De novo generation of an axially vascularized processed bovine cancellous-bone substitute in the sheep arteriovenous-loop model.

BACKGROUND/AIMS: The aim of this study was to generate an axially vascularized bone substitute. The arteriovenous (AV)-loop approach in a large-animal model was applied in order to induce axial vascularization in a clinically approved processed bovine cancellous bone (PBCB) matrix of significant volume with primary mechanical stability and to assess the course of increasing axial vascularization. METHODS: PBCB constructs were implanted into 13 merino sheep together with a microsurgically created AV loop in an isolation chamber. The vascularization process was monitored by sequential magnetic resonance imaging (MRI) scans. Explants were subjected to micro-computed tomography (micro-CT) analysis, histomorphometry and immunohistochemistry for CD31 and CD45. RESULTS: Increasing axial vascularization in PBCB constructs was quantified by histomorphometry and visualized by micro-CT scans. Intravital sequential MRI scans demonstrated a significant progressive increase in perfused volume within the matrices. Immunohistochemistry confirmed endothelial lining of newly formed vessels. CONCLUSION: This study demonstrates successful axial vascularization of a clinically approved, mechanically stable bone substitute with a significant volume by a microsurgical AV loop in a large-animal model. Thus microsurgical transplantation of a tissue-engineered, axially vascularized and mechanically stable bone substitute with clinically relevant dimensions may become clinically feasible in the future.

Thalidomide produces transfusion independence in long-standing refractory anemias of patients with myelodysplastic syndromes.

Thalidomide was administered to 83 patients with myelodysplastic syndrome (MDS), starting at 100 mg by mouth daily and increasing to 400 mg as tolerated. Thirty-two patients stopped therapy before 12 weeks (minimum period for response evaluation), and 51 completed 12 weeks of therapy. International Working Group response criteria for MDS were used to evaluate responses. Intent-to-treat (ITT) analysis classified all off-study patients as nonresponders. Off-study patients belonged to a higher risk category (P =.002) and had a higher percentage of blasts in their pretherapy bone marrow than patients who completed 12 weeks of therapy (P =.003). No cytogenetic or complete responses were seen, but 16 patients showed hematologic improvement, with 10 previously transfusion-dependent patients becoming transfusion independent. Responders had lower pretherapy blasts (P =.016), a lower duration of pretherapy platelet transfusions (P =.013), and higher pretherapy platelets (P =.003). Among responders, 9 had refractory anemia (RA); 5 had RA with ringed sideroblasts; and 2 had RA with excess blasts. By ITT analysis, 19% of patients (16 of 83) responded, and when only evaluable patients were analyzed, 31% (16 of 51) responded. It was concluded that thalidomide, as a single agent, is effective in improving cytopenias of some MDS patients, especially those who present without excess blasts. (Blood. 2001;98:958-965)

Poor prognosis acute myelogenous leukemia: 1 - response to treatment with high dose cytarabine/mitoxantrone/ethyol @ (Amifostine).

Twenty patients with poor prognosis AML and four patients in the blastic phase of a myeloproliferative disorder were treated with two 'pulses' of therapy each consisting of two doses of high dose araC (separated by 12 h) followed by a single dose of mitoxantrone. The pulses were separated by 96 h. Amifostine was then administered tiw. The median age of the population was 68 years with 88% of patients having had either a prior MDS, MPD or toxic exposure. The acute leukemia of 58% of patients either entered a CR or reverted to preleukemic state. For patients under 70 years of age, treatment produced 62% CRs with a leukemia free decision marrow in 77%. For patients over 70 years the CR rate was 27% with 36% of patients having a leukemia free decision marrow.

Patients with myelodysplastic syndromes benefit from palliative therapy with amifostine, pentoxifylline, and ciprofloxacin with or without dexamethasone.

