Evolution in health and medicine Sackler colloquium: Making evolutionary biology a basic science for medicine.

New applications of evolutionary biology in medicine are being discovered at an accelerating rate, but few physicians have sufficient educational background to use them fully. This article summarizes suggestions from several groups that have considered how evolutionary biology can be useful in medicine, what physicians should learn about it, and when and how they should learn it. Our general conclusion is that evolutionary biology is a crucial basic science for medicine. In addition to looking at established evolutionary methods and topics, such as population genetics and pathogen evolution, we highlight questions about why natural selection leaves bodies vulnerable to disease. Knowledge about evolution provides physicians with an integrative framework that links otherwise disparate bits of knowledge. It replaces the prevalent view of bodies as machines with a biological view of bodies shaped by evolutionary processes. Like other basic sciences, evolutionary biology needs to be taught both before and during medical school. Most introductory biology courses are insufficient to establish competency in evolutionary biology. Premedical students need evolution courses, possibly ones that emphasize medically relevant aspects. In medical school, evolutionary biology should be taught as one of the basic medical sciences. This will require a course that reviews basic principles and specific medical applications, followed by an integrated presentation of evolutionary aspects that apply to each disease and organ system. Evolutionary biology is not just another topic vying for inclusion in the curriculum; it is an essential foundation for a biological understanding of health and disease.

Risk-adjusted therapy of acute lymphoblastic leukemia can decrease treatment burden and improve survival: treatment results of 2169 unselected pediatric and adolescent patients enrolled in the trial ALL-BFM 95.

The trial ALL-BFM 95 for treatment of childhood acute lymphoblastic leukemia was designed to reduce acute and long-term toxicity in selected patient groups with favorable prognosis and to improve outcome in poor-risk groups by treatment intensification. These aims were pursued through a stratification strategy using white blood cell count, age, immunophenotype, treatment response, and unfavorable genetic aberrations providing an excellent discrimination of risk groups. Estimated 6-year event-free survival (6y-pEFS) for all 2169 patients was 79.6% (+/- 0.9%). The large standard-risk (SR) group (35% of patients) achieved an excellent 6y-EFS of 89.5% (+/- 1.1%) despite significant reduction of anthracyclines. In the medium-risk (MR) group (53% of patients), 6y-pEFS was 79.7% (+/- 1.2%); no improvement was accomplished by the randomized use of additional intermediate-dose cytarabine after consolidation. Omission of preventive cranial irradiation in non-T-ALL MR patients was possible without significant reduction of EFS, although the incidence of central nervous system relapses increased. In the high-risk (HR) group (12% of patients), intensification of consolidation/reinduction treatment led to considerable improvement over the previous ALL-BFM trials yielding a 6y-pEFS of 49.2% (+/- 3.2%). Compared without previous trial ALL-BFM 90, consistently favorable results in non-HR patients were achieved with significant treatment reduction in the majority of these patients.

Stem cell transplantation for polycythemia vera.

Polycythemia vera (PV) is a rare disease in children. A 9-year-old male was diagnosed following laboratory results acquired because of an acute appendicitis. Regular phlebotomy was performed for over 2 years followed by alpha-interferon treatment. At the age of 12 years, HLA-matched unrelated stem cell transplantation including T-cell depletion was done. The conditioning regimen consisted of busulfan, cyclophosphamide, and ATG. Chimerism was monitored during the whole post-transplant period. A single dose of donor T-lymphocytes was given at month 3. One year after transplantation, chimerism was complete. The patient is in complete remission and shows no signs of transplant-related morbidity at month 78.

Outcome of allogeneic stem cell transplantation in children with non-malignant diseases.

BACKGROUND AND OBJECTIVES: After allogeneic stem cell transplantation treatment failures are mostly caused by graft rejection or graft-versus-host disease (GVHD). T-cell depletion is an appropriate tool to prevent GvHD. However, it might be associated with an increased risk of graft rejection, which can be recognized by serial and quantitative characterization of chimerism. Thus, pre-emptive immunotherapy might be helpful to avoid graft rejection. DESIGN AND METHODS: We present the outcome of 56 transplants performed in 53 children with non-malignant diseases. T-cell depletion was conducted in 27/56 grafts. When increasing mixed chimerism over 30% autologous cells occurred low dose donor lymphocyte transfusions (DLT) were performed. RESULTS: During the course of the follow-up 29 out of 53 patients achieved complete chimerism or low mixed chimerism (0-1%) and 28/29 remained in continuous complete remission. Donor engraftment failed in 2/53 patients who died of serious infection. Increasing mixed chimerism was found in 19 out of 56 transplantations. Fifteen of these 19 patients received additional immunotherapy with DLT. Eleven out of the 15 remained in complete remission. One of the 15 patients developed GvHD grade III that turned out to extensive chronic GvHD. The 3-year overall survival was 100% for patients transplanted from matched related or unrelated donors and 75% for patients transplanted from mismatched donors. INTERPRETATION AND CONCLUSIONS: We demonstrated that children transplanted for non-malignant diseases have an excellent overall survival. T-cell depletion is associated with an increased risk of graft rejection. