is useful for understanding the mechanism underlying G-CSF-associated hematopoietic cell differentiation and activation in the patient with MDS.