Search

Labeling index (LI), apoptosis, levels of 2 pro-apoptotic cytokines tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta(TGF-beta), and the number of monocyte/macrophage cells that are the likely source of the cytokines were simultaneously measured in plastic-embedded bone marrow (BM) biopsy sections of 145 patients with myelodysplastic syndromes (MDS). TNF-alpha was correlated with TGF-beta (P = .001) and with monocyte/macrophage cells (P = .003). Patients with excess blasts in their marrows had a higher TGF-beta level (P = .01) and monocyte/macrophage number (P = .05). In a linear regression model,TGF-beta emerged as the most significant biological difference between patients who have excess of blasts and those who do not (P = .01). We conclude that in addition to TNF-alpha, TGF-beta also plays a significant role in the initiation and pathogenesis of MDS, and that a more precise definition of its role will likely identify better preventive and therapeutic strategies.

Mitochondria (mt) play an important role in both apoptosis and haem synthesis. The present study was conducted to determine DNA mutations in mitochondrial encoded cytochrome c-oxidase I and II genes. Bone marrow (BM) biopsy and aspirate, peripheral blood (PB) and buccal smear samples were collected from 20 myelodysplastic syndrome (MDS) patients and 10 age-matched controls. Cytochrome c-oxidase I (CO I) and II (CO II) genes were amplified using polymerase chain reaction and sequenced. CO I mutations were found in 13/20 MDS patients and the CO II gene in 2/10 normal and 12/20 MDS samples, irrespective of MDS subtype. Mutations were substitutional, deletional and insertional. CO I mutations were most common at nucleotide positions 7264 (25%) and 7289 (15%), and CO II mutations were most common at nucleotide positions 7595 (40%) and 7594 (30%), suggesting the presence of potential 'hot-spots'. Mutations were not found in buccal smears of MDS patients and were significantly higher in MDS samples compared with age-matched controls in all cell fractions (P < 0.05), with bone marrow high-density fraction (BMHDF) showing a higher mutation rate than other fractions (P < 0.05). MDS marrows showed higher levels of apoptosis than normal controls (P < 0.05), and apoptosis in BMHDF was directly related to cytochrome c-oxidase I gene mutations (P < 0.05). Electron microscopy revealed apoptosis affecting all haematopoietic lineages with highly abnormal, iron-laden mitochondria. These results suggest a role for mt-DNA mutations in the excessive apoptosis and resulting cytopenias of MDS patients.

Transmission electron microscopic (TEM) studies have not been reported in bone marrow (BM) biopsies of patients with myelodysplastic syndromes (MDS) owing to failure to overcome the technical impediment of maintaining ultrastructural detail in decalcified tissue. Using a modified technique to physically separate pieces of bone from marrow tissue under a dissecting microscope, and embedding the material directly for TEM, ultrastructural studies were performed in 15 MDS patients and four normal BM biopsies. Biopsy tissue was also used to initiate long-term in vitro cultures and 12-week plates were sacrificed for TEM analysis. Features noted in freshly obtained decorticated tissue included an excessive apoptosis in both haematopoietic and stromal cells, ringed sideroblasts with iron-laden mitochondria and highly active, enormously increased phagocytosis. In addition, type IV nuclear inclusion body variants (NIB-v) and confronting cylindrical cisternae (CCC) were readily identified in up to 40% of stromal cells in vivo, providing an important footprint of a possible infectious agent in the pathology of MDS. Cultured stromal cells did not show excessive apoptosis and only 2-4% fibroblasts showed the presence of NIB-v or CCC, underscoring the artificial nature of ex vivo systems. We conclude that ultrastructure studies using decorticated tissue can be a powerful tool to investigate the biology and aetiology of a variety of haematopoietic disorders as it enables the direct examination of BM biopsies with their intimate stromal parenchymal cell associations preserved intact.

Thirty patients with myelodysplastic syndromes (MDS) were treated with thalidomide at 100 mg/d p.o., increased as tolerated to 400 mg/d for 12 weeks. Levels of apoptosis, macrophage number, microvessel density (MVD), tumour necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGF-beta), interleukin 6 (IL-6), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were determined in the serum, bone marrow (BM) plasma and BM biopsies before and after therapy. Pretherapy biological characteristics of MDS patients were compared with similar studies performed in 11 normal volunteers. Ten patients demonstrated haematological improvement in the erythroid series, six becoming transfusion independent. Responders had a higher pretherapy platelet count (P < 0.048) and lower BM blasts (P < 0.013). Median time to response was 10 weeks, and four remain in remission beyond a year. Pretherapy MDS BMs showed higher MVD (P < 0.001) and TGF-beta (P < 0.03) and higher serum TNF-alpha (P < 0.008) compared with normal control subjects. After therapy, only BM TGF-beta decreased significantly (P < 0.002). Pretherapy haemoglobin was directly related to serum VEGF (P < 0.001) in responders and inversely related in non-responders (P < 0.05), suggesting the possibility that angiogenesis may be a primary pathology in the former and a consequence of anaemia-induced hypoxia in the latter. We conclude that thalidomide has important clinical and biological effects in at least a subset of MDS patients, but the precise mechanism of its action remains unknown and requires further study including a larger number of patients.

