Clinical significance of lymphocyte subpopulations and cytokine-producing activity of blood cells in patients with primary myelodysplastic syndrome

Lymphocyte subpopulation composition and spontaneous production of tumor necrosis factor-alpha (TNF-alpha) by blood cells were studied in 22 and 15 patients with myelodysplastic syndrome (MDS), respectively, who were under immunosuppressive therapy (IST). Patients with hematological response revealed a significantly increased cytotoxic CD8+ lymphocyte concentration and TNF-alpha production. A direct correlation between TNF-alpha production rate and CD8+ lymphocyte level was established. Maximum cytokine-producing activity of blood cells was identified in MDS patients with enhanced erythroid response. The study established a great diversity of MDS, its hypoplastic variants and the prognostic value of spontaneous production of tumor necrosis factor-alpha (TNF-alpha) by blood cells as marker of IST efficacy.     

Generation of progenitor-cell precursors in long-term bone-marrow cultures after marrow purging with ether lipids

Alkyl-lysophospholipid derivates (ALP) are currently being tested as bone marrow (BM) purging agents prior to autologous BM transplantation in different malignancies. We evaluated the toxicity of the ALP ET-18-OCH3 (ET-18; Edelfosine, 1-0-octadecyl-2-0-methyl-rac-glycero-3-phosphocholine) towards early hematopoietic precursors by testing progenitor regeneration of non-purged and ET-18-purged BM (75 mu g and 125 mu g ET-18/ml/2x10(7) BM cells) in autologous long-term bone marrow cultures (LTBMC) from 3 different patients in complete remission. LTBMC feeder layers were irradiated with 875 rad for complete elimination of hematopoietic progenitors and recharged with cryopreserved purged and non-purged BM. In weekly intervals, adherent layer and supernatant LTBMC cells were completely removed and evaluated in colony forming unit (CFU)-assays. We have seen sufficient CFU-regeneration out of ET-18-purged BM up to 8 weeks of LTBMC (>40 CFU/flask). Total CFU-counts from LTBMC with purged BM were slightly reduced compared to non-purged control. High dose purging with 125 mu g ET-18/ml partly inhibited initial CFU-proliferation, but demonstrated elevated CFU-counts after 4 and 8 weeks of LTBMC compared to control. In conclusion, in our LTBMC series ET-18-purging yielded tolerable toxicity towards committed BM-progenitors, but no remarkable decline of early hematopoietic precursors regenerating CFU-progenitors for up to 8 weeks of culture.     

骨髓增生异常综合征中CD34+细胞CXCR4的表达及其与细胞迁移的相关性

  目的  探讨不同危险度骨髓增生异常综合征（myelodysplasticsymdromes,MDS）中骨髓CD34+细胞CXCR4的表达情况及其与细胞迁移率的相关性。   方法  收集40例骨髓增生异常综合征患者的骨髓标本,根据IPSS积分系统进行危险度分组。低危组20例:IPSS积分0~1.5分;高危组20例:IPSS积分≥1.5分;同时采集10例健康者的骨髓标本作为对照。分离纯化骨髓CD34+细胞,通过流式细胞术检测CXCR4膜蛋白的表达;研究SDF-1α趋化作用下CD34+细胞的迁移率及CD34+细胞对骨髓基质细胞的迁移率。   结果  高危组MDS患者CD34+细胞CXCR4的表达率明显高于低危组和正常对照组（P < 0.000 1）;低危组和正常对照组之间CXCR4的表达率无显著性差异（P>0.05）。高危组CD34+细胞对SDF-1α及骨髓基质细胞的迁移率显著高于低危组及正常组（均P < 0.000 1）,且其对骨髓基质细胞的迁移率与CXCR4的表达呈正相关（P=0.000 1）。   结论  高危组MDS患者CD34+细胞CXCR4的表达量及其对骨髓基质细胞的迁移率均明显高于低危组患者,且其迁移率随CXCR4表达量的增加而升高,不同风险组的MDS患者存在SDF-1及其受体CXCR4表达和功能上的差异,SDF-1及其受体CXCR4在MDS发病中具有重要作用。      

Analysis of hematopoietic progenitor cells in patients with myelodysplastic syndromes according to their cytogenetic abnormalities

The present work analyzes the hematopoietic progenitor cells (HPC) in myelodysplastic syndrome (MDS) patients using both an immunophenotypical and a functional approaches in order to know whether they are similar in patients with or without cytogenetic abnormalities. Among CD34+ HPC, the proportion of myeloid committed progenitors was higher in patients with an abnormal karyotype. Ninety MDS patients were studied. Patients with abnormal karyotype showed a similar platting efficiency than patients with normal cytogenetics. Trisomy 8 and 5q- showed a significant higher P.E. than patients with normal karyotype or monosomy 7. We observed that when the most immature HPC were studied, the total number of granulo-monocytic colonies produced by LTBMC was higher in the normal karyotype group. In summary, the present study shows that in MDS the HPC are impaired; this impairment is deeper in patients with abnormal karyotype.     

