Saleem Dar

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-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.

The clinical and biological effects of thalidomide in patients with myelodysplastic syndromes

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‐α), transforming growth factor beta (TGF‐β), 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‐β (P < 0·03) and higher serum TNF‐α (P < 0·008) compared with normal control subjects. After therapy, only BM TGF‐β 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.

Increased incidence of mitochondrial cytochrome c‐oxidase gene mutations in patients with myelodysplastic syndromes

Summary.  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.

Sequential Activation of Caspase-1 and Caspase-3-like Proteases During Apoptosis in Myelodysplastic Syndromes

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.

Biological characteristics of myelodysplastic syndrome patients who demonstrated high versus no intramedullary apoptosis.

Abstract:  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.

Biologic characteristics of patients with hypocellular myelodysplastic syndromes

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 (cellularity < 30%) 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.

Biologic Characteristics of 164 Patients with Myelodysplastic Syndromes

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.

Pilot Study of Pentoxifylline and Ciprofloxacin with or without Dexamenthasone Produces Encouraging Results in Myelodysplastic Syndromes

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.

Pentoxifylline, Ciprofloxacin and Dexamethasone Improve the Ineffective Hematopoiesis in Myelodysplastic Syndrome Patients

Twenty-five patients with a diagnosis of myelodysplastic syndromes (MDS) were randomized to either begin therapy with pentoxifylline, ciprofloxacin and dexamethasone (PCD) immediately (10 patients) or after a 12 week observation period (control arm, 15 patients). PCD was administered with the goal of suppressing cytokine-induced excessive intramedullary apoptosis of hematopoietic cells. No marked fluctuations of blood counts were noted during the period of observation. Twenty-two patients completed at least 12 weeks of therapy: 18/22 showed some type of hematologic response, 9/18 showing an improvement in absolute neutrophil count only (p = < 0.001) and 9/18 showing multi-lineage responses. No unique category of MDS responded better, however 19/25 patients had refractory anemia (RA)/RA with ringed sideroblasts. The median time to response was 6 weeks and 3/18 responding patients maintained their responses beyond a year. We conclude that hematologic improvement in response to PCD therapy supports the validity of this unique anti-cytokine approach. Future trials should combine PCD therapy with established approaches (growth factors/chemotherapy) and also should focus on identifying more effective ways of suppressing the pro-apoptotic cytokines in MDS.

P1-013: Impact of amyloid deposition upon cortical and striatal cholinergic interneuronal survival in APPswe/PS1ΔE9 transgenic mice

Background: The presence of the amyloid in the hippocampus and cortex which receive their primary cholinergic innervation from acetylcholine containing neurons in the basal forebrain may underlie the selective vulnerability of these long projection cells in Alzheimer’s disease (AD). By contrast, the effect of amyloid (A ) deposition upon the survival of local cholinergic interneurons remains unclear. Objective: To evaluate the impact of A deposition on cholinergic local interneurons we performed qualitative and quantitative analyses of cholinergic cortical and striatal interneurons in young (3-6 months old) and old (10-16 months old) amyloid over expressing APPswe/PS1 E9 transgenic and age-matched wild type mice. Methods: Tissue was immunostained for choline acetyltransferase (ChAT) and histochemcically reacted for acetylcholinesterase (AChE) using the Karnovsky and Roots method. Results: APPswe/ PS1 E9 transgenic mice displayed an age-dependent increase in A deposition in the cortex and striatum as well as ChAT and AChE positive dystrophic neurites in close apposition to A plaques in these structures. Interestingly, the overall distribution and the density of ChAT and AChE positive fibers in the cortex appeared similar in these mutant mice at each age examined. The cortical bipolar intrinsic ChAT positive and AChE negative neurons cortex did not show morphological alterations despite being in close proximity to A plaques. In the striatum there was no detectable cell shrinkage or alteration in ChAT optical density measurements in these cholinergic interneurons in the APPswe/PS1 E9 transgenic compared with wild-type mice. Furthermore, stereologic counts did not reveal changes in number of intrinsic striatal or cortical ChAT-immunoreactive neurons between young and old APPswe/PS1 E9 transgenic compared to wild-type mice. Conclusions: These results suggest that amyloid deposition does not impact the survival of cortical and striatal cholinergic interneurons in APPswe/PS1 E9 transgenic mice.