We used bone marrow biopsies instead of mononuclear cells to maintain long-term cultures from 103 patients belonging to all five sub-categories of myelodysplastic syndromes (MDS), as well as 12 normal controls. By week 4, 30-50% confluency was reached and could be maintained for up to 12 weeks with 100% confluency. The four prominent cells were fibroblasts, macrophages, endothelial cells and adipocytes. Immunohistochemical and electron microscopic studies provided lineage confirmation. Normal hematopoiesis was well supported by MDS stroma. Neither the FAB nor cytogenetics was co-related with the potency of growth. MDS stroma appears to be both morphologically and functionally normal.

The bone marrow (BM) in myelodysplastic syndromes (MDS) undergoes pathobiological changes that mimic an inflammatory process, and hence, an infectious etiology was suspected in these disorders. In the present report, we examined the bone marrow mononuclear cells (BMMNC) of 19 MDS patients and seven normal donors for the expression of one latency-related (Latency membrane protein 1 (LMP-1) and immediate early protein (IEP)) and one activation-related (BZLF and DNA-Pol) m-RNA each for two herpes viruses, Epstein-Barr virus (EBV) and cytomegalovirus (CMV), respectively. Reverse transcriptase polymerase chain reaction was used for this purpose. The latency-related transcripts (EBV-LMP-1 and CMV-IEP) were present in all the MDS and normal specimens. Intriguingly, 10/19 MDS specimens ( approximately 53%) and 2/7 normal donors ( approximately 28%) were positive for active EBV-BZLF (P=0.0067), while 2/19 MDS specimens ( approximately 11%) with 1/7 normal ( approximately 14%) showed active CMV-DNA-Pol (P=0.1588). Later, from another set of MDS patients (n=7) and normal donors (n=4), BM stromal cultures were established, which, at a 75% confluency, were overlaid with cord blood mononuclear cells (CBMNC). IEP was detectable in the CBMNC before and after co-incubation with MDS, as well as normal stroma. So, it was also present both in MDS and normal stromal cells. The other three were absent both in MDS and normal stromal layers. In CBMNC though, active EBV-BZLF and CMV-DNA-Pol m-RNA were detectable in one of seven MDS co-cultures each, albeit from different patients. None of the normal co-cultures showed active virus, either in stroma or CBMNC. Thus, the present report demonstrates, for the first time, the presence of active herpes viruses in the BMMNC of MDS patients and reveals the ability of the MDS stroma to support the viral activation.

A higher percentage of apoptotic cells (apoptotic index or AI) is consistently found in bone marrow (BM) biopsies compared to BM aspirates of patients with myelodysplastic syndrome (MDS). Most studies have only investigated the low-density fraction (LDF) mononuclear cells from BM aspirates following density separation for AI determination. In the present study, both LDF and high-density fraction (HDF) cells for AI were examined by electron microscopy (EM) in 10 MDS patients and 4 healthy donors. Matched BM biopsies were subjected to AI detection by in situ end labeling (ISEL) of fragmented DNA. The results indicate that in LDF and HDF cells, AI is consistently higher in MDS patients (8.5% vs 1.5%, respectively; P = .039) compared to healthy donors (27% vs 4%, respectively; P = .004). The BM biopsy AI was also higher in MDS patients than in healthy donors (3+ vs 0+, respectively; P = .036). In addition, in MDS patients, more apoptotic cells were found in HDF cells than in LDF cells (27% vs 8.5%, respectively;P = .0001). All stages of maturation, ranging from blasts to terminally mature cells belonging to all 3 lineages, were represented in the dying cells in both compartments. Using EM, typical Pelger-Huett–type cells appeared to be apoptotic granulocytes. Both LDF and HDF cells should be examined for an accurate estimation of apoptotic cells because AI would be underestimated if only the LDF cells were studied. Ultrastructural studies consistently show a higher AI in BM biopsies compared to BM aspirates despite the correction factor of HDF cells provided by AI. This may represent the actual extant state, which could conceivably be due to a higher concentration of proapoptotic signals in the biopsies.

