Production of myeloid dendritic cells (DC) pulsed with tumor-specific idiotype protein for vaccination of patients with multiple myeloma.

BACKGROUND: Immunotherapy of cancer with DC vaccines has produced encouraging results in clinical trials. Antigen (Ag)-pulsed DC have elicited CD4+ and CD8+ T-cell immunity and tumor regression in humans. However, there is no standard method of DC production. The DC phenotype, number and Ag-loading process used in these studies have varied, making comparisons between trials difficult. METHODS: In the present report a reproducible method was developed for the production of a DC-based vaccine. Monocytes were enriched by adhesion from healthy donor apheresis products and cultured with growth factors for maturation into DC. The cells were loaded with the tumor Ag idiotype proteins from patients with multiple myeloma. DC culture and Ag loading were performed in an automated and closed system. The DC product was characterized for phenotype by flow cytometry and for function in Ag uptake and Ag presentation. RESULTS: These monocyte-derived DC expressed high levels of costimulatory molecules (CD80/86). Ag-pulsed DC functioned to induce allogeneic proliferative lymphocyte responses and Ag-specific cytotoxic T lymphocyte (CTL) responses. The DC viability, phenotype and function were well preserved following prolonged frozen storage. Aliquots from the product of a single DC preparation could be used for sequential vaccinations without batch to batch variability. DISCUSSION: Ag-pulsed DC can be reproducibly generated for clinical use. These standardized methods are now being employed for a clinical trial to evaluate idiotype-pulsed DC vaccine therapy following non-myeloablative transplant for the treatment of multiple myeloma.

Idiotype vaccination using dendritic cells after autologous peripheral blood progenitor cell transplantation for multiple myeloma.

The idiotype (Id) determinants on the multiple myeloma immunoglobulin can serve as tumor-specific antigens. An anti-Id immune response may stem the growth of the malignant clone. We report on 26 patients treated at our institution with high-dose chemotherapy and peripheral blood progenitor cell transplantation (PBPCT) and vaccinated with the Id protein. The patients received chemotherapy and PBPCT to establish a minimal residual disease state. After high-dose therapy, the patients received a series of monthly immunizations consisting of 2 intravenous infusions of dendritic cells (DCs) pulsed with either Id protein or Id coupled with keyhole limpet hemocyanin (KLH) as an immunogenic carrier protein, followed by subcutaneous boosts of Id-KLH conjugates. DCs were obtained in all patients from a leukapheresis product 3 to 9 months after PBPCT. Patients were observed for toxicity, immune responses, and tumor status. The DC infusions and the administration of Id-KLH boosts were well tolerated, with patients experiencing only minor and transient side effects. Of the patients, 24 of 26 generated a KLH-specific cellular proliferative immune response. Only 4 patients developed an Id-specific proliferative immune response. Three of these immune responders were in complete remission at the time of vaccination. A total of 17 patients are alive at a median follow-up of 30 months after transplantation. Id vaccination with autologous DCs is feasible for myeloma patients after transplantation. Id-specific cellular responses can be induced in patients who are in complete remission. Further studies are needed to increase the rate of anti-Id immune responses in patients who do not achieve complete remission.

The findings and impact of nonrehydrated guaiac examination of the rectum (FINGER) study: a comparison of 2 methods of screening for colorectal cancer in asymptomatic average-risk patients.

BACKGROUND: Testing stool for occult blood at the time of digital rectal examination (DRE) has been discouraged because it is thought to increase the number of false-positive test results. OBJECTIVE: To compare the diagnostic yield of colonoscopy and the cost per cancer detected in asymptomatic patients with a positive fecal occult blood test result obtained by DRE with that obtained from spontaneously passed stool (SPS) samples. METHODS: We reviewed the medical records of consecutive asymptomatic patients at average risk for colorectal cancer who were referred for colonoscopy to evaluate a positive fecal occult blood test result obtained by DRE (n = 282) or SPS samples (n = 390). The cost of colonoscopy was estimated by adding the physician fee under Medicaid reimbursement, the facility fee for endoscopy, and the pathology fee for the biopsy specimens. RESULTS: During the 5-year study period, 672 patients were evaluated and a colonic source of occult bleeding was identified in 145 patients (21.6%). The predictive value of a positive fecal occult blood test result (22.0% vs 21.3%, P = .85) and the cost per cancer detected ($7604.80 vs $7814.54) were no different in the DRE and SPS groups, with carcinomas being detected in 11.7% and 11.3% of patients, respectively. CONCLUSIONS: Testing stool for occult blood at the time of DRE does not increase the number of false-positive test results or the cost per cancer detected in asymptomatic patients at average risk for colorectal cancer. In this patient population, all individuals should be evaluated by full colonoscopy regardless of the method of stool collection.

