Constitutively activating mutation in WASP causes X-linked severe congenital neutropenia.

The Wiskott-Aldrich syndrome protein (WASP; encoded by the gene WAS) and its homologs are important regulators of the actin cytoskeleton, mediating communication between Rho-family GTPases and the actin nucleation/crosslinking factor, the Arp2/3 complex. Many WAS mutations impair cytoskeletal control in hematopoietic tissues, resulting in functional and developmental defects that define the X-linked Wiskott-Aldrich syndrome (WAS) and the related X-linked thrombocytopenia (XLT). These diseases seem to result from reduced WASP signaling, often through decreased transcription or translation of the gene. Here we describe a new disease, X-linked severe congenital neutropenia (XLN), caused by a novel L270P mutation in the region of WAS encoding the conserved GTPase binding domain (GBD). In vitro, the mutant protein is constitutively activated through disruption of an autoinhibitory domain in the wild-type protein, indicating that loss of WASP autoinhibition is a key event in XLN. Our findings highlight the importance of precise regulation of WASP in hematopoietic development and function, as impairment versus enhancement of its activity give rise to distinct spectra of cellular defects and clinical phenotypes.

Activity of oral fludarabine phosphate in previously treated chronic lymphocytic leukemia.

PURPOSE: A prospective, multicenter, open-label phase II clinical trial was conducted to assess the efficacy and safety of oral fludarabine phosphate. Reference to an historical group of patients treated with the intravenous (IV) formulation allowed the investigators to compare the two formulations. PATIENTS AND METHODS: Efficacy was assessed using the International Workshop on Chronic Lymphocytic Leukemia (IWCLL) and National Cancer Institute (NCI) criteria for complete remission (CR), partial remission (PR), stable disease, or disease progression. Safety monitoring included World Health Organization (WHO) toxicity grading for all adverse events. RESULTS: Seventy-eight (96.3%) of 81 recruited patients with previously treated B-cell chronic lymphocytic leukemia (CLL) received 10-mg tablets of fludarabine phosphate to a dose of 40 mg/m(2)/d for 5 days, repeated every 4 weeks, for a total of six to eight cycles. According to IWCLL criteria, the overall remission rate was 46.2% (CR, 20.5%; PR, 25.6%). The comparative figures using NCI criteria were 51.3% (CR, 17.9%; PR, 33.3%). Overall, 30 incidents of severe adverse events were reported for 22 patients. WHO grade 3 or grade 4 hematologic toxicities included granulocytopenia (53.8%), leukocytopenia (28.2%), thrombocytopenia (25.6%), and anemia (24.4%). Gastrointestinal adverse events were more common with the oral formulation than previously reported with IV fludarabine phosphate. However, these events were generally mild to moderate. CONCLUSION: This study demonstrates that oral fludarabine phosphate has similar clinical efficacy to the IV formulation and a safety profile that is both predictable and essentially similar to that of the IV formulation.

Economic analysis of a randomized clinical trial to compare filgrastim-mobilized peripheral-blood progenitor-cell transplantation and autologous bone marrow transplantation in patients with Hodgkin's and non-Hodgkin's lymphoma.