Myelodysplastic syndromes (MDS) have previously been reported to show competitively high rates of apoptosis and proliferation in the bone marrow (BM). Using a double-labelling technique in the present study, we demonstrated that a significantly high number of S-phase cells were simultaneously apoptotic (signal antonymy; SA) in MDS (mean ± s.e.m. 53·5 ± 6·7%, n = 24, P

Myelodysplastic syndromes (MDS) are a group of hematopoietic disorders characterized by the concomitant presence of peripheral cytopenias and normocellular to hypercellular BM. This paradox has been proposed to be due to the presence of excessive proliferation matched by excessive intramedullary apoptosis of hematopoietic cells. When cultured in vitro MDS BM mononuclear cells (BMMC) undergo apoptosis within 4 h. We measured caspase-1-like and caspase-3-like activity in 22 MDS and 4 normal BM immediately following cell separation or after 4 h culture. When cultured in vitro, MDS BMMC demonstrated an increased apoptotic index within 4 h as measured by in situ end-labeling of fragmented DNA that was matched by a concurrent increase in caspase-3-like specific activity, and the two were significantly correlated. During the 4 h culture, a sequential activation of caspase-1-like and caspase-3-like activities was detected. Caspase-1-like specific activity was detected early and transiently at approximately 15 min, followed by a gradual increase in caspase-3-like-specific activity peaking at 2 h. When the broad-spectrum caspase inhibitor, Z-VAD.FMK, was included in the MDS BM aspirate 4 h culture, apoptosis was attenuated. We conclude that sequential activation of caspase-1-like and caspase-3-like activities may form the central biochemical pathway of apoptosis in BMMC from some MDS patients, and prevention of this process by caspase inhibitors may be of significant therapeutic value for these patients, in whom supportive care continues to be the mainstay of therapy.

Increased intramedullary apoptotic death of hematopoietic cells is thought to contribute to the ineffective hematopoiesis in myelodysplastic syndromes (MDS). Furthermore, high amounts of tumor necrosis factor alpha (TNF alpha) have previously been correlated with apoptosis in MDS marrows. The present studies were undertaken to examine the status of two key downstream effectors of TNF alpha signaling, i.e. Caspase 1 and Caspase 3 enzymes, using a fluorometric assay in the bone marrow aspirate mononuclear cells (BMMNC) in relation to apoptotic DNA fragmentation detected by in situ end-labeling (ISEL) of DNA and with localization of TNF alpha in the corresponding biopsies from 14 MDS patients. Both Caspase 1 and 3 were detectable in freshly harvested BMMNC, albeit median Caspase 3 levels (47.5 units/mg protein) being almost 10 times higher than Caspase 1 (4.0 units/mg protein). Upon short-term culture for 4 h in a serum-supplemented medium in vitro a significant increase was seen in Caspase 3 activity (58.8 +/- 13.9 at 0 h vs. 177.8 +/- 55.2 units/mg protein at 4 h, n = 14, P = 0.017) and in percent cells labeled by ISEL (apoptotic index or AI%: 0.76% +/- 0.25% vs. 3.99% +/- 1.1%, n = 14, P = 0.004, respectively). Caspase 1 activity increased after 15 min in culture. Interestingly, TNF alpha levels measured by immunohistochemistry correlated with the net increase in Caspase 3 activity after 4 h (p = 0.517, n = 13, P = 0.07) and the starting levels of Caspase 1 at 0 h correlated with the Caspase 3 levels attained at 4 h (p = 0.593, n = 13, P = 0.033). Additionally when TNF alpha-positive bone marrows (8/14) were compared with the negative marrows (6/14) the Caspase 3 levels were significantly higher in the TNF alpha-positive marrows (189.6 +/- 66.2 vs. 25.0 +/- 14.6 units/mg protein, respectively, P = 0.043). The increase in AI%, though not statistically significant, was also higher in the TNF alpha-positive marrows. Finally in HL60 cells the effects of different Caspase inhibitors and pentoxifylline (PTX) (interferes with lipid signaling of cytokines) on TNF alpha-induced apoptosis were evaluated. TNF alpha treatment significantly increased AI% (P < 0.003) as compared to the untreated controls. A co-treatment with three Caspase inhibitors, zVAD.FMK (inhibitor of Caspases 1 and 3, 10 microM/l), Ac.YVAD.FMK (Caspase 1 inhibitor, 1 microM/l), Ac.DEVD.FMK (Caspase 3 inhibitor, 10 microM/l) as well as PTX (250 microM/l) significantly curtailed the AI% induced by TNF alpha. The present studies thus identify the downstream effectors of TNF alpha-inducible apoptosis in MDS and so also the suppressors of TNF alpha apoptotic signaling. These results may have significant clinical implications in the therapy of MDS in the future.

