Marrow transplantation from hepatitis C virus seropositive donors: transmission rate and clinical course

Bone marrow transplant recipients are at risk for acquiring hepatitis C infection from the donated marrow. Twelve patients who were hepatitis C virus (HCV) RNA-negative pretransplant received marrow from anti-HCV seropositive donors. HCV RNA was present in the sera of seven of these donors. After transplant, serial serum specimens were obtained from all marrow recipients for determination of HCV RNA and aminotransferase levels. All seven recipients of marrow from HCV RNA-positive donors were HCV RNA-positive after marrow infusion; none cleared virus from the serum. All five recipients of marrow from anti-HCV seropositive, HCV RNA-negative donors remained free of HCV RNA in serum up to day 100. Abnormal serum aminotransferases were common in both HCV RNA- negative and HCV RNA-positive marrow recipients. One HCV-infected recipient developed marked elevation in aminotransferases after immunosuppressive drugs were stopped. We conclude that the presence of HCV RNA in the serum of marrow donors is an accurate predictor of HCV infection in marrow recipients. The acute infection was subclinical in all patients. The long-term risk of chronic hepatitis C virus infection in these patients remains to be determined.     

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.     

Detection of clonal excess in lymphoproliferative disease by kappa/lambda analysis: correlation with immunoglobulin gene DNA rearrangement

Previous studies have suggested that analysis of the distribution of surface immunoglobulin light chain isotypes by flow cytometry provides evidence for monoclonality of B cell tumors and may detect populations of circulating tumor cells in patients with lymphoproliferative disease. We have used simultaneous flow cytometry and DNA restriction enzyme analysis on 58 samples of tissue and blood to determine whether lymphocyte populations detected by "kappa/lambda" analysis are indeed monoclonal. In greater than 90% of cases, abnormalities detected by flow cytometry correlated with monoclonal rearrangements of immunoglobulin genes as detected by Southern blot analysis. By analyzing tissue and blood from the same patients, we have also demonstrated that monoclonal circulating cells detected by flow cytometry reflect peripheral circulating tumor cells, since DNA from these cells shows the same immunoglobulin rearrangement as DNA from the original tumors in these patients. Although mixing studies suggested that DNA rearrangement studies were more sensitive than was flow cytometry in detecting minor populations of monoclonal lymphocytes, we found only one case in which this affected the diagnostic accuracy of the kappa/lambda analysis, with one notable exception, that of detection of a monoclonal proliferation of B cells that did not express surface immunoglobulin. The kappa/lambda test thus offers a powerful diagnostic tool in the evaluation of lymphoproliferative disease.     

Diagnostic and prognostic significance of Sezary cells in peripheral blood smears from patients with cutaneous T cell lymphoma

Blood smears stained with Wright-Giemsa were obtained from 124 patients with pathologically confirmed cutaneous T cell lymphoma (CTCL), 70 patients with various other cutaneous disorders, and ten healthy adult volunteers. These were examined in a blinded fashion for atypical lymphocytes with cerebriform nuclei (CLs), which were characterized further according to cell diameter. CLs, comprising up to 15% of lymphocytes in smears, were observed in 20% of the patients with benign dermatitis. CLs, comprising up to 89% of lymphocytes in smears, were found in 22%, 30%, 50%, and 96% of patients with patch, plaque, tumor, and erythrodermic CTCL, respectively. Large-diameter CLs (15 to 20 micron) were observed only in smears from patients with CTCL. Total CL counts above 15 per 100 lymphocytes and/or the presence of large CLs occurred in 33 of 49 (67%) patients with erythrodermic disease and in only two patients with other skin manifestations. Blood smears obtained at the time of cytogenetic studies indicated that a total CL count above 15% was the smear criterion that correlated best with the demonstration of a chromosomally abnormal malignant clone in the blood. The presence of large CLs per se, although also predictive of a malignant clone, was less useful. Multivariate survival analysis showed that the duration of disease before the blood smear and the proportion of large CLs within the total CL population were the covariates that correlated most significantly with survival. We speculate that the reduced survival of patients with increased proportions of large CLs in smears reflects the presence of polyploid malignant lymphocytes in the blood.     

Effect of phagocytosis on the reduced soluble sulfhydryl content of human granulocytes

The role of reduced glutathione in relation to hexose monophosphate shunt activity and peroxide detoxification has been well established in human erythrocytes. Less is known about the content of reduced glutathione in phagocytic leukocytes and the changes that occur during functional activity. We have measured the reduced sulfhydryl content of normal resting human granulocytes and of cells isolated from a patient with chronic granulomatous disease. Normal cells and those from the patient with chronic granulomatous disease contained similar concentrations of reduced sulfhydryls. Stimulation of a phagocytic response by incubation with opsonized zymosan particles resulted in prompt and nearly complete depletion of intracellular glutathione from normal granulocytes. This fall in reduced glutathione concentration was dependent on the phagocytic load. Exposure of chronic granulomatous disease granulocytes to a similar phagocytic load resulted in a slower and less complete fall in reduced glutathione. In normal cells, those from the chronic granulomatous disease patient, and those from an obligate carrier of the disease, the decrement in reduced glutathione during phagocytosis was correlated with oxidation of 14C-1-glucose and 14C-formate, nitroblue tetrazolium reduction, and the chemiluminescence phenomenon.     

