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Obstructive lung disease (OLD) has been described as a significant complication after allogeneic bone marrow transplantation (BMT). The incidence of OLD in adults appears to be low (approximately 3%), but there is little data for children. We analyzed 89 consecutive pediatric allogeneic BMTs, > or = 1.5 years post-BMT, performed at British Columbia's Children's Hospital from 1980 to 1992 for evidence of OLD. Diagnosis of OLD was based on clinical findings (nonproductive cough, wheezing, and dyspnea with no evidence of infection), pulmonary function tests (FEV1 < 80% and FEF25–75% < 60% predicted), lung biopsy, and computed tomography scan. Sixty-seven of the 89 children evaluated survived > or = 90 days and were classified as at risk for OLD. Thirteen of 67 (19.4%), developed OLD, 3 of which were transient. The development of OLD was strongly associated with the following high-risk groups: chronic graft-versus-host disease (GVHD) (37.1% OLD), increased donor age, acute GVHD, and either mismatched related or matched unrelated donor transplants. No correlation was found with methotrexate prophylaxis for GVHD, total body irradiation, or cytomegalovirus reactivity in either donor or recipient and the development of OLD. Further analysis of only children with chronic GVHD showed that liver involvement by GVHD before the onset of OLD (57.9%) was the only other significant predictive factor. We observed an overall increased prevalence of OLD in children compared with that previously reported in adults. Further studies are required to confirm whether age is a risk factor for development of OLD after allogeneic BMT.

Polymerase chain reaction (PCR) amplification of the t(14;18) has been shown to be a highly sensitive method to detect minimal residual disease in patients with non-Hodgkin's lymphoma (NHL) whose tumors bear this translocation. The ideal tissue source to detect residual lymphoma would be from a previously involved lymph node. However, lymphoid tissue is rarely available once patients achieve complete remission. Although PCR amplification has been used to detect residual lymphoma cells in both bone marrow (BM) and peripheral blood (PB) of patients in complete remission, it is presently unknown whether BM and PB are equivalent tissue sources to detect residual disease. In the present study, we compared the clinical utility of the detection of residual lymphoma in both the BM and the PB of patients with advanced-stage non- Hodgkin's lymphoma before, at the time of, and after high-dose therapy and autologous BM transplantation (ABMT). The detection of residual lymphoma in either the BM or PB was associated with decreased disease- free survival. However, in the present study, 44% of patients who relapsed had no evidence of circulating lymphoma cells in their PB. At the time of BM harvest, PCR-detectable residual lymphoma cells were detected in 211 of 212 patients; although, in a subset of these patients analyzed, lymphoma cells were detected in the peripheral blood of only 49% of patients. When residual lymphoma cells within the autologous BM are infused into the patient these cells are rapidly detectable circulating in the PB in the patient. These cells continue to circulate during the immediate posttransplant period and be detectable in the PB in the majority of patients who are infused with marrow containing residual lymphoma. We conclude that BM is a more informative tissue source than PB in detecting minimal residual disease at the time of and after ABMT, and that contamination of PB early after ABMT appears to be the consequence of reinfusion of lymphoma cells within autologous marrow.