Rates of proliferation and apoptosis as well as expression of tumor necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGF-beta) and the number of macrophages were measured in bone marrow (BM) biopsies of 33 patients who presented with hypocellular (cellularity30%) myelodysplastic syndromes (MDS). Results showed that 2/3 of the patients had high apoptosis, high cytokine levels and large number of macrophages in their biopsies while 1/3 did not. Apoptosis and TNF-alpha levels were directly related (r = 0.583, P = 0.003, n = 24) as was apoptosis and the degree of anemia (P = 0.033, n = 18). A subgroup of patients with abnormalities of chromosomes 5 or 7 had higher platelets (P = 0.026) and higher apoptosis (P = 0.038) when compared with the rest of the group. Eight patients had no evidence of apoptosis and almost no detectable TNF-alpha in their biopsies. We conclude that within the hypocellular variant of MDS, there may be two distinct sub-groups of patients, one who present with high cytokine-mediated intramedullary apoptosis and the other who may be better characterized as having a stem-cell failure defect since they showed no evidence of apoptosis.

We previously reported excessive apoptosis and high levels of tumor necrosis factor-alpha (TNF-alpha) in the bone marrows of patients with myelodysplastic syndromes (MDS), using histochemical techniques. The present studies provide further circumstantial evidence for the involvement of TNF-alpha in apoptotic death of the marrow cells in MDS. Using our newly developed in situ double-labeling technique that sequentially employs DNA polymerase (DNA Pol) followed by terminal deoxynucleotidyl transferase (TdT) to label cells undergoing apoptosis, we have characterized DNA fragmentation patterns during spontaneous apoptosis in MDS bone marrow and in HL60 cells treated with TNF-alpha or etoposide (VP16). Clear DNA laddering detected by gel electrophoresis in MDS samples confirmed the unique length of apoptotic DNA fragments (180-200 bp). Surprisingly, however, phenotypically heterogeneous population of MDS cells as well as the homogenous population of HL60 cells showed three distinct labeling patterns after double labeling--only DNA-Pol reaction, only TdT reaction, and a combined DNA Pol + TdT reaction, albeit in different cohorts of cells. Each labeling pattern was found at all morphological stages of apoptosis. MDS mononuclear cells, during spontaneous apoptosis in 4 hr cultures, showed highest increase in double-labeled cells (DNA Pol + TdT reaction). Interestingly, this was paralleled by TNF-alpha-induced apoptosis in HL60 cells. In contrast, VP16 treatment of HL60 cells led to increased apoptosis in cells showing only TdT reaction. The double-labeling technique was applied to normal bone marrow and peripheral blood mononuclear cells after treatment with known endonucleases that specifically cause 3' recessed (BamHI), 5' recessed (PstI), or blunt ended (DraI) double-stranded DNA breaks. It was found that the DNA-Pol reaction in MDS and HL60 cells corresponds to 3' recessed DNA fragments, the TdT reaction to 5' recessed and/or blunt ended fragments, and a combined "DNA Pol + TdT reaction" corresponds to a copresence of 3' recessed with 5' recessed and/or blunt ended fragments. Clearly, therefore, apoptotic DNA fragments, in spite of a unique length, may have differently staggered ends that could be cell (or tissue) specific and be selectively triggered by different inducers of apoptosis. The presence of TNF-alpha-inducible apoptotic DNA fragmentation pattern in MDS supports its involvement in these disorders and suggests that anti-TNF-alpha (or anticytokine) therapy may be of special benefit to MDS patients, where no definitive treatment is yet available.

Rates of proliferation, apoptosis and cytokine expression were measured in bone marrow (BM) biopsies of 164 myelodysplastic syndrome (MDS) patients. There were 107 males and 57 females. Median age was 69 years and 101 had refractory anemia (RA), 17 RA with ringed sideroblasts (RARS), 38 with RA and excess blasts (RAEB) and 8 with RAEB in transformation (RAEB-t). Apoptosis measured by in-situ end labeling (ISEL) was directly related to the number of macrophages (p = 0.028, n = 83). Mean tumor necrosis factor alpha (TNF-alpha) and ISEL positivity were higher in RAEB + RAEB-t patients (p = 0.0554 and p = 0.06 respectively) while hemoglobin was higher for RA + RARS group (p = 0.0472). Patients with high apoptosis had lower white blood cell counts (p = 0.0009), lower percentage of blasts (p = 0.0009) and higher number of macrophages (p = 0.0086). We conclude that measurements of apoptosis, proliferation and cytokine expression provide important biological information which helps to distinguish RA + RARS patients from RAEB + RAEB-t patients, and may be of additive prognostic significance.