Thirty-five patients with myelodysplastic syndrome (MDS) were registered on protocol MDS 96-02 and were receiving continuous therapy with pentoxifylline 800 mg 3 times a day and ciprofloxacin 500 mg twice a day by mouth; dexamethasone was added to the regimen for the partial responders and the nonresponders after 12 weeks at a dose of 4 mg by mouth every morning for 4 weeks. Amifostine was administered intravenously 3 times a week at 3 dose levels (200 mg/M(2), 300 mg/M(2), and 400 mg/M(2)) to cohorts of 10 patients each. Therapy has been continued for 1 year in responders. Twenty-nine have completed at least 12 weeks of therapy and are available for response evaluation. Of the 21 men and 8 women (median age, 67 years), 20 had refractory anemia (RA), 3 had RA with ringed sideroblasts (RARS), 5 had RA with excess blasts (RAEB), and 1 had chronic myelomonocytic leukemia (CMMoL). Five had secondary MDS. No differences were noted in response rates among the 3 dose levels. Seven patients did not respond at all, and 22 showed an improvement in cytopenias (76%). Three had a triple lineage response, 10 had a double lineage response, and 9 had a single lineage response (8 of 9 in absolute neutrophil count [ANC] and 1 had more than a 50% reduction in packed red blood cell transfusions). Fifteen patients responded only after the addition of dexamethasone, whereas 7 responded before. When examined by lineage, 19 of 22 showed improved ANC, 11 of 22 demonstrated more than 50% reduction in blood transfusions, improved Hb levels, or both, and 7 of 22 showed improvement in platelet counts. Interestingly, the responses were frequently slow to appear, and continued improvement in counts was seen up to 12 months of therapy and beyond. This study supports the feasibility of treating patients with MDS with the unique approach of cytoprotection and anticytokine therapies as well as the principle that prolonged commitment to treatment is desirable when noncytotoxic agents are administered. (Blood. 2000;95:1580-1587)

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.

Marrow cytokine transcripts and the secondary hematologic disorders.

A comparison was made of the cytokine transcripts in normal, monoclonal, MDS, and AML marrow aspirates. While both normal and monoclonal marrow aspirates contain transcripts for SCF, few MDS or AML marrow aspirates contain these transcripts. Similarly, IL1ra transcripts are found with reduced frequency in MDS and AML marrow aspirates. The fall in SCF transcripts between monoclonal and MDS marrow aspirates parallels the appearance of apoptosis and the reduced in vitro proliferative ability which are characteristics of MDS marrow aspirate cells. The frequent IL1beta production by MDS and AML marrow aspirate cells, with few marrow aspirates producing IL1ra transcripts, suggests that unbalanced IL1beta effects may contribute to the proliferative advantage of MDS and AML cells over their normal counterparts.

Cytokine gene activity in AML cells in vivo in patients.

The proliferation of acute myelogenous leukemia cells is dependent upon cytokine stimulation. Additionally, there is a body of literature which reports that leukemia cells produce GMCSF, IL6, and other cytokines. The study reported here, using an rt-multiplex polymerase method, determined the presence or absence of transcripts in freshly obtained AML cells for the following cytokine or cytokine-related genes: IL 1beta, IL1ra, TNF alpha, GMCSF, IL6, flt 3, and hSCF. This demonstrated that leukemia cell populations usually contain transcripts for IL1beta, TNF alpha, flt 3 and flt 3 ligand in vivo and that transcripts for the other cytokines only appear after the leukemia cells are processed in vitro. The presence of TNF alpha transcripts appears to be associated with resistance to remission induction therapy. Furthermore, the transcript profile of the leukemia cells can change during remission induction therapy. The data also demonstrate the assessment of cytokine production by leukemia cells after in vitro manipulation should not be extrapolated to the in vivo situation.

Does low individual operator coronary interventional procedural volume correlate with worse institutional procedural outcome?