Activity of combination therapy with interferon alfa-2b plus ribavirin in chronic hepatitis C patients co-infected with HIV.

The hepatitis C virus (HCV) and the human immunodeficiency virus (HIV) often co-infect the same individuals because they share comparable routes of transmission. Co-infection with HIV in those patients infected with HCV influences the accuracy of HCV diagnostic testing, levels of HCV viremia, severity of liver histopathology, and rate of progression to cirrhosis. By contrast, the effect of HCV co-infection on HIV disease is unclear. Nevertheless, the combination therapy containing recombinant interferon alfa-2b (rIFN-alpha 2b) plus ribavirin has been shown to be efficacious in the treatment of chronic hepatitis C, whereas alpha interferon monotherapy has been shown to be efficacious in patients co-infected with HCV and HIV. It is therefore logical to propose and test the hypothesis that combination rIFN-alpha 2b/ribavirin therapy will also benefit patients who are co-infected with HCV and HIV. A double-blind, placebo-controlled study is presently under way to investigate this hypothesis.

Is upper gastrointestinal endoscopy indicated in asymptomatic patients with a positive fecal occult blood test and negative colonoscopy?

PURPOSE: There are no recommendations as to whether endoscopic evaluation of the upper gastrointestinal tract is indicated in asymptomatic patients who have a positive fecal occult blood test and a negative colonoscopy. SUBJECTS AND METHODS: All asymptomatic patients with a positive fecal occult blood test who were referred for diagnostic endoscopy were identified. Patient charts, endoscopy records, and pathology reports were reviewed. RESULTS: During the 5-year study period, 498 asymptomatic patients with a positive fecal occult blood test and negative colonoscopy were evaluated. An upper gastrointestinal source of occult bleeding was detected in 67 patients (13%), with peptic ulcer disease being the most common lesion identified (8%). Four patients were diagnosed with gastric cancer and 1 had esophageal carcinoma. In addition, 74 patients (15%) had lesions that were not considered a source of occult bleeding; these findings prompted a change in management in 56 patients (11%). Anemia was the only variable significantly associated with having a clinically important lesion identified (multivariate odds ratio = 5.0; 95% confidence interval 2.9 to 8.5; P <0.001). CONCLUSIONS: Upper gastrointestinal endoscopy yields important findings in asymptomatic patients with a positive fecal occult blood test and negative colonoscopy. Our data suggest that endoscopic evaluation of the upper gastrointestinal tract should be considered, especially in patients with anemia.

Altered oncoprotein expression and apoptosis in myelodysplastic syndrome marrow cells.

Ineffective hematopoiesis with associated cytopenias and potential evolution to acute myeloid leukemia (AML) characterize patients with myelodysplastic syndrome (MDS). We evaluated levels of apoptosis and of apoptosis-related oncoproteins (c-Myc, which enhances, and Bcl-2, which diminishes apoptosis) expressed within CD34+ and CD34- marrow cell populations of MDS patients (n = 24) to determine their potential roles in the abnormal hematopoiesis of this disorder. Marrow cells were permeabilized and CD34+ and CD34- cells were separately analyzed by FACS to detect: (1) a subdiploid (sub-G1) DNA population, and (2) expression of Bcl-2 and c-Myc oncoproteins. Within the CD34+ subset, a significantly increased percentage of cells demonstrated apoptotic/sub-G1 DNA content in early (ie. refractory anemia) MDS patients compared with normal individuals and AML patients (mean values: 9.1% > 2.1% > 1.2%). Correlated with these findings, the ratio of expression of c-Myc to Bcl-2 oncoproteins among CD34+ cells was significantly increased for MDS patients compared to those from normal and AML individuals (mean values: 1.6 > 1.2 > 0.9). Bcl-2 and c-Myc oncoprotein levels were maturation stage-dependent, with high levels expressed within CD34+ marrow cells, decreasing markedly with myeloid maturation. Treatment of seven MDS patients with the cytokines granulocyte colony-stimulating factor plus erythropoietin was associated with decreased levels of apoptosis within CD34+ marrow cells and may contribute to the enhanced hematopoiesis in vivo that was shown. These findings are consistent with the hypothesis that altered balance between cell-death (eg, c-Myc) and cell-survival (eg, Bcl-2) programs were associated with the increased degrees of apoptosis present in MDS hematopoietic precursors and may contribute to the ineffective hematopoiesis in this disorder, in contrast to decreased apoptosis and enhanced leukemic cell survival in AML.