PURPOSE: High-dose chemotherapy (HDC) with peripheral-blood progenitor cell (PBPC) and autologous bone marrow (ABM) transplant (T) has documented survival benefits for relapsed Hodgkin's disease (HD) and non-Hodgkin's lymphoma (NHL). Treatment costs associated with HDC and its supportive care have restricted its use both on and off clinical trial. In a prospective randomized clinical trial, filgrastim-mobilized PBPCT resulted in faster recovery of bone marrow function, with less hospitalization and supportive care than ABMT. This study was undertaken to analyze the costs of the two strategies using prospectively collected data from a randomized clinical trial that compared filgrastim-mobilized PBPCT versus ABMT. PATIENTS AND METHODS: Clinical results and resource utilization from a randomized clinical trial that compared filgrastim-mobilized PBPCT versus ABMT following carmustine, etoposide, cytarabine, and melphalan (BEAM) HDC for HD and NHL are presented. The trial was performed in six centers in Germany, the United Kingdom, and Belgium. Resource utilization data were used to project costs and Massay Cancer Center (MCC) in the United States incurred the cost of treating the cohort. Costs were projected to the United States, because the economic implications to United States centers are significant, costs of care vary markedly among countries but resource utilization on this trial did not, and a randomized trial is unlikely to be performed in the United States. RESULTS: Fifty-eight patients with relapsed HD or NHL underwent HDC with BEAM. The PBPCT and ABMT groups had similar short-term survival after BEAM. PBPCT patients had a shorter hospitalization (median, 17 v 23 days; P = .002), neutrophil recovery (11 v 14 days; P = .005), platelet recovery to > or = 20 x 10(9)/L (16 v 23 days; P = .02), and days of platelet transfusions (6 v 10; P < .001). Estimated costs were $8,531 for ABM harvest and $5,760 for PBPC collection, including filgrastim mobilization. The total estimated average cost was $59,314 for each ABMT patient versus $45,792 for each PBPCT patient. Cost savings of $13,521 (23%) were due to shorter hospitalizations with less supportive care. CONCLUSION: PBPCT is as safe and more effective than ABMT for HD and NHL in the short term. PBPCT represents a significant cost savings due to lower autograft collection costs, shorter hospital stays, and less supportive care. The savings exceed the costs for filgrastim mobilization and PBPC collection. Actual savings will vary depending on local practice patterns, charges, and costs.

Value of different modalities of granulocyte-macrophage colony-stimulating factor applied during or after induction therapy of acute myeloid leukemia.

PURPOSE: The hematopoietic growth factors (HGFs) introduced into induction chemotherapy (CT) of acute myeloid leukemia (AML) might be of benefit to treatment outcome by at least two mechanisms. HGFs given on days simultaneously with CT might sensitize the leukemic cells and enhance their susceptibility to CT. HGFs applied after CT might hasten hematopoietic recovery and reduce morbidity or mortality. MATERIALS AND METHODS: We set out to evaluate the use of granulocyte-macrophage colony-stimulating factor (GM-CSF; 5 microg/kg) in a prospective randomized study of factorial design (yes or no GM-CSF during CT, and yes or no GM-CSF after CT) in patients aged 15 to 60 years (mean, 42) with newly diagnosed AML. GM-CSF was applied as follows: during CT only (+/-, n = 64 assessable patients), GM-CSF during and following CT (+/+, n = 66), no GM-CSF (-/-, n = 63), or GM-CSF after CT only (-/+, n = 60). RESULTS: The complete response (CR) rate was 77%. At a median follow-up time of 42 months, probabilities of overall survival (OS) and disease-free survival (DFS) at 3 years were 38% and 37% in all patients. CR rates, OS, and DFS did not differ between the treatment groups (intention-to-treat analysis). Neutrophil recovery (1.0 x 10(9)/L) and monocyte recovery were significantly faster in patients who received GM-CSF after CT (26 days v 30 days; neutrophils, P < .001; monocytes, P < .005). Platelet regeneration, transfusion requirements, use of antibiotics, frequency of infections, and duration of hospitalization did not vary as a function of any of the therapeutic GM-CSF modalities. More frequent side effects (eg, fever and fluid retention) were noted in GM-CSF-treated patients predominantly related to the use of GM-CSF during CT. CONCLUSION: Priming of AML cells to the cytotoxic effects of CT by the use of GM-CSF during CT or accelerating myeloid recovery by the use of GM-CSF after CT does not significantly improve treatment outcome of young and middle-aged adults with newly diagnosed AML.

Detection of P-glycoprotein with a rapid flow cytometric functional assay using Fluo-3: evaluation of sensitivity, specificity and feasibility in multiparametric analysis.

