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Background — Despite the common occurrence of aortic stenosis, the cellular causes of the disorder are unknown, in part because of the absence of experimental models. We hypothesized that atherosclerosis and early bone matrix expression in the aortic valve occurs secondary to experimental hypercholesterolemia and that treatment with atorvastatin modifies this transformation. Methods and Results — To test this hypothesis, we developed an experimental hypercholesterolemic rabbit model. New Zealand White rabbits (n=48) were studied: group 1 (n=16), normal diet; group 2 (n=16), 1% (wt/wt) cholesterol diet; and group 3 (n=16), 1% (wt/wt) cholesterol diet plus atorvastatin (3 mg/kg per day). The aortic valves were examined with hematoxylin and eosin stain, Masson trichrome, macrophage (RAM 11), proliferation cell nuclear antigen (PCNA), and osteopontin immunostains. Cholesterol and highly sensitive C-reactive protein (hsCRP) serum levels were obtained by standard assays. Computerized morphometry and digital image analysis were performed for quantifying PCNA (% area). Electron microscopy and immunogold labeling were performed for osteopontin. Semiquantitative RT-PCR was performed for the osteoblast bone markers [alkaline phosphatase, osteopontin, and osteoblast lineage-specific transcription factor ( Cbfa-1 )]. There was an increase in cholesterol, hsCRP, PCNA, RAM 11, and osteopontin and osteoblast gene markers (alkaline phosphatase, osteopontin, and Cbfa-1 ) in the cholesterol-fed rabbits compared with control rabbits. All markers except hsCRP were reduced by atorvastatin. Conclusions — These findings of increased macrophages, PCNA levels, and bone matrix proteins in the aortic valve during experimental hypercholesterolemia provide evidence of a proliferative atherosclerosis–like process in the aortic valve associated with the transformation to an osteoblast-like phenotype that is inhibited by atorvastatin.

Rituximab is a chimeric murine/human monoclonal antibody that binds to CD20 on B lymphocytes. Although binding of the Fab domain may induce apoptosis, the Fc domain recruits immune effector functions to mediate cell lysis. Interleukin-12 (IL-12) facilitates cytolytic T-cell responses, enhances the lytic activity of natural killer (NK) cells, and induces the secretion of interferon γ (IFN-γ) by both T and NK cells. Therefore, the hypothesis was considered that combining IL-12 with rituximab would augment the immune-mediated cell lysis induced by rituximab. A phase 1 study of IL-12 in combination with rituximab was conducted in 43 adults with B-cell lymphoma to determine the optimal immunologic dose of this combination. Rituximab was administered at a dose of 375 mg/m2 by intravenous infusion weekly for 4 weeks, and IL-12 was given subcutaneously twice weekly. The starting dose of IL-12 was 30 ng/kg and this was escalated to 500 ng/kg. Constitutional symptoms and liver enzyme elevations at 500 ng/kg of IL-12 were dose limiting. A greater than 20-fold increase in the serum levels of IFN-γ and a 2.5- to 5-fold increase in inducible protein 10 (IP-10) levels was seen at IL-12 doses of 100 ng/kg or greater. Objective responses occurred in 29 of the 43 patients (69%), with 8 of 11 complete responses seen at IL-12 doses of 300 ng/kg or greater. The optimal immunologic dose of IL-12 in combination with rituximab was determined to be 300 ng/kg subcutaneously twice weekly starting on day 2. These data suggest that IL-12 and rituximab is an active combination and further studies of this combination in B-cell non-Hodgkin lymphoma are warranted.

Primary systemic amyloidosis (AL) is a plasma cell (PC) dyscrasia with clinical similarities to multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS), but its molecular basis is poorly understood. Translocations at the immunoglobulin heavy-chain (IgH) locus, 14q32, are likely early genetic events in both MM and MGUS and involve several nonrandom, recurrent, partner chromosomes such as 11q13, 16q23, and 4p16.3. Given the similarities between MM, MGUS, and AL, bone marrow clonal PCs were evaluated in 29 patients with AL using interphase fluorescence in situ hybridization (FISH) combined with immunofluorescence detection of the cytoplasmic light-chain (cIg-FISH) for the presence of 14q32 translocations and the t(11;14)(q13;q32). Of 29 patients studied, 21 (72.4%) showed results compatible with the presence of a 14q32 translocation, and 16 (76.2%) of those had translocation (11;14)(q13;q32) for an overall prevalence of the abnormality of 55%. IgH translocations are common in AL, especially the t(11;14)(q13;q32).

AbstractDT388–GM-CSF, a targeted fusion toxin constructed by conjugation of human granulocyte-macrophage colony-stimulating factor (GM-CSF) with the catalytic and translocation domains of diphtheria toxin, is presently in phase I trials for patients with resistant acute myeloid leukemia. HL-60/VCR, a multidrug-resistant human myeloid leukemia cell line, and wild-type HL-60 cells were used to study the impact of DT388–GM-CSF on metabolism of ceramide, a modulator of apoptosis. After 48 hours with DT388–GM-CSF (10 nM), ceramide levels in HL-60/VCR cells rose 6-fold and viability fell to 10%, whereas GM-CSF alone was without influence. Similar results were obtained in HL-60 cells. Examination of the time course revealed that protein synthesis decreased by about 50% and cellular ceramide levels increased by about 80% between 4 and 6 hours after addition of DT388–GM-CSF. By 6 hours this was accompanied by activation of caspase-9, followed by activation of caspase-3, cleavage of caspase substrates, and chromatin fragmentation. Hygromycin B and emetine failed to elevate ceramide levels or induce apoptosis at concentrations that inhibited protein synthesis by 50%. Exposure to C6-ceramide inhibited protein synthesis (EC50∼5 μM) and decreased viability (EC50 ∼6 μM). Sphingomyelinase treatment depleted sphingomyelin by about 10%, while increasing ceramide levels and inhibiting protein synthesis. Diphtheria toxin increased ceramide and decreased sphingomyelin in U-937 cells, a cell line extremely sensitive to diphtheria toxin; exposure to DT388–GM-CSF showed sensitivity at less than 1.0 pM. Diphtheria toxin and conjugate trigger ceramide formation that contributes to apoptosis in human leukemia cells through caspase activation and inhibition of protein synthesis.

AbstractCurrent therapeutic options for myeloid metaplasia with myelofibrosis (MMM) are limited. A pilot study was conducted of autologous peripheral blood stem cell (PBSC) collection in 27, followed by transplantation in 21 patients with MMM. The median age was 59 (range 45-75) years. PBSCs were mobilized at steady state (n = 2), after granulocyte colony-stimulating factor (G-CSF) alone (n = 17), or after anthracycline-cytarabine induction plus G-CSF (n = 8). A median of 11.6 × 106 (range 0 to 410 × 106) CD34+ cells per kilogram were collected. Twenty-one patients then underwent myeloablation with oral busulfan (16 mg/kg) and PBSC transplantation. The median times to neutrophil and platelet recovery after transplantation were 21 (range 10-96) and 21 (range, 13 to ≥ 246) days, respectively. Five patients received back-up PBSC infusion because of delayed neutrophil or platelet recovery. The median follow-up is 390 (range 70-1623) days after transplantation, and the 2-year actuarial survival is 61%. After transplantion, 6 patients died: 3 of nonrelapse causes (1 within 100 days of PBSC infusion) and 3 of disease progression. Erythroid response (hemoglobin ≥ 100 g/L [10 gm/dL] without transfusion for ≥ 8 weeks) occurred in 10 of 17 anemic patients. Four of 8 patients with a platelet count less than 100 × 109/L (100 000/μL) responded with a durable platelet count more than 100 × 109/L (100 000/μL). Symptomatic splenomegaly improved in 7 of 10 patients. It is concluded that (1) PBSC collection was feasible and stable engraftment occurred after transplantation in most patients with MMM, (2) myeloablation with busulfan was associated with acceptable toxicity, (3) a significant proportion of patients derived clinical benefit after treatment, and (4) further investigation of this novel approach is warranted.

AbstractAutologous stem cell transplantation (ASCT) improves survival in patients with previously untreated multiple myeloma (MM) and relapsed, chemotherapy-sensitive, aggressive non-Hodgkin lymphoma (NHL). Lower relapse rates seen in allogeneic stem cell transplantation have been related to early absolute lymphocyte count (ALC) recovery as a manifestation of early graft-verus-tumor effect. In ASCT, the relation between ALC recovery and clinical outcomes in MM and NHL was not previously described. This is a retrospective study of patients with MM and NHL who underwent ASCT at the Mayo Clinic between 1987 and 1999. The ALC threshold was determined at 500 cells/μL on day 15 after ASCT. The study identified 126 patients with MM and 104 patients with NHL. The median overall survival (OS) and progression-free survival (PFS) times for patients with MM were significantly longer in patients with an ALC of 500 cells/μL or more than patients with an ALC of fewer than 500 cells/μL (33 vs 12 months,P < .0001; 16 vs 8 months, P < .0003, respectively). For patients with NHL, the median OS and PFS times were significantly longer in patients with an ALC of 500 cells/μL or more versus those with fewer than 500 cells/μL (not reached vs 6 months, P < .0001; not reached vs 4 months,P < .0001, respectively). Multivariate analysis demonstrated day 15 ALC to be an independent prognostic indicator for OS and PFS rates for both groups of patients. In conclusion, ALC is correlated with clinical outcome and requires further study.

Acquired pure red cell aplasia (PRCA) can be associated with lymphoproliferative disease of granular T lymphocytes (T-LDGL), also known as T-cell large granular lymphocyte leukemia. Fifteen adult patients with PRCA associated with T-LDGL comprise this study. Neutropenia and rheumatoid arthritis were uncommon. All patients responded to immunosuppressive therapy. The 2 most commonly used treatments were prednisone and cyclophosphamide ± corticosteroids, producing overall response rates of 50% and 60%, respectively. Treatment with cyclophosphamide was associated with a more durable remission (median, 60 versus 7.5 months). After a median follow-up of 67 months, 2 patients died of treatment-related complications, one from myelodysplasia and another from cyclosporine-induced renal failure. The clinical course and treatment responses of PRCA associated with T-LDGL in this series were similar to the general group of PRCA. Because T-LDGL is frequently underdiagnosed, it is likely that a significant proportion of idiopathic or primary PRCA is in fact secondary to T-LDGL.

AbstractLipoprotein (a) [Lp(a)] has been associated with both anti-fibrinolytic and atherogenic effects. However, no direct link currently exists between this atherogenic lipoprotein and intravascular coagulation. The current study examined the binding and functional effects of Lp(a), its lipoprotein constituents, apoliprotein (a) [apo(a)] and low-density lipoprotein (LDL), and lysine-plasminogen (L-PLG), which shares significant homology with apo(a), on tissue factor pathway inhibitor (TFPI), a major regulator of tissue factor-mediated coagulation. Results indicate that Lp(a), apo(a), and PLG but not LDL bound recombinant TFPI (rTFPI) in vitro and that apo(a) bound to a region spanning the last 37 amino acid residues of the c-terminus of TFPI. The apparent binding affinity for TFPI was much higher for Lp(a) (KD ∼150 nM) compared to PLG (KD ∼800 nM) and nanomolar concentrations of apo(a) (500 nM) inhibited PLG binding to TFPI. Lp(a) also inhibited in a concentration-dependent manner rTFPI activity and endothelial cell surface TFPI activity in vitro, whereas PLG had no such effect. Moreover physiologic concentrations of PLG (2 μM) had no effect on the concentration-dependent inhibition of TFPI activity induced by Lp(a). In human atherosclerotic plaque, apo(a) and TFPI immunostaining were shown to coexist in smooth muscle cell–rich areas of the intima. These data suggest a novel mechanism whereby Lp(a) through its apo(a) moiety may promote thrombosis by binding and inactivating TFPI.

AbstractCurrent information on Waldenström macroglobulinemia (WM) is based on retrospective or single-institution studies of patients requiring therapy. Between 1992 and 1998, 231 patients with WM were enrolled in a prospective observational multicenter clinical trial. Of these, 182 patients with symptomatic or progressive disease were treated with 4 to 8 cycles of therapy with a purine nucleoside analogue, fludarabine (FAMP; 30 mg/m2 of body-surface area daily for 5 days every 28 days). A serum β2-microglobulin (β2M) level below 3 mg/L and a hemoglobin level of at least 120 g/L (12 g/dL) at presentation predicted a lower likelihood of requiring therapy. The overall rate of response to FAMP therapy was 36% (95% confidence interval, 29%-44%), with 2% complete remissions. Patients who were 70 years old or older had a substantially lower likelihood of response (odds ratio, 0.34; P = .004) than younger patients. On multivariate analysis, a serum β2M level of 3 mg/L or higher, hemoglobin level below 120 g/L, and serum IgM level below 40 g/L [4 g/dL] were significant adverse prognostic factors for survival. We developed a simple staging system for WM by using these variables and identified 4 distinct subsets of patients with estimated 5-year overall survival rates of 87%, 64%, 53%, and 22%, and 5-year progression-free survival rates of 83%, 55%, 33%, and 12%. Prognosis in WM is highly variable and serum β2M was the dominant predictor of a need for therapy and of survival. FAMP has activity against WM. Our staging system may provide guidance for a risk-based approach to the treatment of WM.

AbstractPreviously, it was reported that patients with multiple myeloma (MM) who have higher baseline levels of blood CD4+or CD19+ cells have longer survival. This article extends the analysis of immune cell levels and survival in a large cohort (N = 504) of patients with MM entered on Eastern Cooperative Oncology Group (ECOG) phase 3 trial (9486). Newly diagnosed patients with MM received 2 cycles of vincristine, bischloroethylnitrosourea, melphalan, cytoxan, prednisone (VBMCP) and were treated on one of 3 randomized arms: VBMCP with either interferon or high-dose cyclophosphamide, or VBMCP alone. Blood immune cell levels were studied at trial entry (baseline), after 2 cycles of chemotherapy, after 2 years of therapy, and at relapse. Baseline CD3+, CD4+, CD8+, CD19+, and CD4+ subset cell levels were all positively associated with survival (P = .0087 toP < .0001). A multivariate analysis incorporating CD4+ and CD19+ cell levels defined 3 separate groups of patients with MM to survival outcome. Higher CD19+ blood levels were positively associated with MM-patient survival at entry to the study, at year 2, and at relapse (P < .0001 at all 3 timepoints). Patients with MM had evidence of immune cell reconstitution after 2 years of therapy, but the rate and extent of recovery was greater for CD8+, which was greater than CD4+, which was greater than CD19+. This latter data affirms the positive relationship between the quantitative status of the blood immune system in MM and survival. In addition, the importance of the CD19+ blood cells to survival is evident throughout the course of MM. Therapeutic efforts to maintain an intact immune system may be crucial in maximizing chemotherapeutic and/or immunotherapy efforts in this disease.