Presence of activation-related m-RNA for EBV and CMV in the bone marrow of patients with myelodysplastic syndromes.

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.

Signal antonymy unique to myelodysplastic marrows correlates with altered expression of E2F1.

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 < 0.001). In contrast, SA was negligible in all other specimens studied, including normal control BM (n = 13) from non-Hodgkin's lymphoma (NHL) patients, BM from patients with de novo acute myelogenous leukaemia (1'AML; n = 5), or secondary AML that had transformed from MDS (2'AML; n = 10), or the solid tumours from patients with NHL (n = 9) or head and neck squamous cell carcinoma (HNSCC; n = 10). Subsequently, the expression of a transcription factor, E2F1, was studied in density-separated BM aspirate mononuclear cells from MDS patients (n = 9) and a normal control. Two separate sets of primers were used that recognized the regulatory retinoblastoma (Rb) protein-binding region and the functional DNA-binding region of E2F1. Interestingly, although the latter manifested the expected band (280 bp) in all samples, the Rb-specific primers showed the expected band (380 bp) in the normal and in 4/9 MDS specimens. Two other MDS specimens also showed a smaller band ( approximately 325 bp), whereas 3/9 MDS patients showed exclusively the smaller band. The levels of SA were significantly higher in those MDS cases that showed the smaller Rb-specific band either alone or in addition to the expected band (median 19.5%, n = 4, P = 0.037) than in those showing exclusively the expected band (median 0.4%, n = 3). Our present studies show SA as a characteristic feature of MDS and, importantly, demonstrate its link with an altered expression of E2F1 in some MDS patients.

Spontaneous down-regulation of Fas-associated phosphatase-1 may contribute to excessive apoptosis in myelodysplastic marrows.

In this study, we examined the role of Fas-signaling in the apoptotic pathway in myelodysplastic syndromes (MDS). Ficoll-separated mononuclear cells from 18 bone marrow aspirate specimens obtained from 17 MDS patients, 4 normal healthy donors, and 3 acute myeloid leukemia patients transformed from MDS (t-AML) were studied for mRNA expression of Fas-L, Fas, and the effectors of their signaling, Caspase 1 and Caspase 3, using reverse transcriptase polymerase chain reaction. Fas-L, Fas, and Caspase 1 were detectable in all of the samples in the three groups. Caspase 3 was detectable both in MDS and t-AML specimens but was negligible in normal cells. The apoptotic index (AI%) determined by in situ end labeling of fragmented DNA in 4-hour cultures of mononuclear cells was significantly higher in MDS cells compared to normal or t-AML cells (mean +/- SEM: 2.3% +/- 0.4% in MDS, n = 10 vs. 0.6% +/- 0.2%, n = 4, P = 0.014 in normal cells, and 0.2% +/- 0.2%, n = 3, P = 0.007 in t-AML cells). Treatment of MDS cells with anti-Fas-L antibody suppressed apoptosis (AI%: 2.1% +/- 0.6% in untreated vs. 1.37% +/- 0.5% in treated, n = 6, P = 0.02), indicating functional participation of Fas-signaling in MDS. Further, it was found that Fas-L, Fas, and Caspase 1 mRNA expression remained unchanged in 4 hours. Caspase 3 expression appeared in normal cells after 4 hours and was present at both 0 and 4 hours in MDS and t-AML cells. In contrast to persistent expression in normal and t-AML cells, cells from the 5 MDS patients studied consistently showed significantly lowered or undetectable expression of a negative regulator of Fas, called Fas-associated phosphatase-1 (Fap-1) after 4 hours. Thus, the high AI% in MDS corresponds to a rapid decline in Fap-1. Furthermore, in tumor necrosis factor alpha (TNF-alpha) treated HL60 promyelocytic cells, a definite periodicity in the expression of different mRNAs was observed with upregulation of TNF-alpha itself at 30 minutes, increased expression of Fas and the appearance of Fas-L after 2 hours, and a decrease in Fap-1 expression after 8 hours. These results suggest that TNF-alpha not only induces the effectors of Fas-signaling but also may downregulate the inhibitor. We conclude that a spontaneous and rapid down-regulation of Fap-1, possibly induced by TNF-alpha, a cytokine shown to be present in excess in MDS marrows, may underlie the increased apoptotic death of hematopoietic cells in these patients. Interference with Fap-1 turnover may provide a new therapeutic modality for MDS.

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

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-alpha) 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-alpha (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.

Evidence for involvement of tumor necrosis factor-alpha in apoptotic death of bone marrow cells in myelodysplastic syndromes.

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.

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.

Cell cycle kinetic studies in 68 patients with myelodysplastic syndromes following intravenous iodo- and/or bromodeoxyuridine.

Sixty-eight patients with myelodysplastic syndromes (MDS) received sequential infusions of iodo- and/or bromodeoxyuridine for cell kinetic analysis. Bone marrow biopsy sections were treated by appropriate antibodies and a labeling index (LI), duration of S-phase (Ts), and total cell cycle time (Tc) of myeloid cells were determined. The mean LI was 28.4%, Ts was 11.8 hours and Tc was 40.7 hours. The %LI decreased as the disease evolved from refractory anemia toward transformation to acute leukemia (p = 0.04). Double-labeling of biopsy sections for apoptosis and proliferation showed that 30-90% of S-phase cells in MDS patients were simultaneously apoptotic or "antonymous." We conclude that MDS are highly proliferative disorders in which the ineffective hematopoiesis is probably the result of excessive apoptosis rather than slow proliferation.

Indication of an involvement of interleukin-1 beta converting enzyme-like protease in intramedullary apoptotic cell death in the bone marrow of patients with myelodysplastic syndromes.

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.

A double-blind controlled trial of two dose levels of factor VIII in the treatment of high risk haemarthroses in haemophilia A.

One hundred and thirteen haemarthroses involving the knees, ankles and elbows of 29 severe haemophiliacs presenting with at least two of the risk factors, pain, tenderness, loss of more than 50% of movement and a delay of more than 3 h in treatment were studied. Each was given either a 20% or 40% dose of factor VIII and progress was then reviewed by medical staff unaware of the initial dosage. There was no significant effect on the retransfusion rate nor on the time to complete resolution. However, the difference between the percentage of patients showing residual movement restriction was significantly in favour of the high dose at 24, 36 and 48 h when all the bleeds were pooled and at 48 h for elbow bleeds.