The specificity and sensitivity of a flow cytometric assay simultaneously measuring expression and transport function of the multidrug resistance associated P-glycoprotein (Pgp) was evaluated. The monoclonal antibody (mAb), MRK16 was used to detect phenotypic Pgp expression while Fluo-3-AM was used as a fluorescent substrate in a Pgp functional transport assay. The specificity of the functional assay was examined in two vinblastine selected human leukemic cell lines (K562/VLB2.5 and CCRF-CEM/VLB50) with acquired Pgp overexpression. Downmodulation of Pgp function in these cell lines could be demonstrated with different substances (verapamil, vinblastine, trifluoperazine, cyclosporin A, progesterone and quinidine) and was proven to be consistently higher in the vinblastine selected cells than in their non-selected drug sensitive counterparts. Unexpectedly, modulator activity was also observed in drug sensitive K562 and CCRF-CEM cell lines despite the inability to detect Pgp in those cells by MRK16 flow cytometrically. Low level expression of the MDR1 gene encoding Pgp in sensitive K562 cells was however demonstrated with a sensitive RT-PCR procedure. The small effect of Pgp modulators in non-drug selected cells could therefore be attributed to low level basal expression of Pgp and illustrates the sensitivity of the functional assay. Also, the effect of various Pgp modulators on Pgp function was more pronounced in a subpopulation of Pgp expressing lymphocytes than in lymphocytes which did not express Pgp. Finally, a correlation was found between discrete variations in Pgp expression and Pgp function of CD4+ lymphocytes, underscoring the feasibility of the functional assay in a triple parametric procedure. The triple parametric assay holds promise to detect Pgp expression and function in clinical samples containing mixtures of malignant and non-malignant cells.

Measurement of serum cytokine levels in patients with myelodysplastic syndromes.

Sera of 25 healthy controls and 75 patients suffering from myelodysplastic syndromes (MDS) were investigated for serum concentration of interleukin-1 alpha (IL-1 alpha), IL-3, IL-6, granulocyte-colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), erythropoietin (Epo), and tumor necrosis factor-alpha (TNF-alpha). According to French-American-British (FAB) classification, 21 refractory anemia (RA), seven refractory anemia with ring sideroblasts (RARS), 15 chronic myelomonocytic leukemia (CMML), 12 refractory anemia with excess of blasts (RAEB), and 20 RAEB in transformation (RAEBt) were examined. TNF-alpha levels were inversely correlated with lower levels of hemoglobin concentration (r = -0.31, p = 0.005), irrespective of the requirements for transfusion in anemic MDS patients. Significant differences in TNF-alpha levels between CMML (26.2 +/- 5.9 pg/ml) and the FAB subgroups (16.1 +/- 1.6 pg/ml) were detected. There was an overall inverse relationship between the level of erythropoietin and the degree of anemia, but a wide range of Epo response between patients with similar hemoglobin concentrations. Serum levels of IL-1 alpha and GM-CSF were undetected in most of the patients. In 57% of the samples there were detectable levels of G-CSF, without a correlation of the serum levels with blood cell counts, nor with any of the FAB subcategories. Overall, 29% and 25% of the patient sera exhibited elevated IL-3 and IL-6 levels, respectively. There was no correlation of the serum levels with any of the blood counts, other cytokines, nor FAB subcategories. In conclusion, simple negative feedback mechanism between a specific cytokine and the production of blood cells seems not to be the case in MDS, except for red cell production and erythropoietin concentration. Our data may suggest the involvement of TNF-alpha in the pathogenesis of anemia in MDS.

A single course of remission reinduction chemotherapy for acute myelogenous leukemia relapsing after allogeneic bone marrow transplantation is complicated by graft-versus-host disease and followed by sustained complete remission.

Graft-versus-host disease (GVHD) remains a major immunological complication after allogeneic bone marrow transplantation (allo-BMT), but also favors development of the beneficial graft-versus-leukemia (GVL) effect. A patient with AML-M4 (inv (16)) is described, who was given non-myeloablative remission reinduction therapy for leukemic relapse (inv (16), trisomy 8) diagnosed on day 184 after HLA-compatible sibling BMT. On day 236, ie about 6 weeks after completion of this course, a clinical syndrome suggestive of acute GVHD grade 3 had developed. Skin biopsy confirmed the clinical diagnosis of GVHD, with a compatible liver biopsy. Transfusion-associated GVHD was ruled out by analysis of short tandem repeat (STR) alleles in the skin biopsy, revealing alleles from donor and recipient but not from third party origin. Cyclosporin A (CsA) therapy, which had been tapered between days 150 and 175, was resumed, resulting in a favorable response and gradual transition to limited chronic GVHD. The patient has since remained in complete remission with an excellent performance status for more than 40 months, without further chemotherapy. Thus this biopsy proven case of GVHD was induced by marrow donor lymphocytes more than 200 days after transplantation and apparently triggered by remission reinduction chemotherapy. The case indicates that intensive non-myeloablative chemotherapy can cure AML relapsing after allo-BMT. The therapeutic effect in this case probably involved a direct pharmacological suppression of the leukemic clone followed by a GVL effect initiated by donor-derived alloreactive T lymphocytes.

