A paradigm shift in myelodysplastic syndromes.

A poorly defined transforming event(s) affects the pluripotential bone marrow (BM) stem cell in myelodysplastic syndromes (MDS), conferring a growth advantage upon it which leads eventually to monoclonal hematopoiesis. The progeny of this transformed ancestor undergo recognizable albeit dysplastic maturation. We propose that this picture is further complicated by a variety of cytokines, tumor necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGF-beta) and interleukin 1beta (IL-1beta) which exert a dual effect on the diseased cells. The immature CD34+ cells are stimulated to proliferate, while their later differentiated daughters are induced to undergo apoptosis accounting for the clinical syndrome of pancytopenia despite hypercellular BMs. Studies directed at measuring the rates of proliferation and apoptosis as well as the levels of TNF-alpha, TGF-beta and IL-1beta confirm this hypothesis and are presented in greater detail. A novel approach towards MDS therapy emerges as a result of this paradigm shift based upon the premise that anti-cytokine therapy would prevent excessive intramedullary apoptosis and result in improved cytopenias as well as cause a slowing down of the diseased precursor cell proliferation resulting in resumption of polyclonal hematopoiesis. Because a number of cytokines function through common lipid second messengers, interruption of this pathway should theoretically cause disruption in the signalling of a cascade of cytokines.

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.

Successful establishment of long-term bone marrow cultures in 103 patients with myelodysplastic syndromes.

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.

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.

Novel insights into the biology of myelodysplastic syndromes: excessive apoptosis and the role of cytokines.

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.

Excessive proliferation matched by excessive apoptosis in myelodysplastic syndromes: the cause-effect relationship.

The paradox of pancytopenia despite cellular bone marrows (BM) was investigated in 120 patients with myelodysplastic syndromes (MDS). Detailed cell cycle kinetics were examined following in vivo infusions of iodo--and/or bromodeoxyuridine (IUdR/BrdU), while the incidence of apoptosis was measured by in situ end labeling (ISEL) of fragmented DNA. Results showed that MDS are highly proliferative disorders with an equally high incidence of apoptotic intramedullary cell death accounting for the paradox of cellularity/cytopenia. By double-labeling BM biopsy sections for ISEL/BrdU we found the peculiar situation of "signal antonymy" where S-phase cells were frequently apoptotic, a phenomenon so far only seen in MDS biopsies. The cause-effect relationship of this excessive proliferation/apoptosis is discussed at length.

The mycelial status and reversibility in Histoplasma capsulatum.

The highly variable mycelial phase cultures of Histoplasma capsulatum are generally described as brown to albino colonies with the albino type overtaking the brown in a unidirectional and mainly irreversible process. In this study it was found that cultures could be reversed from albino to brown type by manipulation of substrates. As the cultural morphology and stability were shown to be essentially substrate-dependent, the mycelial status of this organism was reassessed. It is proposed that albino and intermediate variants are usually artificially induced by conventional refined carbohydrate media, while crude guano substrates tend to promote cultures of a brown wild type.

Long-term outcome in systemic lupus erythematosus membranous glomerulonephritis. Lupus Nephritis Collaborative Study Group.

The pathology of membranous glomerulonephritis (MGN) in patients with systemic lupus erythematosus (SLE) may be complicated by superimposed glomerular inflammation and/or necrosis. This retrospective study examined whether various histologic patterns of glomerulonephritis (GN) seen on renal biopsy impact upon the prognosis of these patients. Clinical parameters at the time of biopsy were also studied, to determine which might serve as risk factors associated with renal and patient outcome. On the basis of renal biopsy findings, patients were stratified into three pathological study groups by using the World Health Organization (WHO) classification (11). Thirty-six patients had "pure' SLE MGN without (WHO Va) or with (WHO Vb) mesangial hypercellularity. Fifteen patients had SLE MGN with segmental endocapillary proliferation and/or necrosis in < 50% of glomeruli (WHO Vc < 50%). Twenty-eight patients had SLE MGN with endocapillary proliferation and/or necrosis in > or = 50% of glomeruli (MGN with segmental proliferation and/or necrosis in > 50%, WHO Vc > or = 50%, or MGN with superimposed diffuse endocapillary proliferation, WHO Vd). There were no significant differences in sex, age, or race among patients in the three study groups. There was a trend for increasing serum creatinine concentration, urine protein excretion, and diastolic blood pressure, and decreasing the third component of serum complement (C3) to be associated with increasing glomerular inflammation, and these differences were significant when Vc > or = 50% and Vd was compared with Va and Vb (P < 0.05). The 5- and 10-yr actuarial survival rates without reaching the study outcomes of death or renal replacement therapy for the three study groups were 86, 72, and 49% and 72, 48, and 20% respectively, and the differences between Va and Vb and Vc > 50% and Vd were significant (P < 0.05). SLE MGN has a heterogeneous course and outcome, and this variability is related to the extent and degree of glomerulonephritis seen on renal biopsy. The only clinical factor with an independent risk of reaching a study outcome was elevation of the initial serum creatinine concentration (Cox regression analysis). This predictive value appears to apply only to patients with the most severe forms of glomerulonephritis, suggesting that they may have a different natural history and/or a response to therapy than SLE MGN with less widespread glomerular inflammation and/or necrosis.