Modulation of apoptosis in human myeloid leukemic cells by GM-CSF.

Apoptosis (programmed cell death) regulates cell population size. To determine the mechanisms whereby hematopoietic growth factors (HGFs) modulate apoptosis in human myeloid leukemic cells, we evaluated the roles of protein and mRNA synthesis for altering apoptosis in growth factor-stimulated vs. quiescent leukemic TF1 cells. Lysates of cells from the granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent myeloid leukemic cell line TF1 were separated into high molecular weight (HMW) pellets of intact DNA and supernatants of fragmented low MW (LMW) DNA, and the DNA purified from these fractions was quantified. In the absence of both GM-CSF and fetal bovine serum (FBS), 70% of the DNA was fragmented after 3 days in culture, with a characteristic apoptotic ladder-like pattern on agarose gel electrophoresis, whereas this proportion had initially been < 5%. In contrast, less than 5% of the DNA was fragmented in cells incubated with GM-CSF plus FBS or GM-CSF alone. Delayed addition of GM-CSF, but not FBS, permitted partial rescue of the cells, inhibiting increasing rates of accumulation of fragmented DNA. When the macro-molecular synthesis inhibitor cycloheximide (CHX) or actinomycin D (Act D) was present for 26 hours in the absence of GM-CSF and FBS, apoptosis was inhibited. In contrast, in the presence of GM-CSF or FBS, apoptosis was enhanced upon addition of CHX or Act D. The latter effect persisted even with the late addition of CHX. These findings indicate that disparate mechanisms of enhancing or inhibiting apoptosis exist in myeloid leukemic cells related to environmental conditions, including HGF-regulated cellular synthesis of distinct proteins and mRNA.

Pre-steady-state kinetics reveal a slow isomerization of the enzyme-NAD complex in the NAD-malic enzyme reaction.

Stopped-flow experiments obtained in the pre-steady-state time scale of the NAD-malic enzyme reaction exhibit a lag prior to the attainment of steady state. Previous results from isotope effect studies in which the deuterium isotope effect on Vmax decreases to a value of 1 at low pH have been interpreted as suggesting a slow release of NADH [Kiick, D. M., Harris, B. G., & Cook, P. F. (1986) Biochemistry 25, 227-236]. The latter, however, requires a burst in the pre-steady-state time course, and thus the previous data have been reinterpreted in view of the observed lag. Preincubation with NAD and/or Mg increases the lag rate, with the latter having the greater effect, while preincubation with Mg and malate (or a malate analog) eliminates the lag. Data suggest a slow isomerization of E:NAD that is increased by addition of malate prior to NAD in the presence of Mg. The lag is also eliminated at low pH as a result of the overall rate being limited by the isomerization; that is, the isomerization is pH-dependent. Fumarate, an activator of the NAD-malic enzyme, when preincubated with enzyme also eliminates the lag, suggesting that the activator preferentially binds the isomerized form of the enzyme or increases the isomerization rate, or both. Stopped-flow data are corroborated by circular dichroism experiments. The unliganded enzyme is approximately 50% alpha-helix on the basis of secondary structural analysis. Binding of NAD and Mg exhibits a substantial change, with a further change observed upon binding the malate analog tartronate.