CD40 triggering of chronic lymphocytic leukemia B cells results in efficient alloantigen presentation and cytotoxic T lymphocyte induction by up-regulation of CD80 and CD86 costimulatory molecules.

Freshly collected chronic lymphocytic leukemia B cells (B-CLL cells) are known to be inefficient at stimulating allogeneic T cells, and to lack significant expression of B7 (CD80 and CD86) costimulatory molecules. We investigated the potential of CD40 triggering to up-regulate the expression of adhesion and costimulatory molecules on B-CLL cells, and to enhance their immunogenicity towards allogeneic T cells. B-CLL cells cocultured with human CD40 ligand-expressing mouse fibroblasts rapidly up-regulated CD54 and CD58 adhesion molecules and B7-1 (CD80) and B7-2 (CD86) costimulatory molecules, and acquired a strong stimulatory capacity towards CD4+ as well as isolated CD8+ allogeneic T cells. Costimulation by both CD80 and CD86 proved critical for allogeneic T cell proliferation and CD25 and HLA-DR expression, since these were strongly inhibited by anti-CD80 or anti-CD86 monoclonal antibodies, and completely abrogated by CTLA4-Ig fusion protein, which blocks both CD80 and CD86. B7 costimulation also proved critical for restimulation of primed B-CLL-reactive T cells. Most importantly, priming of purified CD8+ T cells with CD40-triggered allogeneic B-CLL cells resulted in cytotoxic activity against the unstimulated B-CLL cells. These findings raise the possibility that CD40 triggering of B-CLL cells might be exploited in immunotherapeutic protocols.

CD57+/CD28- T cells in untreated hemato-oncological patients are expanded and display a Th1-type cytokine secretion profile, ex vivo cytolytic activity and enhanced tendency to apoptosis.

Three-color flow cytometry immunophenotyping revealed significant increases of CD57+ and CD28- cells among both circulating CD4+ and CD8+ T lymphocytes of untreated hemato-oncological patients (n = 54) as compared to healthy donors (n = 55), with CD57 and CD28 expression on the patients' T cells being largely reciprocal. Marked expansion of CD57+ cells among circulating CD4+ T lymphocytes was frequently detected in patients with chronic leukemia of B cell origin (B-CLL, hairy cell leukemia) but not in patients with chronic myeloid leukemia, suggesting a causal relation with the tumor's major histocompatibility complex class II expression. Using immunomagnetic separation techniques, we further demonstrate that the patients' CD57+/CD28- T cells display a typical Th1-type cytokine secretion profile upon anti-CD3 stimulation, with a markedly higher secretion of the Th1-type cytokines IL-2, IFN-gamma, and TNF-alpha than their CD57-/CD28+ counterparts. Cytotoxic activity of circulating CD8+ T lymphocytes, measured ex vivo in an anti-CD3-redirected assay, was almost exclusively exerted by the CD57+/CD28- subset. Moreover, a marked cytotoxic activity was detected within CD4+CD57+ T cells from some B-CLL patients. Finally, the patients' CD57+/CD28- T cells displayed an increased tendency to apoptosis in culture. Collectively, our results indicate that the expanded CD57+/CD28- T cells in hemato-oncological patients represent differentiated effector cells, similar to their (quantitatively minor) counterpart in healthy donors. The reason for their expansion and their pathophysiologic significance, however, remains unclear.

The myelodysplastic syndromes.