OBJECTIVES: To assess the relation between individual operator coronary interventional volume and incidence of complications, the in-hospital outcome at a single, moderate volume urban academic center was prospectively collected over a 3-year period. BACKGROUND: A minimum of 75 coronary interventions/operator per year may be required in the future to obtain formal certification. However, few data exist regarding individual operator volumes and procedural outcome. METHODS: Between January 1993 and December 1995, 1,389 consecutive procedures were performed or supervised by nine geographic full-time operators: 171 (12.3%) utilized various devices, and 350 (25.2%) involved multivessel coronary intervention. Left ventricular ejection fraction was 59 +/- 15% (mean +/- SD), and there were 1.7 +/- 0.7 vessels diseased (with > or = 70% stenosis). Clinical indications included stable angina in 22.5% of cases, unstable angina in 31.9%, acute myocardial infarction (MI) in 2.9%, post MI in 20.6%, shock or acute heart failure in 3.0% and restenosis in 19.1%. In the last consecutive 857 lesions in 655 cases, 20.7% type A, 55.5% type B and 23.8% type C lesions were categorized before coronary intervention. RESULTS: Average yearly operator volume ranged from 26 to 83 cases (mean 51 +/- 26). Each operator has performed a total of 590 +/- 268 coronary interventions, with 10.0 +/- 4.3 years of coronary interventional experience. The mean angioplasty volume rating for the nine operators was 180 +/- 37 (> 170 considered adequate). The in-hospital major complication rate was 1.4% (95% confidence interval 0.7% to 1.893%) for all coronary interventions, including death in 3 patients, bypass surgery in 13, arrhythmia in 3 and Q wave MI in 2. To ascertain how these outcomes compared with standard measures of coronary interventional outcome, four previously published registries were reanalyzed in a similar manner. The rate of complications in the present study was found to be significantly lower than that of the 1992-1993 Society for Cardiac Angiography and Intervention registry (1.9%, n = 19,594, p < 0.05 [excludes ventricular arrhythmias]), the 1994 American College of Cardiology database (3.9%, n = 38,963, p = 0.001), the Mid-America Heart Institute outcome in 1988 (2.3%, n = 5,413, p = 0.02) and the 1985-1986 National Heart, Lung, and Blood Institute Registry (7.2%, n = 1,801, p = 0.001). Odds ratios and 95% confidence intervals showed the outcome in the current study to be at least comparable to the standard registries. CONCLUSIONS: Despite individual operator volumes below those currently being considered for credentialing, the overall institutional outcome was excellent in a diverse and complex patient population.

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.

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.

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.

A multivariate study of non Hodgkin's lymphoma involving proliferation, apoptosis, bcl-2 and the microenvironment.

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.

Novel in situ double labeling for simultaneous detection of proliferation and apoptosis.

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.

Splanchnic transplantation. An approach to the infant dependent on parenteral nutrition who develops irreversible liver disease.

Two infants with short-bowel syndrome and liver failure associated with obligatory parenteral nutrition received a composite allograft that consisted of en bloc liver, stomach, duodenum, pancreas, jejunum, and ileum. Solutions to the fatal complications in the first case resulted in a functioning composite splanchnic system in the second case. Despite a number of early complications, the small intestine and liver developed near-normal function until a monoclonal, malignant, B-cell lymphoproliferative disorder appeared. The analysis of these two cases supports three summary observations: the operative procedure can be safely performed in a metabolically compromised infant; intestinal allograft rejection, in this model, is controllable with existing immunosuppressive drugs; and this procedure appears to be associated with a uniquely high incidence of lymphoma. Since transplantation is a feasible solution to this devastating infantile disease, further development of this therapy must incorporate means of preventing lymphoma.

Bilateral trephine bone marrow biopsy for staging non-Hodgkin's lymphoma--a second look.