Focal segmental glomerular sclerosis in adults: presentation, course, and response to treatment.

The authors performed a retrospective clinicopathologic study in 81 patients with primary focal segmental glomerular sclerosis (FSGS) to determine whether they could identify clinical or histologic features at presentation that could be predictive of outcome and response to therapy. Males constituted 58% of patients, and 53% were black. At biopsy the patients were 40 +/- 17 years old; 74% were nephrotic, and renal insufficiency was present in 62%. The average time from presentation to biopsy was 16 months, and the average total follow-up was 62 months. Nephrotic patients had a significantly poorer prognosis as compared with nonnephrotic patients (5- and 10-year survivals of 76% and 57% v 92% and 92%; P < 0.05). A multivariate analysis was done on histologic and clinical features at biopsy, assessing for risk factors leading to end-stage renal disease, showing only the serum creatinine and the degree of interstitial fibrosis to have a significant correlation. Thirty nephrotic patients received prednisone, with a treatment time of 5.5 +/- 4 months and a total dose of 5.9 +/- 2.9 g per course of treatment. Fifteen of these patients (50%) achieved a remission by 3.7 +/- 2 months (10 complete remission and 5 partial remissions), with all patients responding within 9 months. Only two patients had spontaneous remissions (both partial). The 5- and 10-year survival for patients in remission were both 100% as compared with 66% and 41% (P < 0.01), respectively, for nephrotic patients not in remission. No clinical feature at presentation of biopsy was predictive of response to therapy when a multivariate analysis was performed.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary focal segmental glomerular sclerosis in adults: prognostic value of histologic variants.

Primary focal segmental glomerular sclerosis (FSGS) is a clinicopathologic syndrome in which variable amounts of proteinuria are associated with the renal biopsy finding of segmental glomerular scarring in some, but not all, of the glomeruli. Additional histologic features have been described in FSGS, including the position of the scar relative to the vascular and tubular pole of the glomerulus, foam cells, hyalinosis, mesangial deposits of immunoglobulin M, diffuse mesangial hypercellularity, glomerular visceral epithelial cell hyperplasia and hypertrophy, and the extent of associated interstitial fibrosis and tubular atrophy. We performed a retrospective study on 81 patients with biopsy-proven, primary FSGS to determine whether any of the histologic features of FSGS correlated with renal function at the time of biopsy and the incidence of end-stage renal disease at follow-up. Sixty patients were nephrotic and 21 had nonnephrotic proteinuria. Only the degree of interstitial fibrosis correlated with the initial serum creatinine (r = 0.536) and none of the histologic features predicted the presence of nephrotic-range proteinuria at the time of biopsy. Segmental scars involved 21% +/- 14% of the glomeruli per biopsy specimen, but their position within the glomerulus was uniform in only 13% of the cases. Diffuse mesangial hypercellularity was present in 17% of the biopsy specimens, and glomerular epithelial cell lesions were present in 57% of the biopsy specimens. Multivariate analysis showed that only the degree of interstitial fibrosis predicted end-stage renal disease in all 81 patients and in the 60 patients with nephrotic-range proteinuria. The current data do not support different therapeutic approaches in primary FSGS based on histologic subtypes.