The myelodysplastic syndromes constitute a fascinating model for monoclonal premalignant disorders. Haemopoiesis is 'dysplastic' with inefficient maturation of a slowly expanding or sometimes of a stable population, of blood cell precursors. About one third of the patients evolve into acute leukaemia, the result of either a progressive expansion of the original clone or a new mutation producing a more malignant subclone. The majority of patients suffer from the results of bone-marrow insufficiency, with pancytopenia and possibly immune deficiency. Characteristic karyotype anomalies involving mainly chromosomes 5, 7 and 8 are seen in half the patients. These same chromosomes are known to carry different oncogenes. The myelodysplastic syndrome occurs mainly in the aged and there is a moderate male preponderance. The incidence is still unknown but is probably similar to that of acute leukaemia. The etiology is also unknown; however, a secondary myelodysplastic syndrome precedes acute myeloid leukaemia, as a late consequence of chemo- and radio-therapy in treated Hodgkin's disease. This suggests that environmental mutagens might also be involved in primary myelodysplastic syndromes. Treatment remains highly unsatisfactory but a few recent developments improve prognosis, at least in the younger patient.

Fungal cell wall inhibitors: emphasis on clinical aspects.

Invasive fungal infections, mainly caused by Candida and Aspergillus species, are an emerging cause of morbidity and mortality among all categories of immunocompromised patients. Currently available antifungal agents, both polyenes, flucytosine and (tri)azoles are hampered by serious infusion- or drug-related toxicity, by hazardous drug-drug interactions, or by pharmacokinetic problems and by the development of resistance, in vitro as well as in vivo. In recent years, several companies have become interested in antifungal drug development and have launched new compounds into preclinical and clinical trials. Some of these agents target the fungal cell wall in stead of the cell membrane. They exert their fungicidal action through inhibition of the synthesis of critical compounds of that fungal cell wall, not present in mammalian cells. Exciting and promising agents include inhibitors of beta-(1,3)-D-glucan synthase and inhibitors of chitin synthase. These drugs appear well tolerated in Phase I-II studies and will soon enter Phase III studies. This review wants to provide the clinical framework for assessing the utility of these agents compared to existing antifungals, thereby focusing on invasive fungal disease and emphasising the changing fungal epidemiology and susceptibility in immunocompromised hosts.

The changing face of stem cell transplantation by the use of recombinant human granulocyte colony stimulating factor.

The use of peripheral blood progenitor cells (PBPC) for autografting following high-dose chemotherapy in a variety of malignancies has increased markedly with the advent of efficacious and safe myeloid growth factors, which have dramatically improved collection yields. There is still controversy as to whether PBPC should be harvested after a combination of chemotherapy and growth factors, or after growth factors alone. The use of powerful combinations of newer growth factors, such as interleukin-3 with granulocyte-macrophage colony stimulating factor (GM-CSF), or granulocyte colony stimulating factor (G-CSF) with stem cell factor, may obviate the need for chemotherapy. The problem of providing sufficient numbers of PBPC for successful transplantation and sustained engraftment has led to the development of a variety of in vitro expansion systems for stem cells, using cocktails of growth factors. Single blood collections may contain sufficient CD34+ cells to be expanded on a large scale for clinical transplantation, eliminating costly and labour intensive apheresis procedures. PBPC transplants may carry less risk for contamination with malignant cells; however, sensitive detection techniques have revealed that tumour cell contamination of PBPC harvests may be much more prevalent than was previously suspected and both positive and negative selection of CD34+ cells for clinical transplantation is being investigated. PBPC transplantation generally results in more rapid engraftment, with faster recovery of neutrophils and platelets, compared with autologous bone marrow transplants. In most studies, PBPC transplants also resulted in fewer septic episodes, fewer intensive care admissions, fewer transfusions of red blood cells and platelets, reduced antibacterial and parenteral nutrition use, and reduced hospital costs. Dose optimisation and intensification of chemotherapy, relying on repeated administration of growth factor-mobilised PBPC, offers interesting prospects in the treatment of a variety of tumours. However, well-controlled, randomised prospective trials will be needed to prove the real value of PBPC transplants with regard to survival and cure. In the clinical setting, PBPC transplantations are likely to replace autologous marrow transplants in the near future. Manipulation of PBPC in vitro, i.e. expansion or gene transfer, may prove to be the most exciting perspective in the treatment of both malignant and non-malignant haematological conditions.

Cataracts after total body irradiation and bone marrow transplantation in patients with acute leukemia in complete remission: a study of the European Group for Blood and Marrow Transplantation.