High-dose chemotherapy with or without total body irradiation followed by autologous bone marrow and/or peripheral blood stem cell transplantation for patients with relapsed and refractory Hodgkin's disease: results in 85 patients with analysis of prognostic factors

Eight-five consecutive patients with relapsed or refractory Hodgkin's disease (HD) underwent high-dose chemotherapy or chemo/radiotherapy followed by autologous bone marrow (ABMT) and/or peripheral blood stem cell (PBSC) transplantation. Two preparative regimens were used. Twenty- two patients (26%) without prior radiation received fractionated total body irradiation (FTBI) 1,200 Gy in combination with high-dose etoposide (VP-16) 60 mg/kg and cyclophosphamide (CTX) 100 mg/kg. Sixty- three patients (74%) with prior radiotherapy received carmustine (BCNU) 450 mg/m2 instead of FTBI. The median age was 32 years (range, 16 to 56). The median number of prior chemotherapy regimens was three (range, 1 to 7). Forty-three patients (51%) received transplants in first relapse or second complete remission (CR), whereas 33 (39%) received transplants after second or subsequent relapse. All relapsed patients, except one, received conventional salvage chemotherapy and/or radiotherapy in an attempt to reduce tumor bulk before transplant. At the time of analysis in April 1994, fifty-seven patients (67%) are alive, including 44 (52%) in continuous CR, with a median follow-up for the surviving patients of 28 months (range, 7 to 66). Thirty patients (35%) relapsed at a median of 9 months (range, 1 to 43). Eleven patients (13%) died of transplant-related complications including veno- occlusive disease of the liver (VOD) in five, acute and late interstitial pneumonitis in three, graft failure in one, cerebral hemorrhage in one, and therapy-induced myelodysplasia (MDS)/acute leukemia in one patient. At a median follow-up of 25 months (range, 0.6 to 66), the cumulative probability of 2-year overall and disease-free survival (DFS) of all 85 patients is 75% (95% confidence interval [CI] 64% to 84%) and 58% (95% CI 47% to 69%), respectively. Three independent prognostic variables were identified by univariate analysis: number of prior chemotherapy regimens, prior radiotherapy, and extranodal disease at ABMT. Multivariate stepwise Cox regression identified the number of prior chemotherapy regimens as the only significant prognostic factor predicting for both relapse and DFS. There were no significant differences in the outcome of the treatment between the two preparative regimens. Our results confirm that high- dose therapy and ABMT is an effective therapy for patients with relapsed or refractory HD. Earlier transplantation is recommended before the development of drug resistance and end organ damage that results from repeated attempts of salvage therapy.     

Cell death and cytotoxic effects in YAC-1 lymphoma cells following exposure to various forms of mercury

The effects of 1 min-4 h exposures to four Hg compounds (mercuric chloride [HgCl2], methyl mercuric chloride [CH3HgCl], p-chloromercuribenzoate [p-CMB] and thimerosal [TMS; ethylmercurithiosalicylate]) on cell death, microtubules, actin, CD3 receptor expression, protein tyrosine phosphorylation (PTyr-P) and intracellular calcium ([Ca2+]i) levels were investigated in YAC-1 lymphoma cells using flow cytometry. YOPRO-1 (YP) and propidium iodide (PI) dye uptake indicated all forms of Hg tested were toxic at concentrations ranging from 25.8-48.4 microM, with two distinct patterns of effects. Early apoptosis was prolonged for CH3HgCl- and TMS-treated cells, with more than 50% remaining YP+/PI- after 4h. Both CH3HgCl and TMS induced complete loss of beta-tubulin fluorescence, indicative of microtubule depolymerization and inhibition of tubulin synthesis and/or beta-tubulin degradation, while F-actin fluorescence diminished to a lesser degree and only after loss beta-tubulin. CH3HgCl and TMS induced an almost immediate two-fold increase in CD3 fluorescence, with levels returning to baseline within minutes. With continued exposure, CD3 fluorescence was reduced to approximately 50% of baseline values. Both compounds also increased PTyr-P two- to three-fold immediately, with levels returning to baseline at 4h. Similarly, two- to three-fold increases in [Ca2+]i were noted after 1 min exposure. [Ca2+]i increased progressively, reaching levels five- to eight-fold greater than control values. In contrast, dye uptake was delayed with HgCl2 and p-CMB, although cell death proceeded rapidly, with almost all non-viable cells being late apoptotic (YP+/PI+) by 4h. p-CMB produced early reductions in F-actin, and after 4h, complete loss of F-actin with only partial reduction of total beta-tubulin was seen with both p-CMB and HgCl2. HgCl2 reduced CD3 expression and PTyr-P slightly within minutes, while p-CMB produced similar effects on CD3 only at 4h, at which time PTyr-P was increased two- to three-fold. Both compounds increased [Ca2+]i within minutes, though levels remained under twice the baseline concentration after 15 min exposure. With continued exposure, [Ca2+]i increased to levels two- to five-fold greater than control values. These findings indicate the two groups of Hg compounds may induce cell death by distinct pathways, reflecting interactions with different cellular targets leading to cell death.