Iron distribution in Belgrade rat reticulocytes after inhibition of heme synthesis with succinylacetone

We have used succinylacetone (4,6-dioxoheptanoic acid), a specific inhibitor of delta-aminolevulinic acid dehydrase, to gain insight into the defect in iron metabolism in the Belgrade anemia. The Belgrade rat has an inherited microcytic, hypochromic anemia associated with poor iron uptake into developing erythroid cells. Succinylacetone inhibits heme synthesis, leading to nonheme iron accumulation in mitochondria and cytosol of normal reticulocytes. When succinylacetone is used to inhibit Belgrade heme synthesis, iron from diferric transferrin does not accumulate in the stromal fraction that contains mitochondria, nor does 59Fe accumulate in the nonheme cytosolic fraction. Hence, the defect in the Belgrade rat reticulocyte occurs in the endocytic vesicle or in a step subsequent to iron transit from the vesicle but before the nonheme cytosolic or mitochondrial iron fractions. Therefore, the mutation affects either the release of iron from transferrin or iron transport from the vesicle to the mitochondrion.     

Monoclonal antibody 1F5 (anti-CD20) serotherapy of human B cell lymphomas

Four patients with refractory malignant B cell lymphomas were treated with continuous intravenous (IV) infusions of murine monoclonal antibody (MoAb) 1F5 (anti-CD20) over five to ten days. Dose-dependent levels of free serum 1F5 were detected in all patients. Two patients had circulating tumor cells and in both cases 90% of malignant cells were eliminated from the blood stream within four hours of initiation of serotherapy. Antigenic modulation did not occur, and sustained reduction of circulating tumor cells was observed throughout the duration of the infusions. Serial bone marrow aspirations and lymph node biopsies were examined by immunoperoxidase and immunofluorescence techniques to ascertain MoAb penetration into extravascular sites. High doses (100 to 800 mg/m2/d and high serum 1F5 levels (13 to 190 micrograms/mL) were required to coat tumor cells in these compartments in contrast to the low doses that were adequate for depletion of circulating cells. Clinical response appeared to correlate with dose of MoAb administered with progressive disease (52 mg), stable disease (104 mg), minor response (1,032 mg), and partial response (2,380 mg) observed in consecutive patients. The patient treated with the highest 1F5 dose achieved a 90% reduction in evaluable lymph node disease, but the duration of this remission was brief (six weeks). This study demonstrates that high doses of 1F5 can be administered to patients with negligible toxicity by continuous infusion and that clinical responses can be obtained in patients given greater than 1 g of unmodified antibody over a ten-day period.     

Infusional cyclophosphamide, doxorubicin, and etoposide in human immunodeficiency virus- and human T-cell leukemia virus type I-related non-Hodgkin's lymphoma: a highly active regimen

Fourteen patients with poor-prognosis intermediate- to high-grade non- Hodgkin's lymphoma (NHL) associated with human immunodeficiency virus (HIV) infection (12 patients) or human T-cell leukemia virus type I (HTLV-I) infection (two patients) received cyclophosphamide 750 mg/m2, doxorubicin 50 mg/m2, and etoposide 240 mg/m2 administered as a continuous intravenous (IV) infusion over 4 days (infusional CDE); treatment was repeated every 28 or more days for up to six cycles. All HIV-positive patients had at least one poor prognostic feature, which included either extranodal disease (10 patients), Karnofsky performance status less than 70% (six patients), a CD4 count less than 100/microL (six patients), or a prior history of acquired immunodeficiency syndrome (AIDS; one patient). Both HTLV-I-positive patients had an elevated serum lactate dehydrogenase (LDH) level, a poor prognostic feature in that setting. Complete response (CR) occurred in 10 patients (71%; 95% confidence interval, 48% to 95%) and partial response (PR) occurred in three patients (21%), yielding an overall objective response rate of approximately 93%. The estimated Kaplan-Meier median survival was 17.4 months; seven of 12 HIV-positive patients are alive and disease-free with a median follow-up of 15 months (range, 7 to 24 months). Hospitalization was required after 19% of treatment cycles due to fever associated with granulocytopenia. Documented or suspected opportunistic infection occurred in five patients (36%), bacteremia occurred in three patients (21%), and candidemia occurred in one patient (7%). There was one treatment-related death attributable to disseminated aspergillosis. This pilot study suggests that infusional CDE may be a highly active regimen capable of producing durable remissions in a high proportion of patients with HIV-related NHL. Further study is required to confirm this observation.     