We postulated that defective generation of granulocyte colony- stimulating factor (G-CSF) by cells of newborn infants might underlie their deficiencies in upregulating neutrophil production and function during bacterial infection. To test this, we isolated monocytes from the blood of preterm neonates, term neonates, and adults and, after stimulation with various concentrations of interleukin-1 alpha (IL-1 alpha) or lipopolysaccharide (LPS), quantified G-CSF concentrations in cell supernatants and G-CSF mRNA in cell lysates. When stimulated with plateau concentrations of IL-1 alpha for 24 hours, G-CSF concentrations were higher in supernatants of adult cells (8,699 +/- 5,529 pg/10(6) monocytes) than in those from term infants (2,557 +/- 442 pg, P < .05) or from preterm infants (879 +/- 348 pg, P < .05 v adults). When stimulated with plateau concentrations of LPS, supernatants of monocytes from preterm neonates had less G-CSF than did those from term neonates or adults. G-CSF mRNA content was low in cells from preterm infants, higher in those from term infants, and highest in those from adults. On the basis of the in vitro studies, we speculated that serum G-CSF concentrations might be less elevated in neutropenic neonates than in neutropenic adults. Indeed, serum concentrations were relatively low in all nonneutropenic subjects; 92 +/- 34 pg/mL (mean +/- SEM) in 10 preterm neonates, 114 +/- 21 pg/mL in 16 term neonates, and 45 +/- 13 pg/mL in 11 healthy adults. Serum concentrations were not elevated in 7 neutropenic neonates (39 +/- 17 pg/mL) but were in 8 neutropenic adults (2101 +/- 942 pg/mL, P < .05 v healthy adults). Other studies suggested that the lower G-CSF production in neonates is not counterbalanced by a heightened sensitivity of G-CSF--responsive progenitors to G-CSF. Therefore, we speculate that newborn infants, particularly those delivered prematurely, generate comparatively low quantities of G-CSF after inflammatory stimulation, and that this might constitute part of the explanation for their defective upregulation of neutrophil production and function during infection.

Although molecular biologic techniques can now detect minimal numbers of residual cancer cells in patients in complete clinical remission, the clinical significance of minimal residual disease has never been conclusively established. If the detection of minimal residual disease predicts which patients will relapse, then therapy could be altered based upon the detection of these cells. The t(14;18) can be detected by polymerase chain reaction (PCR) amplification in 50% of patients with B-cell non-Hodgkin's lymphoma and allows detection of one lymphoma cell in up to 1 million normal cells. To determine the clinical significance of the detection of minimal residual lymphoma cells in the bone marrow (BM) PCR amplification was used to detect the presence of residual lymphoma cells after autologous BM transplantation (ABMT) in serial BM samples from 134 patients with B-cell lymphoma in whom a bcl- 2 translocation could be detected. PCR analysis was performed on a total of 542 BM samples obtained while these patients were in complete remission. Disease-free survival was markedly increased in patients with no PCR-detectable lymphoma cells in the marrow compared with those in whom residual lymphoma cells were detected (P < .00001), and the presence of detectable lymphoma cells was associated with a 48-fold increase in the risk of relapse. Of the 77 patients (57%) with no PCR- detectable lymphoma cells in their most recent BM sample, none have relapsed. In contrast, all 33 patients (25%) who have relapsed had PCR- detectable lymphoma cells detected in their BM before clinical relapse occurred. In 19 patients (14%), residual lymphoma cells in the BM were detected early following transplantation and subsequently were no longer detectable, although these patients received no further therapy. In these patients, residual lymphoma cells may already have been irreversibly damaged by the high-dose therapy or an endogenous immune mechanism may be capable of eliminating residual lymphoma cells in some patients. Therefore, although the detection of minimal residual disease by PCR following ABMT in patients with lymphoma identifies those patients at high risk of relapse, the presence of residual minimal disease early after transplantation may not be associated with poor prognosis in a small subset of patients. Confirmatory studies will be required to determine more definitively the role of minimal disease detection to identify which patients require additional therapy.

The product of the c-raf-1 proto-oncogene, Raf-1, is a 74,000-dalton cytoplasmic serine/threonine protein kinase that has been implicated as an intermediate in signal transduction mechanisms. In the human factor- dependent myeloid cell line MO7, both granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-3 (IL-3) were found to induce rapid, dose-dependent phosphorylation of Raf-1, which resulted in altered Raf-1 mobility in sodium dodecyl sulfate-polyacrylamide gels. The increase in phosphorylation was due primarily to an increase in phosphoserine, with only a minor component (less than 2%) of phosphotyrosine. PMA (12-phorbol 13-myristic acid) also induced Raf-1 phosphorylation in MO7 cells, but the resulting alteration in electrophoretic mobility was different than that observed after GM-CSF or IL-3. GM-CSF and IL-3 rapidly and transiently increased Raf-1 kinase activity using Histone H1 as a substrate in an immune complex kinase assay in vitro. These results suggest that phosphorylation of Raf-1 could play a role in some aspect of GM-CSF and IL-3 signal transduction.