PURPOSE: Advances in bone marrow transplantation (BMT) have consistently improved long-term survival. Therefore, evaluation of late complications such as cataracts is of paramount importance. METHODS AND MATERIALS: We analyzed data of 2149 patients from the EBMT registry. A cohort of 1063 patients were evaluable for survival and ophthalmologic status after transplant for acute leukemia (AL) in first or second complete remission. Conditioning therapy included either single-dose total body irradiation (STBI) or fractionated TBI (FTBI) grouped in different dose rates (low: LDR < or = 0.04 Gy/min; high: HDR > 0.04 Gy/min). RESULTS: The overall 10-year estimated cataract incidence (ECI) was 50%. It was 60% in the STBI group, 43% in the FTBI group < or = 6 fractions, and 7% in the FTBI group > 6 fractions (p < 10(-4)). It was significantly lower (30%) in the LDR than in the HDR groups (59%;p < 10(-4)). Patients receiving heparin for veno-occlusive disease prophylaxis had fewer cataracts than those who did not (10-year ECI: 33% vs. 53%, respectively;p = 0.04). The 10-year ECI was 65% in the allogeneic vs. 46% in the autologous BMT patients (p = 0.0018). Factors independently associated with an increased risk of cataract were an older age (> 23 years), higher dose rate (> 0.04 Gy/min), allogeneic BMT, and steroid administration (> 100 days). The use of FTBI was associated with a decreased risk of cataract. Heparin administration was a protective factor in patients receiving STBI. In terms of cataract surgery, the unfavorable factors for requiring surgery were: age > 23 yr, STBI, dose rate > 0.04 Gy/min, chronic graft-vs.-host disease (cGvHD), and absence of heparin administration. Among the patients who required cataract surgery (111 out of 257), secondary posterior capsular opacification was observed in 15.7%. CONCLUSION: High dose rate and STBI are the main risk factors for cataract development and the need for surgery, and the administration of heparin has a protective role in cataractogenesis.

Mechanisms of vascular damage in gout and oxalosis: crystal induced, granulocyte mediated, endothelial injury.

Immune triggered granulocyte (PMN)-endothelial interactions have been implicated in the pathogenesis of vascular diseases. While hyperuricemia and gout are associated with an increased risk of atherogenesis, we studied the modulation by monosodium-urate (MSU) crystals of PMN-endothelial interactions in vitro. The relationship between calcium oxalate (COX) crystals - implicated in the vasculitis of primary oxalosis - and immunologically mediated endothelial injury was also explored. Both MSU- and COX-crystal treated sera stimulate PMN to adhere to and induce significant 51Cr-release from endothelial cells in vitro. Platelets significantly increase crystal-triggered PMN endothelial cell adherence and 51Cr-release. This platelet augmenting effect depends on the release of platelet constituents (e.g. serotonin). Microcrystalline material present in vessel walls, thus may cause C-activation and may trigger PMN and platelets to damage endothelium in vitro and in vivo. These findings may have relevance to the understanding of the accelerated atherogenesis of hyperuricemia and the fulminant vasculitis of oxalosis or ethylene glycol poisoning.

Protective effect of vitamin E on immune triggered, granulocyte mediated endothelial injury.

The effect of alfa-tocopherol on the cell-cell interactions at the vessel wall were studied, using an in vitro model of human umbilical vein endothelial cell cultures (HUEC). Immune triggered granulocytes (PMN) will adhere to and damage HUEC and platelets enhance this PMN mediated endothelial injury. When HUEC are cultured in the presence of vitamin E, 51Cr-leakage induced by complement stimulated PMN is significantly decreased and the enhanced cytotoxicity by platelets is completely abolished (p less than 0.001). The inhibition of PMN induced endothelial injury is directly correlated to a diminished adherence of PMN to vitamin E-cultured HUEC (p less than 0.001), which may be mediated by an increase of both basal and stimulated endogenous prostacyclin (PGI2) from alfa-tocopherol-treated HUEC (p less than 0.025). The vitamin E-effect is abolished by incubation of HUEC with the irreversible cyclo-oxygenase inhibitor, acetylsalicylic acid, but the addition of exogenous PGI2 could not reproduce the vitamin E-mediated effects. We conclude that vitamin E exerts a protective effect on immune triggered endothelial damage, partly by increasing the endogenous anti-oxidant potential, partly by modulating intrinsic endothelial prostaglandin production. The failure to reproduce vitamin E-protection by exogenously added PGI2 may suggest additional, not yet elucidated vitamin E-effects on endothelial metabolism.