The racial prevalence of glomerular lesions in nephrotic adults.

We retrospectively evaluated the prevalence of primary glomerular lesions in adults who had a renal biopsy for nephrotic proteinuria. From July 1975 to May 1994, 340 patients undergoing renal biopsies evaluated at Rush-Presbyterian-St Lukes Medical Center had the primary glomerular lesions of minimal-change disease, focal segmental glomerular sclerosis (FSGS), membranous glomerulonephritis (MGN), membranoproliferative glomerulonephropathy, immunoglobulin A nephropathy, and immunotactoid glomerulopathy. The patients had a mean age of 43 +/- 17 years, 57% were male, and 50% were white, 36% were black, 7% were of other race, and 7% were of unknown race. The distribution of lesions for black patients was minimal-change disease 14%, FSGS 57%, MGN 24%, membranoproliferative glomerulonephritis 2%, immunoglobulin A 2%, and immunotactoid glomerulopathy 1%; for white patients, the distribution of lesions was minimal-change disease 20%, FSGS 23%, MGN 36%, membranoproliferative glomerulonephropathy 6%, immunoglobulin A 8%, and immunotactoid glomerulopathy 6%. The prevalence of FSGS was significantly greater (P < 0.0001) and that for MGN, immunoglobulin A, and immunotactoid glomerulopathy was significantly less (P < 0.05) for black patients compared with white patients. In a logistic regression analysis, race remained the only significant predictor of FSGS (P = 0.0001), with black patients four times as likely to have FSGS as white patients. The distribution of lesions among white patients was similar based on gender, age, and biopsy period. For black patients the distribution was also similar based on gender and age, but over 20 years the incidence of FSGS increased from 39% (1975 to 1984) to 64% (1985 to 1994) (P < 0.01). Thus, significant racial differences in the distribution of primary glomerular lesions exists. This has important prognostic and therapeutic implications for nephrotic adults.

Intramedullary apoptosis of hematopoietic cells in myelodysplastic syndrome patients can be massive: apoptotic cells recovered from high-density fraction of bone marrow aspirates.

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. (Blood. 2000;96:1388-1392)

Apoptosis in bone marrow biopsy samples involving stromal and hematopoietic cells in 50 patients with myelodysplastic syndromes.

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.

The clinical and biologic significance of abnormal lipid profiles in patients with myelodysplastic syndromes.

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.

Assessment of small-diameter aortic mechanical prostheses: physiological relevance of the Doppler gradient, utility of flow augmentation, and limitations of orifice area estimation.

BACKGROUND: Noninvasive assessment of functionally stenotic small-diameter aortic mechanical prostheses is complicated by theoretical constraints relating to the hemodynamic relevance of Doppler-derived transprosthetic gradients. To establish the utility of Doppler echocardiography for evaluation of these valves, 20-mm Medtronic Hall and 19-mm St Jude prostheses were studied in vitro and in vivo. METHODS AND RESULTS: Relations between the orifice transprosthetic gradient (equivalent to Doppler), the downstream gradient in the zone of recovered pressure (equivalent to catheter), and fluid mechanical energy losses were examined in vitro. Pressure-flow relations across the 2 prostheses were evaluated by Doppler echocardiography in vivo. For both types of prosthesis in vitro, the orifice was higher than the downstream gradient (P<0.001), and fluid mechanical energy losses were as strongly correlated with orifice as with downstream pressure gradients (r2=0.99 for both). Orifice and downstream gradients were higher and fluid mechanical energy losses were larger for the St Jude than the Medtronic Hall valve (all P<0.001). Whereas estimated effective orifice areas for the 2 valves in vivo were not significantly different, model-independent dynamic analysis of pressure-flow relations revealed higher gradients for the St Jude than the Medtronic Hall valve at a given flow rate (P<0.05). CONCLUSIONS: Even in the presence of significant pressure recovery, the Doppler-derived gradient across small-diameter aortic mechanical prostheses does have hemodynamic relevance insofar as it reflects myocardial energy expenditure. Small differences in function between stenotic aortic mechanical prostheses, undetectable by conventional orifice area estimations, can be identified by dynamic Doppler echocardiographic analysis of pressure-flow relations.