Tumor necrosis factor-alpha (TNF-alpha) levels were measured in the serum (sTNF-alpha) or bone marrow (BM) biopsies of 43 patients with myelodysplastic syndromes (MDS) who subsequently received therapy with a combination of pentoxifylline and ciprofloxacin (PC) with or without dexamethasone (PCD). All 43 patients received only PC therapy for 12 weeks, after which 18 of 36 nonresponders received PCD. A total of 18 of 43 patients showed a hematologic or cytogenetic response or both. BM TNF-alpha levels were semiquantitatively assessed using immunohistochemistry on a scale of 0-8+ and in the serum using enzyme linked immunoassay. The median TNF-alpha for the entire group was 3.0 in BM and 6.9 pg/ml in the serum, and 14 patients had no detectable levels. Responders had higher BM levels (median 3.5 vs. 2.0) than nonresponders, although this was not statistically significant. During PC therapy, a decline in BM TNF-alpha level was seen in the entire group, which was significant at 2 weeks (p = 0.02), 8 weeks (p = 0.001), and 12 weeks (p = 0.0001). Both responders (p = 0.01) and nonresponders (p = 0.03) had a decline at 8 weeks, but at 12 weeks, only the responders continued to show a significant decline (p = 0.03). We conclude that MDS patients with high BM TNF-alpa levels have a better chance of responding to PCD therapy and that the therapy is quite successful in reducing the TNF-alpha levels in a sustained fashion. Future studies need to be directed at identifying agents that would be more potent suppressors of the proapoptotic cytokines in these patients.

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-alpha), transforming growth factor-beta (TGF-beta) 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-alpha and TGF-beta 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-alpha and the incidence of PCD (p= 0.0015). Patients who showed high PCD also had an elevated TNF-alpha level. Thus, the expression of high amounts of TNF-alpha and TGF-beta 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-alpha drugs such as pentoxifylline.

Our previous studies using in situ end labeling (ISEL) of fragmented DNA revealed extensive apoptotic cell death in the bone marrows (BM) of patients with myelodysplastic syndromes (MDS) involving both stromal and hematopoietic cells. In the present report we show greater synthesis of interleukin-1 beta (IL-1 beta) in 4 hour cultures of density separated BM aspirate mononuclear (BMAM) cells from MDS patients as compared to the cultures of normal BM from healthy donors or lymphoma patients (1.7 +/- 0.37 pg/10(5) cells, n = 29 v 0.42 +/- 0.24 pg/10(5) cells, n = 11, respectively, P = .049). Further, these amounts of IL-1 beta in MDS showed a significant correlation with the extent of apoptosis detected by ISEL in corresponding plastic embedded BM biopsies (r = .480, n = 30, P = .007). In contrast normal BMs did not show any correlation between the two (r = .091, n = 12, P = .779). No significant correlation was found between the amounts of IL-1 beta and % S-phase cells (labeling index; LI%) in MDS determined in BM biopsies using immunohistochemistry following in vivo infusions of iodo- and/or bromodeoxyuridine. Neither anti-IL-1 beta antibody nor IL-1 receptor antagonist blocked the apoptotic death of BMAM cells in 4 hour cultures (n = 5) determined by ISEL (apoptotic index; AI%), although the latter led to a dose-dependent accumulation of active IL-1 beta in the culture supernatants. On the other hand, a specific tetrapetide-aldehyde inhibitor of ICE significantly retarded the apoptotic death of BMAM cells at 1 mumol/L in 5/6 MDS cases studied (AI% = 2.99 +/- 0.30 in controls v 1.58 +/- 0.40 with ICE-inhibitor, P = .05) and also reduced the levels of active IL-1 beta synthesized (5.59 +/- 2.63 v 2.24 +/- 0.93 pg/10(6) cells, respectively). In normal cells, neither IL-1 blockers nor the ICE inhibitor showed any effect on the marginal increase in apoptosis observed in 4 hour cultures. Our data thus suggest a possible involvement of an ICE-like protease in the intramedullary apoptotic cell death in the BMs of MDS patients.