Enkephalins modify granulocyte-endothelial interactions by stimulating prostacyclin production.

Granulocyte (PMN)-endothelial interactions have been implicated in the primary events of vascular injury and atherogenesis. We now present data which show that endogenous opioid peptides, e.g. enkephalins (ENK), dampen immune-triggered, granulocyte-induced endothelial damage by enhancing prostacyclin production. Concurrent exposure of human umbilical vein endothelial cells (HUEC) to Met5-enkephalin increased arachidonic acid (AA, 20 muM) and thrombin (T, 10 U/ml) induced 6-keto-PGF1 alpha-release to respectively 197.2 +/- 28.1% and 204.1 +/- 17.8% (mean +/- SEM) of base line stimulation (p less than 0.025). The increases noted were significant at ENK-concentrations as low as 10(-12)M. Simultaneous addition of naloxone with ENK completely abolished the enhanced 6-keto-PGF1 alpha-release. Addition of a protease resistant enkephalin analogue significantly (p less than 0.01 over several different concentrations) reduced PMN adherence to HUEC; concomitantly 51Cr-leakage from HUEC that had been exposed to PMN plus activated serum complement was decreased. The even further enhanced 51Cr-leakage that occurs when platelet release products (e.g. serotonin) are included is also decreased by added enkephalin. These data suggest that endogenous neurotransmitters may affect endothelial prostaglandin metabolism, and by so doing provide a protective effect during in vitro, and perhaps in vivo, PMN mediated endothelial injury. This link between neurohumoral and inflammatory systems might enhance our understanding of stress-related phenomena in inflammation and vascular diseases.

Peripheral blood lymphocyte subset shifts in patients with untreated hematological tumors: evidence for systemic activation of the T cell compartment.

Flow cytometry immunophenotyping of peripheral blood lymphocyte subsets and multivariate data-analytical techniques revealed that among untreated hemato-oncological patients (n = 48) with lymphomas, acute and chronic myeloid and lymphocytic leukemias, monoclonal gammopathy of undetermined significance, and multiple myeloma, 42% had (nonmalignant) lymphocyte profiles clearly distinct from healthy donors. Notably, a similar pattern of increased CD3+ CD57+, CD3+ HLA-DR+, CD3+ CD(16 + 56)+, CD4- CD8+, CD8+ CD57+, CD8+ CD28-, and CD8+ CD62L- subsets was detected. More extensive three-color immunophenotyping on an additional group of 49 untreated patients revealed that both CD4+ and CD8+ T cells displayed significant increases of activation markers: CD69, CD(16 + 56), HLA-DR, CD71, and CD57, and a loss of CD62L and CD28, which is also interpreted as a sign of activation. Consistent with the phenotypical signs of in vivo immune activation, polyclonal cytolytic activity, measured ex vivo in an anti-CD3-redirected assay, was detected within immunomagnetically purified CD4+ T cells of three out of six B-CLL patients investigated, but not within purified CD4+ T cells of five healthy donors. The purified CD8+ T cells of patients (n = 28) and donors (n = 5) on the other hand displayed similar polyclonal cytotoxic activities at the various effector:target ratios investigated. Tumor-directed cytotoxic activity of purified CD4+ (n = 6) and/or CD8+ T cells (n = 15) against freshly isolated autologous tumor cells was not detected in any of the experiments. Collectively, our results demonstrate systemic T cell activation as a common feature in hematological neoplasia, and a markedly enhanced cytolytic activity of the CD4- subset in CLL patients. The reason(s) for this expansion of activated T cells and its pathophysiologic significance, however, remain unclear.

Mucormycosis in allogeneic bone marrow transplant recipients: report of five cases and review of the role of iron overload in the pathogenesis.