Autoreactive T-lymphocytes are implicated in the pathogenesis of bone marrow failure in patients with systemic lupus erythematosus

There is an increasing evidence that the trafficking and homing of autoreactive lymphocytes in the bone marrow (BM) of patients with systemic lupus erythematosus (SLE) may affect the haemopoiesis supporting capacity of BM stroma and may also damage haemopoiesis even at the level of the haemopoietic stem cell not only via direct immune destruction but also by an indirect effect due to the release of pro-inflammatory cytokines rendering stem cells sensitive to Fas-induced apoptosis. We have shown that the low BM CD34+ cell number in SLE is due to--at least in part--Fas up-regulation on these cells and subsequent apoptotic cell death induced by interferon-gamma- and Fas ligand- producing T-cells in the BM microenvironment. In accordance to previous reports demonstrating that co-culture of T-cells from SLE patients with autologous or allogeneic BM mononuclear cells may inhibit their clonogenic potential while removal of T-cells from patient BM samples may improve the in vitro colony formation, we have shown that intensive immunoablation followed by autologous stem cell rescue may restore the number and survival characteristics of BM CD34+ cells and the haemopoiesis supporting capacity of marrow stromal cells in these patients. The consideration of the complex interactions between autoreactive lymphocytes and haemopoietic progenitor cells in the BM may elucidate further the pathogenetic mechanisms underlying cytopenias in SLE and may provide an explanation for the difficulties in the stem cell collection procedure seen in SLE patients undergoing autologous stem cell transplantation.     

Morphologic, immunophenotypic and in vitro growth characteristics of blood and bone marrow associated with stem cell mobilisation in patients with lymphoma

The proportion of CD34+ cells in the bone marrow (BM) is predictive of the size of progenitor cell mobilisation into the blood (PB). To investigate which other PB and BM parameters may be related to mobilisation, we analysed at steady state PB and BM of 23 patients with relapsed or resistant lymphoma before administering high-dose cyclophosphamide and G-CSF Cell morphology, number of CD34+ cells, and growth in clonogenic assay and in long-term cultures (LTC) were determined and then correlated with mobilisation extent (CD34+ and GM-CFC) and quality (growth of harvested cells in LTC). We found that the good mobilising patients (CD34 > 50 x 10(3)/ml, n=10) had several baseline BM characteristics (number of CD34+ MNC, GM-CFC, BFU-E, production of CFCs in LTC) similar to a group of 12 healthy controls, while patients with reduced mobilisation (CD34 < 50 x 10(3)/ml, n=13) had clearly reduced BM progenitors and LTC growth (p< 0.05). In a multivariate analysis including baseline clinical, blood and bone marrow characteristics, the most significant PB and BM factors independently associated with a higher number and/or quality of mobilised cells were a higher number of CD34+ and GM-CFC in the BM and a higher baseline haemoglobin, platelet, and CD34+ blood count. The capacity to release progenitor cells into the circulation is therefore not predicted by the distribution of morphologically distinguishable cells, marginally predicted by the BM content of highly undifferentiated cells (growth in long term culture), while it is proportional to the number of BM progenitors (CD34+, GM-CFC and BFU-E).     

A practical miniature long-term bone marrow culture system for investigating early myelodysplasia.

A method was devised to grow haemopoietic cells in long-term bone marrow culture (LTBMC) which requires only 1 x 10(6) cells/culture. Such miniature cultures were used to study growth patterns of marrow from patients with myelodysplastic syndromes (MDS). Consistent differences in LTBMC cellularity and cellular composition were noted between MDS and normal marrow. These differences were accentuated by rGM-CSF. The criteria which distinguished between and MDS marrows were: cell count at weeks 1 and 4, % neutrophils and % blasts. In 10 patients with unexplained macrocytosis or pancytopenia miniature LTBMC results clearly segregated into either 'normal' or 'MDS' growth patterns. Miniature LTBMC with rGM-CSF may therefore be a useful diagnostic test for early MDS.     