Cell-cycle kinetics were measured in situ after infusions of iododeoxyuridine and/or bormodeoxyuridine in 50 patients with myelodysplastic syndromes (MDS) and the median labeling index in bone marrow (BM) biopsy samples was 28.6%. Unfortunately, 26 of 50 patients showed that > or = 75% of hematopoietic cells of all three lineages were undergoing programmed cell death (PCD) in their biopsy samples as shown by the in situ end labeling (ISEL) technique. Ten patients had 1/3 and eight had 2/3 ISEL+ cells. Stromal cells were frequently ISEL+ and often S-phase cells were also found to be simultaneously ISEL+. Nucleosomal DNA fragments as a ladder in agarose gel were present in BM aspirates of four patients who showed high ISEL and were absent in two who had no ISEL staining in biopsy samples, but only when DNA was extracted after a 4-hour in vitro incubation in complete medium. Therefore, laddering data confirmed the ISEL findings that the majority of hematopoietic cells in MDS are in early stages of PCD. We conclude that extensive intramedullary cell death may explain the paradox of pancytopenia despite hypercellular marrows in MDS patients. Investigating approaches that protect against PCD in some MDS subsets would be of interest.

We describe a novel double-labeling method to simultaneously investigate proliferation and apoptosis from plastic-embedded biopsy specimens (PEBs). Infusions of bromo- and/or iododeoxyuridine (BrdU/IudR) were given to 10 patients, five with acute myeloid leukemia (AML) and five with myelodysplastic syndromes (MDS), and S-phase cells were measured in PEBs using a monoclonal anti-IudR/BrdU antibody. Apoptosis was measured by in situ end-labeling (ISEL) of DNA. The results demonstrate that both AML and MDS are highly proliferative disorders but that there is almost no apoptosis in the former, whereas extensive apoptosis was observed in the latter. Double labeling revealed that large numbers of S-phase cells in MDS were simultaneously undergoing apoptosis. We conclude that the high cell death in MDS cancels the high cell birth, resulting in a functionally aplastic marrow and thus accounting for the observed ineffective hematopoiesis. On the other hand, AML is rapidly fatal, probably owing to high cell birth with no or minimal cell death. Therapeutic strategies to prevent intramedullary programmed cell death of hematopoietic precursors should be evaluated in MDS, and efficacy of chemotherapy in AML can be assessed by measuring the induction of apoptosis in post-treatment biopsy specimens.

Huet first observed an abnormal hyposegmented neutrophil granulocyte in 1928, and a year later Pelger reported an additional case. Such a cell later became known as Pelger-Huet anomaly. In 1932 Huet pointed out its hereditary and systemic character.[1][1] The Pelger-Huet anomaly is a familial

Myelodysplastic syndromes (MDS) are considered to be stem cell disorders. A number of cytogenetic abnormalities have been demonstrated in these patients. In the last decade it has become apparent that besides the cytogenetic and molecular abnormalities at the individual cell level, the global alterations of the bone marrow (BM) microenvironment brought about by shifts in the cytokine milieu may play a crucial role in defining the pathobiology of these disorders. The combination of factors produced by adherent stromal cells and hematopoietic cells constitute the web of cytokines meshing a variety of biological processes including survival, proliferation and differentiation of the hematopoietic cells. Imperatively thus, hematopoiesis in these patients is a result of a critical balance between the stimulatory and inhibitory signals transduced by these cytokines. Primarily effects of these cytokines could be chemotatic, stimulatory or inhibitory to hematopoiesis.

MDS is a heterogeneous clonal stem cell disorder with trilineage dysplasia resulting in ineffective, monoclonal hematopoiesis ([Resegotti et al., 1993]). Extensive studies over the decade have led to better understanding of cell proliferation, differentiation, cytokine production and apoptosis in MDS ([Greenburg et al., 1987]; [Shetty et al., 1996]; [Clark et al., 1990]; [Yoshida et al., 1993]; [Raza et al., 1995]). However there has been limited information in the literature regarding long term bone marrow cultures and ultrastructural studies performed on MDS. In the past, it has been difficult to grow MDS bone marrow (BM) cells as the progenitor cells have a great propensity to undergo programmed cell death ([Ohmori et al., 1991]; [Aizawa et al., 1999]; [Raza et al., 1996]). On the other hand the role of electron microscopy has not been clearly established in MDS either, other than few individual cases on BM aspirate because of the failure to overcome the technical impediment of maintaining the ultrastructural details in decalcified tissues ([Guccion et al., 1992]; [Gerrad et al., 1992]; [Cohen et al., 1997]; [Payne et al., 1987]). As a result very little is known about the association between the hematopoietic cells and their microenvironment especially in a disease like MDS where both the parenchymal and stromal cells may be involved in the disease process ([Coutinho et al., 1990]; [Sawada et al., 1998])). The present chapter focuses on novel techniques where primarily the MDS core bone marrow biopsy was used to maintain long term bone marrow cultures followed by ultrastructural studies, hence preserving the BM architecture and the geographical distribution of the hematopoietic and stromal cells as they existin vivo.A number of novel and interesting observations were made which are summarized here.