In a 10-year consecutive series of 263 allogeneic bone marrow transplant recipients, we identified five cases (1.9%) of invasive mucormycosis. Only one infection occurred within the first 100 days after transplantation, while the remainder complicated the late post-transplant course (median day of diagnosis: 343). Sites of infection were considered 'non-classical' and included pulmonary, cutaneous and gastric involvement. No case of fungal dissemination was observed. Mucormycosis was the primary cause of death in three of the five patients. Corticosteroid-treated graft-versus-host disease, either acute or chronic, or severe neutropenia were present in all cases. However, compared with a matched control population, the most striking finding was the demonstration of severe iron overload in each of the mucormycosis patients. The mean level of serum ferritin, transferrin saturation and number of transfused units of red cells (2029 microg/l, 92% and 52 units, respectively) in the study group is significantly higher compared with the control group (P < 0.05). The difference with other risk groups for mucormycosis, including deferoxamine-treated dialysis patients and acidotic diabetics, was analyzed in view of the possible pathogenic role of iron. Although these infections are often fatal, limited disease may have a better prognosis if diagnosed early and treated aggressively.

Treatment of patients with myelodysplastic syndromes with allogeneic bone marrow transplantation from genotypically HLA-identical sibling and alternative donors.

Between December 1981 and March 1994, 24 patients with a myelodysplastic syndrome (MDS) underwent allogeneic bone marrow transplantation (BMT) for RA with trilineage dysplasia (n = 4), CMML (n = 1), RAEB (n = 4), RAEBt (n = 9) and AML following MDS (n = 6). Fifteen patients (two RAEB, seven RAEBt and six sAML) received chemotherapy before BMT resulting in complete remission in 10 patients (six RAEBt and four sAML) at the time of BMT. Sixteen marrow donors were genotypically HLA-identical siblings. Remaining donors were other family members (five) or unrelated donors (three). The status of the underlying disease at the time of conditioning was the major factor determining long-term survival. The disease-freed survival of RA patients and patients presenting with RAEB, RAEBt and AML but transplanted in complete remission, was respectively 50 and 60%. On the contrary, none of the nine high-risk MDS patients transplanted with persistent disease, survived. Outcome after transplantation with alternative donors was inferior with one long-term survivor, mainly related to the high incidence of severe acute GVHD and its accompanying infectious complications. Six patients relapsed resulting in an actuarial probability of relapse of 28%. Twelve patients died of transplant-related complications leading to a non-relapse mortality at 5 years of 50%. At present eight patients are alive and disease-free 20 to 132 months post-transplantation resulting in an actuarial 5-year disease-free survival of 40.7%. Our results suggest that allogeneic bone marrow transplantation is a feasible treatment option for patients with MDS. However, improvement in GVHD prophylaxis and supportive care to reduce transplant-treated mortality and improved relapse prevention are imperative.

Peripheral blood stem cell transplantation as an alternative to autologous marrow transplantation in the treatment of acute myeloid leukemia?

The clinical use of autologous marrow transplantation in acute myeloid leukemia (AML) has been hampered by the inability to collect adequate numbers of cells after remission induction chemotherapy and the notably delayed hematopoietic regeneration following autograft reinfusion. Here we present a study in which the feasibility of mobilizing stem cells was investigated in newly diagnosed AML. Among 96 AML patients, 76 patients (79%) entered complete remission. Mobilization was undertaken with low dose and high dose schedules of G-CSF in 63 patients, and 54 patients (87%) were leukapheresed. A median of 2.0 x 10(6) CD34+ cells/kg (range 0.1-72.0) was obtained in a median of three leukaphereses following a low dose G-CSF schedule (150 microg/m2) during an average of 20 days. Higher dose regimens of G-CSF (450 microg/m2 and 600 microg/m2) given during an average of 11 days resulted in 28 patients in a yield of 3.6 x 10(6) CD34+ cells/kg (range 0-60.3) also obtained following three leukaphereses. The low dose and high dose schedules of G-CSF permitted the collection of 2 x 10(6) CD34-positive cells in 46% and 79% of cases respectively (P = 0.01). Twenty-eight patients were transplanted with a peripheral blood stem cell (PBSC) graft and hemopoietic repopulation was compared with the results of a previous study with autologous bone marrow. Recovery of granulocytes (>0.5 x 10(9)/l, 17 vs 37 days) and platelets (>20 x 10(9)/l; 26 vs 96 days) was significantly faster after peripheral stem cell transplantation compared to autologous bone marrow transplantation. These results demonstrate the feasibility of PBSCT in the majority of cases with AML and the potential advantage of this approach with respect to hemopoietic recovery.