Several reports have suggested that bilateral bone marrow biopsy is better than unilateral biopsy in staging non-Hodgkin's lymphomas. Therefore since 1975, bilateral iliac crest bone marrow biopsy has been part of our standard initial staging investigation of non-Hodgkin's lymphomas. The present study is to determine the relative value of bilateral marrow biopsy compared to the unilateral procedure. We studied 176 patients who had evaluable adequate bilateral biopsies and tumor histology. Among 57 patients with low-grade lymphoma, 7 (12%) were unilaterally positive for lymphoma and 25 (44%) were bilaterally positive. Among 119 patients with intermediate and high-grade lymphoma, 6 biopsies (5%) were unilaterally positive and 10 (8%) were bilaterally positive for lymphoma. Doing the second (contralateral) biopsy changed the disease stage as assessed by bone marrow biopsy in 6.1% of patients with favorable histologies and in 2.5% of patients with unfavorable histologies. These changes are small. Moreover, treatment would not have been significantly altered in the majority of patients as a result of the changes. We conclude that the routine use of bilateral trephine biopsy in initial staging of non-Hodgkin's lymphomas needs to be reappraised. We believe that its use should be limited to selected patients in whom the finding of bone marrow involvement may influence the choice of treatment.

Cat scratch disease. Identification of bacteria in seven cases of lymphadenitis.

A retrospective study of lymph node biopsy specimens from nine patients with the clinical findings and histologic features of cat scratch disease was undertaken to determine whether the recent report by Wear et al. that pleomorphic bacteria are present in the lymph nodes of cat scratch disease could be confirmed. In seven of our nine cases, pleomorphic bacteria were demonstrated with the Warthin-Starry (WS) silver stain. These were gram-negative with the Brown-Hopps tissue Gram stain and were almost at the limit of microscopic resolution. Lymph node specimens from 13 additional patients with nonspecific lymphadenitis who had neither clinical nor histologic findings of cat scratch disease were studied similarly; in none of these were bacteria demonstrated with the WS silver stain. After examining the distribution of the organisms and the related morphologic features in cat scratch disease, we conclude that demonstration of pleomorphic, gram-negative, WS-positive bacteria in the appropriate clinical and histologic setting can firmly establish the diagnosis of cat scratch disease.

Red cell comets: ultrastructure of axial elongation of the membrane skeleton.

In order to study intact red cell membrane skeletons, we developed two methods for the electron microscopic study of stress-ruptured and partially opened glutaraldehyde pre-fixed Triton shells of red cell ghosts. Both methods showed continuous networks of fine filaments decorated and apparently crosslinked by elongated particles. Our electron micrographs show a filamentous component that is morphologically consistent with F-actin. Quantitative considerations, including the dimensions of the elongated particles and published estimates of the number of particles in situ, suggest that compact spectrin decamers or dodecamers may exist in intact red cells. Long filaments consistent with F-actin appear to be decorated with the putative spectrin particles.

Burkitt cell acute lymphoblastic leukemia with partial expression of T-cell markers and subclonal chromosome abnormalities in a man with acquired immunodeficiency syndrome.

A 45-year-old white male, bisexual, with a 2-year history of acquired immunodeficiency syndrome (AIDS) prodrome, developed a Burkitt cell-like acute lymphoblastic leukemia (ALL). Marker studies of marrow blasts show an unusual and possibly unique pattern, in that an unequivocal monoclonal B cell leukemia, having K-IgM with HLA-DR and B cell subset antigen (BA-1) expression, was superimposed with a mature suppressor T cell marker profile (pan-T, mature T, and suppressor/cytotoxic T antigens). The leukemic blasts were totally negative for terminal deoxynucleotidyl transferase (TdT), human T cell leukemia-lymphoma virus (HTLV) p19 antigen, and other immunoglobulin isotypes. Chromosome analysis of marrow cells disclosed that 70% of the cells had 47,XY, + 12,t(8;14)(q24;q32) chromosome complement, and 30% of the cells had a 47,XY, + 12,dup1q + (q22-31),t(8;14)(q24;q32). The consistent finding of the specific chromosome rearrangement (8/14 translocation) in all abnormal cells suggests that the cells were derived from a common precursor. With regard to the partial T cell marker expression, the significance of these markers in B cell leukemia is unclear, as is their relation to the additional chromosome abnormalities that apparently developed in the process of clonal evolution.