Failure in generating hemopoietic stem cells is the primary cause of death from cytomegalovirus disease in the immunocompromised host.

We have shown in a murine model system for cytomegalovirus (CMV) disease in the immunocompromised host that CMV infection interferes with the earliest detectable step in hemopoiesis, the generation of the stem cell CFU-S-I, and thereby prevents the autoreconstitution of bone marrow after sublethal irradiation. The antihemopoietic effect could not be ascribed to a direct infection of stem cells. The failure in hemopoiesis was prevented by adoptive transfer of antiviral CD8+ T lymphocytes and could be overcome by syngeneic bone marrow transplantation. CD8+ T lymphocytes and bone marrow cells both mediated survival, although only CD8+ T lymphocytes were able to limit virus multiplication in host tissues. We concluded that not the cytopathic effect of virus replication in host tissues, but the failure in hemopoiesis, is the primary cause of death in murine CMV disease.

Modulation of p145c-kit function in cells of patients with acute myeloblastic leukemia.

The function of the steel factor receptor, p145c-kit, in patient-derived acute myeloblastic leukemia (AML) cells was investigated. Steel factor stimulation of AML cells coexpressing p145c-kit and the progenitor cell antigen CD34 resulted in complete receptor down-regulation, a marked decrease of CD34 antigen expression, and the induction of the granulocyte lineage antigen CD15. These changes in surface marker expression paralleled morphological differentiation to granulated blasts and promyelocytes. Interestingly, the same phenotype was achieved by IL-3 stimulation of AML cells. p145c-kit extracellular domain-specific antibodies had either blocking or enhancing effects on ligand binding, receptor phosphorylation and down-regulation, and induction of cell proliferation. Correlations of these phenomena with distinct effects of antibody stimulation on cell substrate phosphorylation provide clues for the dissection of the p145c-kit signal and the analysis of its relevance for AML.

Dose escalation of cyclophosphamide in patients with breast cancer: consequences for pharmacokinetics and metabolism.

PURPOSE: The alkylating anticancer agent cyclophosphamide (CP) is a prodrug that undergoes a complex metabolism in humans producing both active and inactive metabolites. In parallel, unchanged CP is excreted via the kidneys. The aim of this study was to investigate the influence of dose escalation on CP pharmacokinetics and relative contribution of activating and inactivating elimination pathways. PATIENTS AND METHODS: Pharmacokinetics of CP were assessed in 12 patients with high-risk primary breast cancer who received an adjuvant chemotherapy regimen that included four courses of conventional-dose CP (500 mg/m2 over 1 hour every 3 weeks) followed by one final course of high-dose CP (100 mg/kg over 1 hour). Plasma concentrations of CP were analyzed by high-performance liquid chromatography (HPLC), 24-hour urinary concentrations of CP, and its inactive metabolites (carboxyphosphamide, dechloroethylcyclophosphamide [dechlorethylCP], ketocyclophosphamide [ketoCP]) were determined by 31-phosphorus-nuclear magnetic resonance (31P-NMR)-spectroscopy. RESULTS: There was no difference in dose-corrected area under the concentration-time curve (AUC) (216 v 223 [mumol.h/[mL.g]), elimination half-life (4.8 v 4.8 hours), systemic clearance (79 v 77 mL/min) and volume of distribution (0.49 v 0.45 L/kg) of CP between conventional- and high-dose therapy, respectively. However, during high-dose chemotherapy, we observed a significant increase in the renal clearance of CP (15 v 23 mL/min; P < .01) and in the formation clearance of carboxyphosphamide (7 v 12 mL/min; P < .05) and dechloroethylCP (3.2 v 4.2 mL/min; P < .05), whereas metabolic clearance to ketoCP remained unchanged (1.3 v 1.2 mL/min). Consequently, metabolic clearance to the remaining (reactive) metabolites decreased from 52 to 38 mL/min (P < .001). The relative contribution of the different elimination pathways to overall clearance of CP demonstrated wide interindividual variability. CONCLUSION: Overall pharmacokinetics of CP are apparently not affected during eightfold dose escalation. However, there is a shift in the relative contribution of different clearances to systemic CP clearance in favor of inactivating elimination pathways, thereby indicating saturation of bioactivating enzymes during dose escalation. Besides individual enzyme capacity, hydration and concomitant medication with dexamethasone modulated CP disposition.

The product of the proto-oncogene c-kit (P145c-kit) is a human bone marrow surface antigen of hemopoietic precursor cells which is expressed on a subset of acute non-lymphoblastic leukemic cells.

A monoclonal antibody (17F11) was raised by immunization of a Balb/c mouse with leukemic blasts from a patient with acute non-lymphocytic leukemia (ANLL). This antibody recognizes most leukemic blasts of myeloid but not of lymphoid lineage and no peripheral blood cells. By screening NIH-3T3 fibroblasts transfected with the human proto-oncogene c-kit (NIH-3T3/hckit) it could be shown that 17F11 specifically recognizes the gene product P145c-kit. Immunofluorescence analysis on normal hemopoietic cells revealed that 17F11 weakly stains 1-3% of bone marrow mononuclear cells (BMMNC). By FACS sorting and colony assays it could be shown that granulocyte--macrophage progenitor cells could be enriched 10-20-fold, granulocyte progenitors 50-80-fold, and erythroid and multipotential progenitor cells 15-20-fold, in the 17F11 positive fraction. Double fluorescence analysis revealed that P145c-kit is co-expressed on 40-60% of the CD34 positive BMMNC. Finally, these data show that P145c-kit is expressed on blast cells from most patients with ANLL (26/30) and chronic myeloid leukemia in blast crisis (7/9), but is absent on blasts from patients with acute lymphoblastic leukemia expressing the T-, B-lineage, or common ALL phenotypes.

Endoglin is expressed on a subpopulation of immature erythroid cells of normal human bone marrow.

A monoclonal antibody (1G2) was raised by immunization of a Balb/c mouse with the leukemic blasts from a patient suffering from chronic myelogenous leukemia blast crisis (CML-BC). Sequential immunoprecipitation of the protein from human umbilical vein endothelial cells with 1G2 and the endoglin-specific monoclonal antibody 44G4 indicated that both antibodies react with the same molecule, a homodimer of molecular mass 180,000. This protein was first identified on acute lymphoblastic leukemia and was shown to be primarily associated with endothelial cells. In addition, 1G2 and 44G4 identified the same subpopulation of human bone marrow mononuclear cells (BMMNC), as established by two colour immunofluorescence analysis. By cell sorting and colony assays it could be demonstrated that endoglin is not expressed on hemopoietic precursor cells (CFU-G, CFU-GM, CFU-GEMM, BFU-E). May-Grünwald-Giemsa staining of sorted BMMNC revealed that 1G2 recognized immature proerythroblasts and double-fluorescence analysis showed that endoglin is present on a subset of glycophorin A-positive BMMNC. 1G2 was not reactive on bone marrow B-cells (CD19, CD20), T-cells (CD3, CD7), natural killer cells (CD56), myeloid cells (CD13, CD14, CD15, CD33), and on CD34-positive cells. Endoglin contains an arginine-glycine-aspartic acid sequence, a feature generally associated with extracellular matrix proteins which interact with integrins. It is suggested that proerythroblasts may utilize endoglin to interact with integrins in cell-cell adhesion events in the stromal or hemopoietic compartment of the bone marrow.

Intracellular cytosine arabinoside accumulation and cytosine arabinoside triphosphate formation in leukemic blast cells is inhibited by etoposide and teniposide.

Cytosine arabinoside (ara-C) is one of the most active compounds in the treatment of acute leukemias. In the majority of current protocols ara-C is combined with other cytotoxic agents in an attempt to increase antileukemic activity. The present study investigated the impact of etoposide, teniposide, amsacrine, mitoxantrone, anthracyclines, and asparaginase on the cellular accumulation of ara-C and its intracellular metabolism in order to provide a better rationale for combination therapy. Intracellular accumulation and phosphorylation of ara-C were determined in peripheral blast cells from twenty patients with acute leukemias after exposure to 1 and 10 mumol/l ara-C alone and after preincubation with 1 and 10 micrograms/ml etoposide, 10 and 100 micrograms/ml teniposide, 10 mumol/l amsacrine, 500 ng/ml mitoxantrone (or daunorubicin or doxorubicin) or 10 mumol/l asparaginase. Ara-C accumulation at 10 mumol/l was decreased by 1 microgram/ml etoposide (67 +/- 18% of control), 10 micrograms/ml etoposide (30 +/- 22%), 10 micrograms/ml teniposide (12 +/- 23%), 100 micrograms/ml teniposide (10 +/- 18%), and amsacrine (51 +/- 21%). Intracellular ara-CTP formation was determined at an extracellular concentration of 10 mumol/l and preincubation with these drugs. The intracellular formation of ara-CTP was decreased by 1 microgram/ml etoposide (77 +/- 15% of control), 10 micrograms/ml etoposide (32 +/- 22%), 10 micrograms/ml teniposide (10 +/- 9%), 100 micrograms/ml teniposide (0 +/- 0%), but not by amsacrine. These data indicate that prior exposure to etoposide and teniposide influence ara-C metabolism and possibly cytotoxicity, and thus should not immediately precede ara-C administration in clinical trials.

Microimmunohistology of hematopoietic colonies.

A new method is described which allows immunohistological analysis of hematopoietic colonies grown in methylcellulose. With the use of a specific embedding technique, we prepared cryostat sections that facilitated multicell marker studies as well as the evaluation of the in situ organization of individual colonies.

Inhibition of in vitro hematopoiesis by hepatitis A virus.

Inoculation of human bone marrow with hepatitis A virus (HAV) resulted in a dose- and duration-of-incubation-dependent suppression of hematopoietic progenitor (CFU-GM, BFU-E, CFU-Mix) growth in vitro. Monocytic progenitors appeared to be least affected. While HAV inactivation by heat or beta-propiolactone and neutralization by specific antibodies completely abrogated hematopoietic inhibition, depletion of adherent bone marrow cells, and enrichment of progenitors did not alter the pattern of suppression, which also seemed to be independent of HuIFN-alpha, -beta, -gamma, and TNF. These findings support the concept that direct infection of progenitor cells by HAV may be responsible for hematologic changes commonly seen during early phases of infectious hepatitis and possibly for some cases of bone marrow failure.

Selective inhibition of interleukin 2 but not of interferon-gamma, tumour necrosis factor-alpha or granulocyte/macrophage colony stimulating factor secretion by human helper T cell clones after antigen-driven tolerisation.

Alloreactive human T-helper cell clones chronically exposed to specific antigen can become tolerised, i.e. lose their autocrine proliferative capacity and no longer undergo antigen-driven clonal expansion. To study parameters of possible clonal anergy in such cells, lymphokine secretion before and after tolerisation was measured. 10/10 CD4+ clones were found to secrete interleukin 2 (IL-2), interferon gamma, tumour necrosis factor alpha and granulocyte/macrophages colony stimulating factors. Tolerised clones which had lost autocrine proliferative capacity and had lost the ability to secret IL-2 nevertheless retained the ability to secrete the other lymphokines. These results thus show a highly selective lesion in the noncoordinate regulation of lymphokine secretion by tolerised cells.

Regulation of normal myelopoiesis and chronic myelogenous leukaemia cell proliferation through a non-cytotoxic mechanism by a gamma/delta T cell clone.

Regulatory effects on myelopoiesis and myelogenous leukaemia cell proliferation mediated by a human T cell clone (TCC) carrying a gamma/delta receptor have been studied. MHC-unrestricted cytotoxicity could be induced in this clone by culture with IL-2 but not IL-4. Increasing concentrations of IL-2 resulted in increased lysis of natural killer (NK)-susceptible target cells but lysis of NK-resistant targets could not be induced. Moreover, cytotoxicity on fresh chronic myeloid leukaemia cells was not measurable even after culture with 1000 U/ml IL-2. However, NK-resistant targets could be lysed when anti-receptor antibodies (OKT3 or TCR-delta 1) were added to the assay. Clone 290-2 cells secreted lymphokines potentially inhibitory for myelopoiesis (TNF-alpha, IFN-gamma), and their supernatants could inhibit optimally stimulated granulocyte/macrophage colony formation by normal bone marrow. Moreover, 290-2 cells prevented the consistently observed IL-3-stimulated enhancement of proliferation of CML cells, although even IL-3-pretreated leukaemic cells were still resistant to lysis by this clone. Thus, cells of this type, even when not directly cytolytic, could have a role in the regulation of myeloid cell growth.

Mutagenic activity of marihuana smoke condensates.

Smoke condensates prepared from marihuana cigaretts were mutagenic in strain TA98 of the Ames Salmonella/microsome test, a short-term bioassay which estimates the mutagenic and carcinogenic potential of some chemicals. The mutagens in marihuana smoke condensates required liver enzymes to be activated. The specific mutagenic activity of marihuana smoke condensates were similar to that of tobacco smoke condensates prepared from American cigarettes. Fractionation studies of the marihuana smoke condensates showed that basic components accounted for 76% of the recovered mutagenic activity.

Relative cloning efficiencies and long-term propagation capacity for T cell clones of highly purified natural interleukin 2 compared to recombinant interleukin 2 in man.