Serum lipid profiles were obtained in 108 patients with myelodysplastic syndrome (MDS) and compared to 28 healthy volunteers. Serum cholesterol and low-density and high-density lipoproteins (LDL and HDL) were found to be significantly lower in MDS patients than in normals (p = 0.0001, 0.0038 and 0.037, respectively). This difference was significant for all MDS categories. Serum cholesterol and HDL were negatively related to biopsy cellularity (p = 0.001 and 0.0001, respectively), and serum triglycerides were negatively related to labeling index (p = 0.0003). No differences were noted in the lipid profiles of MDS patients with normal versus abnormal karyotypes. However, low-risk MDS patients with abnormal karyotypes had significantly lower triglyceride levels compared with the high-risk patients (p = 0.027), as did low-risk patients with normal cytogenetics (p = 0.015). Serum HDL levels were significantly higher for the low-risk group with normal cytogenetics as well (p = 0.003). We conclude that serum cholesterol, LDL, and HDL are significantly reduced in MDS patients, probably indicating excessive intracellular lipid biosynthesis in the expanding clone. These relatively simple measurements could serve as important prognostic markers and reliable indicators of disease activity in individual patients. Prospective studies to determine their utility as independent variables that guide the need for active therapeutic intervention are warranted.

Spontaneous intramedullary apoptosis was measured in bone marrow (BM) biopsies of 175 patients with myelodysplastic syndromes (MDS) using in situ end-labeling (ISEL) of fragmented DNA. Two groups of high (n = 71) versus low (n = 43) levels of apoptosis were identified while 61 patients were ISEL-negative. Semiquantitative assessment of 3 cytokines, the number of macrophages and in vivo labeling indices (LI) were also determined from consecutive sections of the biopsy. Patients with high apoptosis levels tended to have a high LI (p = 0.013), more macrophages in their BM biopsies (p = 0.006) and higher tumor necrosis factor alpha (TNF-α) levels (not significant) compared to patients with no apoptosis. In addition, low risk MDS patients had significantly lower rates of apoptosis (p = 0.047) and lower levels of TNF-α (p = 0.055) compared to high-risk MDS patients. We conclude that the genesis of cytopenias in MDS is of multifactorial origin and that cytokine-associated apoptosis clearly identifies a distinct biological subgroup of patients who may benefit selectively by use of anti-cytokine therapies.

Forty-three patients with myelodysplastic syndromes (MDS) were treated with a combination of pentoxifylline and ciprofloxacin (PC) with the addition of dexamethasone (PCD) in 18 patients who failed to respond to PC. There were 15 females and 28 males, and the median age was 67 years. A total of 18 patients either showed a hematopoietic improvement, a partial or complete cytogenetic response or a combination of both for an overall response rate of 42%. Seven PC only patients responded, four showing hematologic improvement, two cytogenetic responses and one patient showing a combined response. This 16% response rate to PC was increased to 61% by the addition of dexamethasone with 11/18 patients showing a response. Four of the 7 patients who responded initially to PC were given dexamethasone after at least 12 weeks of PC therapy, and only 1 showed a further improvement in response. Thus, we conclude that the combination of PCD provides an encouraging novel approach to treating MDS. The mechanism of action is probably related to the suppression of a veriety of cytokines which in turn attenuate the excessive intramedullary apoptotic death of hematopoetic cells in MDS, an observation which has been speculated to be the basis of the paradox of variable cytopenias despite cellular marrows in MDS. Larger numbers of patients need to be treated and followed for longer periods to determine the true efficacy of this therapy, especially the nature and duration of the cytogenetic responses.

Myelodysplastic syndromes (MDS) are highly proliferative bone marrow (BM) disorders where the primary lesion presumably affects a CD34+ early progenitor or stem cell. We investigated the proliferative characteristics of CD34+ cells of 33 untreated MDS patients (19 RA, 5 RARS, 7 RAEB, 2 RAEBt) and five patients with acute myeloid leukemia after MDS (sAML). All patients received a 1-h infusion of the thymidine analogue iodoor bromodeoxyuridine intravenously before a BM aspirate and biopsy was taken. A double-labeling immunohistochemistry technique by monoclonal anti-CD34 (QBend/10) and anti-IUdR/BrdU antibodies was developed and performed. By this technique we recognised CD34+ and CD34- cells actively engaged in DNA synthesis or not. As MDS evolves a significant increase occurred in the percentage of CD34+ cells of all myeloid cells (mean value: RA/RARS 1.67%; RAEB(t) 8.68%; sAML 23.83%) as well as in the percentage of proliferating CD34+ cells of all myeloid cells (RA/RARS 0.19%; RAEB(t) 0.43%; and sAML 3.30%). This was associated with a decreasing trend in the overall myeloid labeling index (LI: RA/RARS 25.8%, RAEB(t) 24.6% and sAML 21.5%). This decrease in overall myeloid LI is due to an exponential increase in the proportion of CD34+ cells of the proliferating compartment during MDS evolution (RA/RARS 0.35%, RAEB(t) 1.44% and sAML 11.98% of all S-phase cells). These CD34+ cells appeared to proliferate more slowly than their more mature CD34 negative counterparts, since we found a progressive increment in the mean total cell cycling time (Tc) of all myeloid cells during MDS progression (RA/RARS 39.8, RAEB(t) 45.2 and sAML 65.8 h). This study showed that during MDS evolution to sAML the CD34+ compartment develops a growth advantage leading to apparent expansion.