Fas ligand expression in the bone marrow in myelodysplastic syndromes correlates with FAB subtype and anemia, and predicts survival.

Increased apoptosis in the bone marrow (BM) may contribute to the cytopenias that occur in myelodysplastic syndromes (MDS). The Fas receptor, Fas ligand (FasL) pathway is a major mechanism of apoptosis. Since hematopoietic progenitors can express the Fas receptor, they may be susceptible to apoptosis induced by FasL-expressing cells. We examined FasL expression in the BM of patients with MDS (n = 50), de novo acute myeloid leukemia (AML; n = 10), AML following prior MDS (n = 6), and normal controls (n = 6). Compared to controls, FasL expression was increased in MDS, and was highest in AML. In MDS, FasL expression was seen in myeloid blasts, erythroblasts, maturing myeloid cells, megakaryocytes and dysplastic cells, whereas in AML, intense expression was seen in the blasts. FasL expression correlated with the FAB subtype groups of MDS, and also correlated directly with the percentage of abnormal metaphases on cytogenetic analysis. The FasL expressed in MDS BM inhibited the growth of clonogenic hematopoietic progenitors. This inhibition could be blocked by a soluble recombinant FasFc protein. In MDS, FasL expression in the initial diagnostic BM was higher in patients who were more anemic, correlated directly with red cell transfusion requirements over the subsequent course of the disease, and was predictive of survival. These studies indicate that FasL expression in MDS is of prognostic significance, and suggest that pharmacological blockade of the Fas-FasL pathway may be of clinical benefit.     

Long-term bone marrow cultures: their use in autologous marrow transplantation

When cultured together, hemopoietic cells and marrow stromal cells can support hemopoiesis in vitro in the absence of added growth factors. The continued production of stem cells, progenitor cells, and mature cells within these "long-term bone marrow cultures" (LTBMC) requires their physical association with the stromal cells. For reasons that are not well understood, leukemic cells often fail to survive in such cultures. We and others have undertaken studies to examine whether LTBMC can be exploited as a way of purging the bone marrow of leukemia patients prior to autologous marrow transplantation.     

Early disappearance of murine plasmocytoma stem cells in long-term bone marrow culture.

Long-term bone marrow culture (LTBMC) was evaluated as a purging procedure in the murine plasmocytoma MOPC-315s system. MOPC-315s cells injected in Balb-c mice rapidly proliferate both in marrow and spleen, where macroscopic tumor colonies develop. A linear relationship between the number of injected cells and spleen colonies was observed, consistent with the presence of 1 clonogenic myeloma stem cell out of 1800 cells. In vitro, MOPC-315s cells are easily identifiable as rosette-forming cells (RFC+) with trinitrophenil acid (TNP) coated sheep red blood cells. When bone marrow (BM) cells containing 20-40% RFC+ were seeded in LTBMC, RFC+ rapidly decreased and were no longer detectable by day 14 of culture. Clonal Ig gene rearrangement was evident at time 0, but it was no more detectable later on. In addition, cells taken at days 14 and 21 of culture were no more tumorigenic when injected in vivo. The results suggest the efficacy of the LTBMC for the in vitro elimination of myeloma cells, including the neoplastic stem cells.     

Inappropriate notch activity and limited mesenchymal stem cell plasticity in the bone marrow of patients with myelodysplastic syndromes

Myelodysplastic syndromes (MDSs) are a heterogeneous group of hematological disorders characterized by ineffective hematopoiesis, enhanced bone marrow apoptosis and frequent progression to acute myeloid leukemia. Several recent studies suggested that, besides the abnormal development of stem cells, microenvironmental alterations are also present in the MDS bone marrow. In this study, we have examined the relative frequencies of stem and progenitor cell subsets of MDS and normal hematopoietic cells growing on stromal cell layers established from MDS patients and from normal donors. When hematopoietic cells from MDS patients were co-cultured with normal stromal cells, the frequency of either early or late cobblestone area-forming cells (CAFC) was significantly lower compared to the corresponding normal control values in 4 out of 8 patients. In the opposite situation, when normal hematopoietic cells were incubated on MDS stromal cells, the CAFC frequencies were decreased in 5 out of 6 patients, compared to normal stromal layer-containing control cultures. Moreover, a soluble Notch ligand (Jagged-1 protein) was an inhibitor of day-35-42 CAFC when normal hematopoietic cells were cultured with normal or MDS stromal cells, but was unable to inhibit MDS stem and early progenitor cell growth (day-35-42 CAFC) on pre-established stromal layers. These findings suggest that in early hematopoietic cells isolated from MDS patients the Notch signal transduction pathway is disrupted. Furthermore, there was a marked reduction in the plasticity of mesenchymal stem cells of MDS patients compared with those of normal marrow donors, in neurogenic and adipogenic differentiation ability and hematopoiesis supporting capacity in vitro. These results are consistent with the hypothesis that when alterations are present in the myelodysplastic stroma environment along with intrinsic changes in a hematopoietic stem/progenitor cell clone, both factors might equally contribute to the abnormal hematopoiesis in MDS.     