Highly purified (HP) natural (n) and recombinant (r) interleukin 2 (IL 2) have been compared for their ability to support clonal outgrowth and long-term clonal propagation of alloactivated human T lymphocytes. The frequency of outgrowth of T cell colonies was as high as 1:2 to 1:4 when HPnIL 2 was employed for limiting dilution cloning. Separated CD4+ and CD8+ activated populations produced similar cloning efficiencies. Recloning of established CD4+ lines in HPnIL 2 suggested that a clonable cell frequency of 1:2-1:4 was the maximum possible that could be achieved in this system. In contrast, purified rIL 2 allowed outgrowth of only 1:10-1:20 cells from alloactivated populations. Again, CD4+ and CD8+ fractions generated similar cloning efficiencies. In terms of the fraction of derived clones, which could be propagated to greater than 1 X 10(6) and greater than 1 X 10(8) cells, nIL 2 again proved superior to rIL 2. In either source of IL 2, the proportion of CD4+ clones which could be extensively propagated was greater than in the CD8+ population. Surprisingly, although the addition of PHA to these lectin-free IL 2 preparations reduced the frequency of clonable cells, the proportion of clones which could be extensively propagated was increased. These results suggest that nIL 2, consisting of three differently glycosylated molecular species, may be preferable to rIL 2, which consists only of a single non-glycosylated species, for the cloning and long-term propagation of human T cells. These results may have some bearing on the choice of rIL 2 versus nIL 2 for therapeutic applications.

Plasma levels of granulocyte colony-stimulating factor in patients after allogeneic bone marrow transplantation for chronic myeloid leukemia correlate with engraftment of transplanted marrow.

Granulocyte colony-stimulating factor (G-CSF) is considered to play a pivotal role in hemopoietic regulation. Its pharmacological application is reported to shorten chemotherapy-induced neutropenia as well as time to engraftment in patients after bone marrow transplantation (BMT). In order possibly to establish further rationale for G-CSF treatment strategies in patients undergoing BMT, we evaluated G-CSF plasma levels of 89 patients after allogeneic BMT for chronic myeloid leukemia (CML). EDTA anti-coagulated plasma samples were collected starting on day -1 (before grafting) and thereafter twice weekly for four consecutive weeks. G-CSF levels were estimated by enzyme immunoassay. Patients with late (> 30 days) bone marrow engraftment had consistently higher G-CSF levels at day +1 (after grafting) compared to patients with early (< or = 30 days) engraftment, while all patients had low plasma levels on day -1/0. Mean G-CSF plasma levels and time to engraftment were correlated (r = 0.79). In univariate analyses, high G-CSF levels at days +1, +4, +7, +10 and several clinical variables (such as TBI, unrelated donor transplant, state of disease) were predictive of late engraftment. Further analysis by multivariate Cox regression resulted in the following predictive model: high G-CSF plasma levels at day +7 and +10 (after grafting), in combination with a blastic phase of the disease were highly predictive of late engraftment. The significantly higher G-CSF levels in patients with impaired engraftment may reflect early compensating mechanisms of the hemopoietic system, which should be investigated further.

Melphalan-resistant multiple myeloma: results of treatment according to the M-2 protocol.

A group of 46 patients with melphalan-resistant multiple myeloma was treated according to the M-2 protocol with melphalan, prednisolone, BCNU, cyclophosphamide, and vincristine. According to the Salmon and Durie classification, four patients had stage II A; 36, stage III A; and six, stage III B disease. Treatment resulted in five patients (11%) entering remission, while 21 (46%) had stable and 20 (43%) had progressive disease. The median survival for all patients was 12.5 months, patients in remission surviving longer (median 46 months) than those with stable disease (median 15.4 months) or progressive disease (median 6.9 months). Compared with other treatment regimens used in melphalan-resistant myeloma, the remission rate is low but the median survival exceeds that reported by most other authors.

Fractionated administration of high-dose cyclophosphamide: influence on dose-dependent changes in pharmacokinetics and metabolism.