The study was carried out on 22 patients with non-Hodgkin's lymphoma (NHL) who had received sequential infusions of two thymidine analogues iododeoxyuridine (IUdR) and bromodeoxyuridine (BrdU). Cell cycle kinetic studies seemed to differentiate distinctly between low grade lymphoma (n = 8, LI = 2.6%) compared to that of intermediate grade (n = 9, LI = 13%, p = 0.0001) and high grade NHL (n = 5, LI = 16.3%, p = 0.0062). While the majority of 14 intermediate and high grade lymphomas had a high labeling index there were 3/14 patients with a LI of 5.5%, 5.5% and 4.1% respectively. A decrease in the rate of programmed cell death (PCD) or apoptosis due to the overexpression of bcl-2 has been implicated as the possible pathogenesis for follicular lymphoma. We determined the presence of bcl-2 protein immunohistochemically and apoptosis by in situ end labeling of DNA which detects cells in early stages of PCD not recognized morphologically. Nine NHL patients demonstrated PCD ranging from 1%-40%, while it was undetectable in 13/22 patients. Of these 13 cases, 6 showed the presence of bcl-2 expression. To understand the relationship of the microenvironment to the lymphoma cells, the presence of transforming growth factor beta (TGF-beta) was determined immunohistochemically. TGF-beta was present in all the cases where bcl-2 was present, except one. This study highlights some of the key biological features of NHL cells and their microenvironment.

Despite hypercellular bone marrows (BM), peripheral cytopenias are the rule in patients with myelodysplastic syndromes (MDS). This study examined the roles played by cell birth and cell death rates in generating this paradox. Cell kinetics from BM biopsies of 35 MDS patients were measured using intravenous infusions of either iododeoxyuridine or bromodeoxyuridine, or both. Degree of apoptosis or programmed cell death (PCD) was estimated using in situ end-labeling of DNA directly from BM biopsies, which were simultaneously double-labeled for proliferation/PCD. MDS were found to be highly proliferative disorders with large numbers of myeloid, erythroid, and megakaryocytic cells synthesizing DNA. Median cycling time (Tc) of myeloblasts was more rapid than that of patients with acute myeloid leukemia (44.1 hr vs. 56.0 hr). Interestingly, most marrow cells of all three lineages in 32 of 34 evaluable cases were undergoing PCD. In 19 of 32 patients, greater than 75% cells were apoptotic. Surprisingly, large numbers of S-phase cells were found to be simultaneously undergoing PCD, as were stromal cells of the BM microenvironment. We conclude that the extensive apoptosis in hematopoietic cells effectively cancels the high birth rate resulting in ineffective hematopoiesis and accounting for deficient bone marrow function. ©1995 Wiley-Liss, Inc.

The paradox of myelodysplastic syndromes (MDS) which present with pancytopenias despite cellular bone marrows (BM) was investigated by conducting detailed studies of proliferation and apoptosis in 89 MDS patients. Our results demonstrated a rapid rate of both proliferation as well as apoptosis. Levels of three cytokines, tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta) and interleukin-1 beta (IL-1 beta) were measured in the same patients. High levels of TNF-alpha were found to correlate with high levels of apoptosis in 83 MDS patients (P = 0.0045). We propose a dual role for TNF-alpha (or other cytokines) in the pathogenesis of MDS. On the one hand, TNF-alpha induces apoptosis in the maturing cells causing pancytopenia while on the other, it stimulates the proliferation of the primitive progenitors accounting for the hypercellular BM frequently seen in MDS. A new model for MDS is presented. The initial abnormality probably affects a primitive hemopoietic progenitor which acquires a growth advantage leading to monoclonal hemopoiesis, which in turn makes these cells susceptible towards acquiring additional mutations and appearance of cytogenetically marked (or unmarked) clones. Cytokines such as TNF-alpha whose source is presently unknown, then contribute towards the clinical syndrome of pancytopenia and hypercellularity.