Immunologic aspects of hypoplastic myelodysplastic syndrome

The pathophysiology of myelodysplastic syndromes (MDS) is multiple, complex, and poorly understood. In some cases of MDS, especially those in which the bone marrow is hypocellular, there is increasing experimental and clinical indication that an immune-mediated damage to hematopoietic precursors and changes in the hematopoiesis-supporting microenvironment contribute to disease development. Increased serum levels of type-1 cytokines, tumor necrosis factor-α (TNF-α), and interferon-γ (INF-γ), and oligoclonal expansion of cytotoxic T cells are observed in human MDS. In some cases, the immunologic attack to the marrow appears to be triggered by MDS-specific antigens, damaging the microenvironment and inducing cell apoptosis especially of normal progenitors. In murine models, dysregulation of osteoprogenitors leads to disrupted hematopoiesis of healthy hematopoietic progenitor and stem cells, eventually resulting in MDS and leukemia. In hypocellular MDS, marrow failure appears to be not only the result of ineffective erythropoiesis of abnormal clones, but also due to inhibition of normal progenitors. Immunosuppressive therapy with cyclosporine, anti-thymocyte globulin, or alemtuzumab may alleviate cytopenias and in some instances induce cytogenetic remission. However, not all patients respond to immunosuppression, and the identification of relevant biomarkers for an immune mechanism is necessary to identify those patients who may benefit from this treatment modality.     

Prognostic effect of epithelial and stromal lymphocyte infiltration in non-small cell lung cancer

PURPOSE: The major value of prognostic markers in potentially curable non-small cell lung cancer (NSCLC) should be to guide therapy after surgical resection. In this regard, the patients' immune status at the time of resection may be important and also measurable. The immune system has paradoxical roles during cancer development. However, the prognostic significance of tumor-infiltrating lymphocytes is controversial. The aim of this study is to elucidate the prognostic significance of epithelial and stromal lymphocyte infiltration in NSCLC. EXPERIMENTAL DESIGN: Tissue microarrays from 335 resected NSCLC, stage I to IIIA were constructed from duplicate cores of viable and representative neoplastic epithelial and stromal areas. Immunohistochemistry was used to evaluate the epithelial and stromal CD4+, CD8+, and CD20+ lymphocytes. RESULTS: In univariate analyses, increasing numbers of epithelial CD8+ (P = 0.023), stromal CD8+ (P = 0.002), epithelial CD20+ (P = 0.023), stromal CD20+ (P < 0.001), and stromal CD4+ (P < 0.001) lymphocytes correlated significantly with an improved disease-specific survival. No such relation was noted for epithelial CD4+ cells. Furthermore, a low level of stromal CD8+ lymphocyte infiltration was associated with an increased incidence of angiolymphatic invasion (P = 0.032). In multivariate analyses, a high number of stromal CD8+ (P = 0.043) and CD4+ (P = 0.002) cells were independent positive prognostic factors for disease-specific survival. CONCLUSIONS: High densities of CD4+ and CD8+ lymphocytes in the stroma are independent positive prognostic indicators for resected NSCLC patients. This may suggest that these cells are mediating a strong antitumor immune response in NSCLC.     

Long-term follow-up of high-dose treatment with autologous haematopoietic progenitor cell support in 693 patients with follicular lymphoma: an EBMT registry study.