Value of beta 2-microglobulin level and plasma cell labeling indices as prognostic factors in patients with newly diagnosed myeloma

Beta 2-microglobulin (beta 2M) has been proposed as a prognostic factor in multiple myeloma (MM), but beta 2M levels are reported to correlate with other prognostic indicators such as stage and creatinine level. This study addressed the independent prognostic values of these and other variables, including plasma cell labeling indices (LI), in patients with newly diagnosed MM. beta 2M levels were measured with an enzyme-linked immunosorbent assay. LI were determined with a [3H]thymidine autoradiography method. By multivariate analysis and Kaplan-Meier survival analysis, the uncorrected beta 2M level remained the most significant prognostic factor after adjustment for age. Stage and creatinine level were closely related to beta 2M level and were no longer predictive of outcome after adjustment for age and beta 2M. Plasma cell LI varied independently of beta 2M level and remained predictive. A subset of patients with plasma-blastic myeloma had poor survival since beta 2M level and plasma cell LI were high. By using beta 2M level and LI, three risk groups were defined: low (beta 2M less than 4 micrograms/mL and LI less than 0.4%, median survival 48 months); intermediate (beta 2M less than 4 micrograms/mL and LI greater than or equal to 0.4%, median survival 29 months); and high (beta 2M greater than or equal to 4 micrograms/mL, median survival 12 months). Such grouping may better identify MM patients who might benefit from new treatment regimens.     

Monoclonal antibodies to human vitamin K-dependent protein S

Monoclonal antibodies to human protein S have been prepared using established hybridoma technology. One antibody was isolated that binds protein S only when Ca2+ is present; others bind antigen equally well in the presence or absence of EDTA. Other antibodies display a diminished affinity for protein S in the presence of EDTA. Purified immunoglobulins from cell lines displaying Ca2+ dependence were immobilized and used to purify protein S from fractions obtained by barium precipitation of citrated plasma, ammonium sulfate fractionation, and chromatography on diethylaminoethanol (DEAE)- Sephadex and dextran sulfate agarose. Essentially homogeneous protein S was isolated from the barium-citrate-adsorbed, 35% ammonium-sulfate- soluble proteins using a totally Ca2+-dependent antibody and EDTA elution. Protein S and several substances of higher mol wt were bound directly from plasma by a partially Ca2+-dependent antibody and were eluted partially with EDTA and NaCl and finally with NaSCN. The largest and most abundant of the high mol wt materials is likely protein S- complement C4b-binding protein complex. The immunoaffinity-isolated protein S was found to be indistinguishable from conventionally isolated protein S in terms of activity, sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) mobility, and by high- performance liquid chromatography (HPLC). These studies establish reagents that can probe the structure of protein S and isolate protein S in its free and complexed forms.     

Human herpesvirus 6: infection and disease following autologous and allogeneic bone marrow transplantation

Human herpesvirus 6 activity (HHV-6) was studied in 15 allogeneic and 11 autologous marrow transplantation patients. After transplantation, HHV-6 was isolated from the peripheral blood mononuclear cells of 12 of 26 patients (6 allogeneic and 6 autologous). All isolates were variant B. Eleven of 26 and 12 of 19 patients showed salivary shedding of HHV-6 DNA before and after transplantation, respectively. The antibody titer increased in 7 of 26 patients. Thus, 23 of 26 patients showed evidence of active HHV-6 infection either by virus isolation, salivary shedding, or increases in antibody titers. The fraction of saliva specimens positive in 19 patients was negatively associated with their antibody titers (P= .005). The proportion of cultures positive increased after transplantation (P = .007). Sinusitis was associated with HHV-6 isolation in autologous recipients (P= .002). In allogeneic patients, active human cytomegalovirus infection was associated with HHV-6 isolation (P = .04). No association was observed between HHV-6 infection and GVHD, pneumonia, delay in engraftment, or marrow suppression. Of the 120 clinical events analyzed in 26 patients, HHV-6 was defined as a probable cause of 16 events in 9 patients based on the propinquity of HHV-6 activity and the clinical event plus the absence of other identified causes of the event.     

Protective effect of prophylactic penicillin on splenectomized mice exposed to an aerosolized suspension of type III Streptococcus pneumoniae

Prophylactic penicillin has been recommended for use in asplenic patients and postsplenectomy patients. A laboratory model using aerosolized pneumococci has been devised to test the effectiveness of prophylactic penicillin in a manner analogous to human experience. There is increased mortality, over time, in asplenic mice exposed to aerosolized type III Streptococcus pneumoniae. One hundred twenty-one male Swiss mice (mean weight 26 g) were divided into four groups: splenectomized, sham-operated, splenectomized + penicillin, and sham- operated + penicillin. After 2 wk the four groups were exposed for 30 min to an aerosolized atmosphere of 2.4 x 10(9) colony-forming units of type III S. pneumoniae using a Tri-R model A-42 airborne infection apparatus. Penicillin was given at a daily intramuscular dosage of 40,000 units procaine penicillin G beginning 2 days prior to exposure and continuing through the third day after exposure. The splenectomized and sham-operated mice given penicillin showed significantly lower mortality (p less than 0.001) than mice not given penicillin.     