Induction of mitochondrial permeability transition (MPT) and cytosolic translocation of cytochrome C are considered essential components of the apoptotic pathway. Hence, there is the realization that mitochondrial-specific drugs could have potential for use as chemotherapeutic agents to trigger apoptosis in tumor cells. Recently, we showed that photoproducts of merocyanine 540 (pMC540) induced tumor cell apoptosis. In this study, we focused on identifying mitochondrial-specific compounds from pMC540 and studied their apoptotic potential. One purified fraction, C5, induced a drop in mitochondrial transmembrane potential and cytosolic translocation of cytochrome C in HL60 human leukemia cells. Moreover, the addition of C5 to purified rat liver mitochondria induced MPT as indicated by mitochondrial matrix swelling, which was completely inhibited by cyclosporin A, an inhibitor of the inner-membrane pore. Supernatant of C5-treated mitochondria showed a dose-dependent increase in cytochrome C, which was also inhibited in the presence of cyclosporin A, strongly indicating a direct effect on the inner-membrane pore. Despite the strong mitochondrial reactivity, C5 elicited minimal cytotoxicity (less than 25%) against HL60 leukemia and M14 melanoma cells because of inefficient caspase activation. However, prior exposure to C5 significantly enhanced the apoptotic response to etoposide or the CD95 receptor. Thus, we demonstrate that MPT induction and cytochrome C release by the novel compound C5, in the absence of effective caspase activation, is insufficient for triggering efficient apoptosis in tumor cells. However, when used in combination with known apoptosis inducers, such compounds could enhance the sensitivity of tumor cells to apoptosis. (Blood. 2000;95:1773-1780)

One hundred and sixty eight adult patients with B-cell non-Hodgkin's lymphoma (NHL) and other hematologic malignancies who underwent autologous or allogeneic bone marrow transplantation (BMT) were investigated for the subsequent development of hemolytic-uremic syndrome (HUS). All patients were conditioned with cyclophosphamide and total body irradiation. When examined at 3-month intervals for the first year post-BMT, all patients had uniform measurements of hematocrit (Hct) and serum creatinine. Sixteen patients who initially exhibited Hct and creatinine values that were normal range for the BMT populations developed a sudden decrease in Hct and increase in creatinine between 3 and 11 months post-BMT and fulfilled the clinical and laboratory criteria for HUS. None of these patients had known active cytomegalovirus infection, graft-versus-host disease, or cyclosporine administration. The degree of decrease in Hct and creatinine elevation ranged from solely laboratory abnormalities to a clinically significant syndrome. Twelve of the 16 patients developed acute clinical complications of congestive heart failure, hypertension (HTN), or peripheral edema. Twelve patients required red blood cell support, whereas only four patients required platelet transfusions. Both hemolytic anemia and thrombocytopenia have resolved in virtually all cases. At a mean follow up of 18 months postdiagnosis, creatinine elevations have persisted along with HTN. All patients have survived without life-threatening long-term sequelae. With the increasing use of BMT as a curative modality for patients with hematologic malignancies, it becomes important to prospectively monitor patients for the development of HUS and its potential long-term impact on renal function.

Thymocytes from normal mice strains as well as from genetically determined stem cell defective W/Wv anemic mice were cocultured with syngeneic (or congeneic) bone marrow cells. We assayed these cocultures for the proliferation of erythroid progenitor cell types (BFU-E and CFU- E) using the plasma clot technique. Results indicate that when concentrations of thymocytes were lower than bone marrow cells, significant suppression of erythroid growth was observed. However, when the concentration of thymocytes exceeded that of the bone marrow cells in culture (greater than 1:1), significant enhancement of erythroid growth was demonstrated. The W/Wv anemic bone marrow appears to respond to this interaction by enhancement at all concentrations of added normal thymocytes. The regulatory functions observed can be diminished by treatment of the thymocytes in vitro with anti-theta serum plus complement. Thus, we establish regulatory functions for anti-theta- sensitive regulatory cells (TSRC) with both positive (enhancement) and negative (suppression) components.