To evaluate the outcome of a large series of patients who received high-dose treatment (HDT) for follicular lymphoma (FL), 693 patients undergoing HDT (total-body irradiation (TBI)-containing regimen: 58%; autologous bone marrow (BM)/peripheral blood progenitor cells (PBPCs): 378/285 patients) were included in the study. A total of 375 patients (54%) developed recurrent lymphoma, 10-year progression-free survival (PFS) being 31%. On multivariate analysis, younger age (P=0.003) and HDT in first complete remission (CR1) (P<0.001) correlated with longer PFS. With a median follow-up of 10.3 years, 330 patients died. Ten-year overall survival (OS) from HDT was 52%. Shorter OS was associated on multivariate analysis with older age (P<0.001), chemoresistant disease (P<0.001), BM+PBPC as source of stem cells (P=0.007) and TBI-containing regimens (P=0.004). Thirty-nine patients developed secondary myelodysplastic syndrome/acute myeloid leukaemia (MDS/AML), in 34 cases having received TBI as the conditioning regimen. The 5-year non-relapse mortality (NRM) was 9%. On multivariate analysis, older age (P<0.001), refractory disease (P<0.001) and TBI (P=0.04) were associated with a higher NRM. This long follow-up study shows a plateau in the PFS curve, suggesting that a selected group of patients might be cured with HDT. On the downside, TBI-containing regimens are associated with a negative impact on survival.     

Bone marrow CD34+ cell count is predictive for adequate peripheral progenitor cell collection

Several studies have investigated the influence of clinical and biological variables on mobilisation of peripheral blood progenitor cells (PBPCs). The aim of this study was to evaluate the role of steady-state bone marrow (BM) CD34+ cells as a predictive parameter of PBPC yield. We studied 90 patients with multiple myeloma (MM) (41 patients), non-Hodgkin's lymphoma (NHL) (25 patients) or acute myeloid leukaemia (AML) (24 patients), mobilised with chemotherapy and growth factor. The median time from first treatment to mobilisation was 5 months. Only one patient was previously exposed to alkylating agents. The median BM CD34+ count at mobilisation was 833 microl(-1) (range: 1.4-15.540) corresponding to 1.51% of mononuclear cells (range: 0.02-8.6). Sixty-six patients (73%) reached the optimal target of 4 x 10(6) kg(-1) CD34+ cells with 1 (18 patients), 2 (42 patients) or 3 leukaphereses (6 patients). Eleven patients (12%) mobilised less than 4 x 10(6) kg(-1) CD34+ cells and 13 (15%) failed mobilisation. Among the laboratory and clinical parameters evaluated at the time of mobilisation, only BM CD34+ count was a predictive factor for adequate collection (P = 0.04), particularly in MM patients (P = 0.003). In this setting, a BM concentration of CD34+ cells lower than 66 microL(-1) was associated with a higher probability of inadequate collection.     

The detection of Philadelphia chromosome negative metaphases in long-term bone marrow cultures of the peripheral blood from patients with chronic myeloid leukemia predicts response to interferon-alpha 2a.

The cytogenetic response of 10 patients with chronic myeloid leukaemia (CML) to human recombinant interferon-alpha 2a (rhIFN alpha 2a) was compared to the Philadelphia chromosome (Ph) status of the pre-treatment peripheral blood cells after in vitro culture under long-term bone marrow culture (LTBMC) conditions. Pre-treatment light density peripheral blood cells were cultured in LTBMC on sex-mismatched irradiated allogeneic stromal layers with weekly cytogenic examination of metaphases in the non-adherent cell fraction. This was correlated with the patients' response to rhIFN alpha. Two groups of patients, five showing a cytogenetic response (responsive) and five who failed to achieve a cytogenetic response (nonresponsive) were studied. At the initiation of the LTBMCs the Ph' was found to be present in 100% of the cells analysed for nine patients and 97% for one patient. Pretreatment peripheral blood from four responsive patients demonstrated a decline in the proportion of Ph'-positive cells (Ph+) after 1 to 2 weeks in LTBMC. In contrast, peripheral blood from all the non-responsive subjects showed persistence of the Ph+ clone in 100% of the cells analysed out to a maximum of 3 to 5 weeks in LTBMC. A significant difference was observed (Fisher exact test, p = 0.023) between the two patient groups in respect to the appearance of normal clones in the nonadherent population. The presence of Ph- metaphases in LTBMC of peripheral blood cells of CML patients may predict their cytogenetic response to rhIFN alpha 2a.     

Prognostic analysis of early lymphocyte recovery in patients with advanced breast cancer receiving high-dose chemotherapy with an autologous hematopoietic progenitor cell transplant.