Role of myocardial scintigraphy with thallium-201 in the characterization of episodes of transient ischemia at rest. Clinical and electrocardiographic correlates

In the past ten years we studied 80 patients with angina at rest by 201-Thallium perfusion scintigraphy. According to ECG changes during episodes of transient ischemia at rest, the patients were divided into three groups. Thirty six patients showed transient ST segment elevation (Group 1); 33 ST segment depression (Group 2) and 11 normalization of negative T waves (Group 3). 201-TI scintigraphy was performed during spontaneous or ergonovine induced episodes of ischemia and at redistribution. Group 1 showed localized and severe perfusion defects, well correlated to the site of ECG changes. Group 2 showed more diffuse and less severe perfusion defects, less correlated to the site of ECG changes. Group 3 showed perfusion defects similar to those observed in Group 1 and associated in 54% with basal perfusion defects due to previous myocardial infarction. In conclusion: A) three main perfusion patterns are associated with the three types of ECG changes; B) relative to ECG, myocardial scintigraphy provides a more accurate definition of the site and extension of ischemia, particularly in Group 2 patients.     

Characteristics of bone marrow fibroblast colony-forming cells (CFU-F) and their progeny in patients with myeloproliferative disorders

Chronic myeloproliferative disorders (MPD) are clonal diseases of the pluripotent hematopoietic stem cell frequently associated with myelofibrosis (MF). There is only indirect evidence indicating that the increased deposition of collagen in bone marrow matrix is a secondary phenomenon. A liquid culture system for cloning and growing bone marrow fibroblasts has permitted us to approach more directly the understanding of the pathogenesis of myelofibrosis by comparing the biophysical, growth, and functional characteristics of fibroblasts from normals, MPD patients without MF, and those with MF. In patients with MF, marrow fibroblast colony (CFU-F) formation could not be studied; fibroblasts were grown from marrow explants. CFU-E from normals and MPD patients exhibited similar cell density distribution and similar cell sedimentation rates. These similarities contrasted sharply with the differences seen when the erythroid and granulocyte-macrophage progenitors were studied by the same methods. There was a marked light density shift and a rapidly sedimenting shift of MPD hematopoietic colony-forming cells. Marrow fibroblasts from MPD patients with and without MF displayed the same in vitro growth characteristics as fibroblasts from normals. Both types of fibroblasts exhibited anchorage and serum dependence, and contact inhibition of growth. Marrow fibroblasts were also characterized for the presence and distribution of fibronectin and collagen types by immunofluorescent staining using monospecific antibodies. Extracellular matrix, membrane-, and cytoplasm- associated fibronectin, type I, type III, and type V collagen showed a similar staining pattern in both normal and myelofibrotic marrow fibroblasts. Plasminogen-dependent fibrinolytic activity elicited from normal and myelofibrotic marrow fibroblasts were equivalent. Chromosomal analysis of hematopoietic cells and marrow fibroblasts from Philadelphia chromosome positive chronic myelocytic leukemia patients with and without MF showed that the Philadelphia chromosome was present only in hematopoietic cells. The results of these studies taken together demonstrate that bone marrow collagen-producing cells from MPD patients with and without MF behave in vitro as do those from normals. These findings support the hypothesis that that the marrow fibrosis observed in patients with MPD results from a reactive process rather than from a primary disorder affecting the marrow collagen-producing cells.     

Peripheral blood monoclonal plasma cells as a predictor of survival in patients with multiple myeloma [see comments]

The purpose of this study was to quantitate the number and labeling index of monoclonal plasma cells in the blood of patients with newly diagnosed multiple myeloma (MM) to learn if these values were independent prognostic factors for survival. Patients were candidates for this study if they had untreated myeloma requiring therapy, were evaluated at our institution between 1984 and 1993, and had a sample of blood analyzed with a sensitive immunofluorescence technique for monoclonal plasma cells and the blood B-cell labelling index (BLI). The % blood monoclonal plasma cells (%BPC) and the BLI were analyzed along with stage, marrow plasma cell LI, % marrow plasma cells, calcium, creatinine, albumin, beta-2-microglobulin, and C-reactive protein as univariate and multivariate factors for survival. Eighty percent of the 254 patients accrued to this study had monoclonal BPC detected. The median % BPC was 6% and 57% (144 of 254) of patients had a high number (> or = 4%). Patients with > or = 4% BPC had a median survival of 2.4 years vs 4.4 years for those with < 4% BPC (P < .001). The BLI was also prognostic (P = .008). In a multivariate analysis, the % BPC, age, albumin, stage, marrow plasma cell LI, and the BLI were independent factors for survival. The %BPC and the marrow plasma cell LI best separated the group into low, intermediate, and high risk myeloma with median survivals of 52, 35, and 26 months, respectively. Patients with high %BPC were less likely to have lytic bone disease from their MM (P = .002). The %BPC and the BLI are independent prognostic factors for survival and are useful in identifying patients as low, intermediate, and high risk. Clonal cells in the blood should be quantified in future clinical trials for myeloma.