PURPOSE: The alkylating agent cyclophosphamide (CP) is a prodrug that is metabolized to both cytotoxic and inactive compounds. We have previously shown that following dose escalation from conventional-dose (CD) to high-dose (HD) levels; the fraction of the dose cleared by bioactivation is significantly decreased (66% versus 48.5%) in favor of inactivating elimination pathways when the HD is given as a single 1-h infusion. Based on the concept of bioactivating enzyme saturation with increasing doses, we investigated the influence of fractionated application of HD-CP on dose-dependent changes in metabolism. PATIENTS AND METHODS: Plasma concentrations of CP (measured by high-performance liquid chromatography, HPLC) and urinary concentrations of CP and its major metabolites (quantified by [31P]-nuclear magnetic resonance spectroscopy; [31P]-NMR spectroscopy), were determined in four patients with high-risk primary breast cancer who received adjuvant chemotherapy including both CD-CP (500 mg/ m2 infused over 1 h) and split HD-CP (50 mg/kg infused over 1 h on each of 2 consecutive days (d): d1 and d2. RESULTS: (Data are given as mean values for CD and d1/d2 of HD, respectively). Systemic clearance (CL) of CP was similar during CD and d1 of HD, but significantly increased on d2 of HD (CL: 83 and 78/115 ml/min; P < 0.01 for d1 versus d2). The latter was translated into an increase in formation CL of both active (+ 16.4 ml/min) and inactive metabolites (+ 17.6 ml/ min) and reflects autoinduction of metabolism. As compared with CD-CP, no statistically significant decrease was observed in the relative contribution of bioactivation CL to overall CL during both days of HD (63% versus 57%/53%). Recovery of intact CP in 24-h urine corresponded to 24%, 29%, 22% of the dose (P < 0.05 for d1 versus d2 of HD). CONCLUSIONS: Following dose escalation of CP, dividing the high dose over 2 days instead of one single infusion may favorably impact the metabolism of CP in terms of bioactivation. In addition, on day 2 of a split regimen, renal elimination of CP is decreased, which implies that more drug is available for metabolism.

Hemophagocytic syndrome. Differential diagnostic aspects in a case of well-differentiated malignant histiocytosis.

Diagnosis of malignant histiocytosis (MH) often resembles the solving of an intricate puzzle consisting of clinical symptoms such as lymphadenopathy, splenomegaly, fatigue, fever, and rapid progression, and hematopathological findings such as the presence of atypical histiocytes, especially in blood and bone marrow smears. The lack of one or more of these criteria may greatly impede diagnosis, as in the case of a 45-year-old male with an unusual hematopathological manifestation of MH. The major clinical findings included panhemocytopenia, splenomegaly, and signs of liver dysfunction with severe jaundice. During life, a definite diagnosis could not be established. Histological and cytological evaluation of the spleen following splenectomy revealed a marked increase in histiocytes/macrophages with pronounced hemophagocytosis. These findings were interpreted as a (benign) hemophagocytic syndrome, possibly related to a viral infection. Extensive serological investigations, however, furnished no evidence of a so-called virus- or infection-associated hemophagocytic syndrome. The patient died 5 months after the onset of disease with symptoms of progressive liver failure. Meticulous histological examination of bone marrow revealed a few patchy tumorous infiltrates consisting of dense pleomorphic histiocytes. Thus, a diagnosis of MH was established. This case of MH was unusual with particular regard to its pronounced hemophagocytosis, slight cytological atypia of the histiocytes, and absence of infiltration of lymph nodes.

[Renal involvement in malignant lymphoma: significance of imaging technics for determining spread]. / Der Nierenbefall bei malignem Lymphom: Bedeutung der bildgebenden Verfahren innerhalb der Ausbreitungsdiagnostik.

Large autopsy series show an incidence of 33 to 62% of renal involvement in all patients with malignant lymphoma; at the time of diagnosis, this is found only very rarely. The present paper describes three patients with renal involvement at the time of diagnosis. The value of sonography, CT and angiography in detecting renal involvement and its clinical significance in the management of malignant lymphoma is discussed.

The monoclonal antibody 11G7 recognizes a novel differentiation antigen expressed on hemopoietic precursor cells.

A monoclonal antibody (11G7) detecting a novel antigen on human hemopoietic progenitor cells (named 11G7R = 11G7 receptor) was raised by immunization of a Balb/c mouse with the leukemic blasts of a patient suffering from chronic myelogenous leukemia blast crisis (CML-BC). The antigen is expressed on most of MHC class II bearing peripheral blood leucocytes (PBL) and on a subpopulation of bone marrow mononuclear cells (BMMNC). By FACS-sorting and colony assays, it could be demonstrated that 11G7R is expressed on myelo-monocytic and myelo-granulocytic bone marrow precursor cells (GM-CFC, G-CFC, M-CFC) but is absent from erythroid precursor cells (BFU-E) and on cells exhibiting the capacity to form mixed colonies (GEMM-CFC). Double-fluorescence analysis on BMMNC revealed that 11G7R is expressed on a subset of B-cells, myeloid cells and cells carrying the HPCA-1 antigen (CD34). It has a similar distribution pattern to the myeloid antigens CD13 and CD33. However, in contrast to these antigens, 11G7R is also expressed on the blasts of several lymphoid leukemias (4/9 B-ALL, 1/2 T-ALL) and therefore it is not restricted to the myeloid lineage.