PURPOSE: The purpose of this study was to evaluate the prognostic effect of early posttransplant lymphocyte recovery in patients with advanced breast cancer receiving high-dose chemotherapy with autologous hematopoietic progenitor cell transplantation. EXPERIMENTAL DESIGN: We analyzed the effect of the absolute lymphocyte count on day +15 posttransplant on freedom from relapse and overall survival in patients with high-risk primary breast cancer or metastatic breast cancer, enrolled between 1990 and 2001 in prospective high-dose chemotherapy trials, using a uniform regimen of cyclophosphamide, cisplatin, and 1,3-bis(2-chloroethyl)-1-nitrosourea. RESULTS: Four hundred and seventy-six patients (264 high-risk primary breast cancer and 212 metastatic breast cancer patients) were evaluated at median follow-up of 8 years (range, 1.5-11 years). The disease-free survival and overall survival rates in the high-risk primary breast cancer group were 67% and 70%, respectively. Patients with metastatic breast cancer patients had 21.8% disease-free survival and 31.5% overall survival rates. Day +15 absolute lymphocyte count correlated with freedom from relapse (P = 0.007) and overall survival (P = 0.04) in the metastatic breast cancer group, but not in the high-risk primary breast cancer group (P = 0.5 and 0.8, respectively). The prognostic effect of absolute lymphocyte count in metastatic breast cancer was restricted to those patients receiving unmanipulated peripheral blood progenitor cells (P = 0.04). In contrast, absolute lymphocyte count had no significant effect in those metastatic breast cancer patients receiving bone marrow or a CD34-selected product. In multivariate analyses, the prognostic effect of day +15 absolute lymphocyte count in metastatic breast cancer was independent of other predictors, such as disease status, pre-high-dose chemotherapy treatment, number of tumor sites, or HER2. CONCLUSIONS: Early lymphocyte recovery is an independent outcome predictor in metastatic breast cancer patients receiving high-dose chemotherapy and an autologous peripheral blood progenitor cell transplant. These observations suggest that immune strategies targeting minimal posttransplant residual disease may prove worthwhile.     

The effects of alpha interferon on human long-term bone marrow culture

The effect of alpha IFN on normal long term bone marrow culture (LTBMC) was assessed by measuring haemopoietic progenitor formation and stromal cell number and composition over the course of 5 weeks. When alpha IFN was added at the initiation of LTBMC, there was a marked inhibition of CFU-GEMM, BFU-E and CFU-GM formation from both adherent and non-adherent compartments of culture. There was a profound inhibition of stromal layer formation, especially the reticulo-fibroblast component, and this was not a result of TNF release from macrophages. When alpha IFN was added to established normal LTBMC, although haemopoietic progenitor formation was inhibited, there was little effect on the stromal layer in terms of number or composition. This suggests that the cytostatic effects of alpha IFN when added at commencement of LTBMC result from the anti-proliferative effects of alpha IFN on these actively dividing cells. It is concluded that in addition to the established inhibitory effects of alpha IFN on haemopoietic progenitors as previously demonstrated in semi-solid culture systems, alpha IFN has profound effects upon the marrow microenvironment. This is of particular relevance to bone marrow transplantation where such cytokines may be considered for clinical use.     

Measurement of apoptosis, proliferation and three cytokines in 46 patients with myelodysplastic syndromes

Extensive apoptosis or programmed cell death (PCD) of both hematopoietic (erythroid, myeloid, megakaryocytic) and stromal cells in myelodysplastic syndromes (MDS) cancels the high birth-rate resulting in ineffective hematopoiesis and has been demonstrated as the probable basis for peripheral cytopenias in MDS by our group. It is proposed that factors present in the microenvironment are inducing apoptosis in all the cells whether stromal or parenchymal. To investigate this hypothesis further, bone marrow biopsies from 46 MDS patients and eight normal individuals were examined for the presence of three cytokines, tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta (TGF-β) and granulocyte macrophage-colony stimulating factor (GM-CSF) and one cellular component, macrophages, by the use of monoclonal antibodies immunohistochemically. Results showed the presence of TNF-α and TGF-β in 41/46 and 40/46 cases of MDS respectively, while only 15 cases showed the presence of GM-CSF. Further a significant direct relationship was found between the degree of TNF-α and the incidence of PCD (p = 0.0015). Patients who showed high PCD also had an elevated TNF-α level. Thus, the expression of high amounts of TNF-α and TGF-β and low amounts of the viability factor GM-CSF may be responsible for the high incidence of PCD leading to ineffective hematopoiesis in MDS. Future studies will be directed at attempting to reverse the lesion in MDS by using anti-TNF-α drugs such as pentoxifylline.