Platelet aggregation requires the binding of adhesive proteins such as fibrinogen to the heterodimer of membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa). Human erythroleukemia (HEL) cells synthesize both GPIIb and GPIIIa. Using poly(A+) RNA purified from HEL cells, we constructed a cDNA library in the lambda gt10 phage vector. This library was screened with a 38mer oligonucleotide derived from a platelet GPIIIa peptide, and three overlapping cDNAs were isolated. The three inserts encompassed 3.5 kilobases (kb), including the entire coding region of mature GPIIIa (2,286 basepairs, bp) and 1.3 kb of 3′ untranslated sequence. All 222 residues determined directly from platelet GPIIIa tryptic peptides exactly matched the HEL cell-deduced amino acid sequence. The HEL cell sequence matched a previously reported endothelial cell cDNA sequence except for eight nucleotides. Five of these nucleotide differences were silent changes consistent with genetic polymorphisms. The other three differences resulted in changes in the deduced amino acid sequence of GPIIIa; reexamination of the endothelial cell cDNA sequence in these three areas revealed that it is actually identical to the HEL cell sequence. The virtual identity of the endothelial and HEL cell cDNA sequences provides direct evidence that GPIIIa is a subunit common to cell-adhesion receptors present in more than one cell type. We localized the gene for GPIIIa to chromosome 17, the same chromosome to which we had previously mapped the gene for GPIIb.

Bone marrow and peripheral blood cells from three newborns with Down's syndrome and transient myeloproliferative disorders were cultured in vitro. In the methylcellulose semiliquid system, normal colony formation with maturation and differentiation into granulocytes and monocyte-macrophages were observed in all three patients. This is different from the growth pattern usually seen in acute nonlymphocytic leukemia. A maternal serum inhibitor of both normal allogeneic GM-CFU and blast cell growth was also demonstrated, but its role in pathogenesis is uncertain. Normal in vitro granulopoiesis may help to differentiate the transient myeloproliferative syndrome from congenital leukemia in newborns with Down's syndrome.

Monoclonal antibody OKT11 was found to compete with sheep red blood cells for binding sites on human lymphocytes. Preincubation of lymphocytes with OKT11 eliminated E-rosette formation. In a study of 142 peripheral blood samples ranging from 1% to over 90% E-rosette- positive cells, comparison to the percent OKT11-positive cells yielded a correlation coefficient of 0.93. In normal donors, subsets of OKT11+ cells were identified using two-color immunofluorescent staining methods with OKT3, OKT4, and OKT8. On the average, approximately 13% of OKT11+ lymphocytes were OKT3- and 13% of OKT11+ lymphocytes were OKT4- and OKT8-. Based on our double antibody fluorescence intensity data, low antigen density OKT11+ lymphocytes were OKT3-. OKT4+ and OKT8+ lymphocytes in normal peripheral lymphocytes have similar OKT11 antigen density.

Tartrate-resistant acid phosphatase (TRAcP) is used as a marker for osteoclasts, which are believed to be derived from phagocytic cells or phagocyte stem cell precursors. To further investigate the relationship between monocytic phagocytes and osteoclasts, acid phosphatase (AcP) activity was measured by three different techniques in human peripheral blood monocytes, monocyte-derived macrophages, and the U937 cell line. We found that cytochemistry and gel electrophoresis led to similar results, but that the colorimetric assay was inconsistent. Normal human peripheral monocytes expressed both tartrate-sensitive and -resistant AcP. In culture these cells formed polykaryons and expressed TRAcP activity that was further identified as an isoenzyme associated with bone tissue. In contrast, the U937 cells did not express TRAcP activity as measured by gel electrophoresis. Both U937 cells and monocytes possess material that interferes with interpretation of the colorimetric assay of AcP. The presence of TRAcP in monocyte-derived macrophages further supports the relationship between phagocytic cells and bone osteoclasts.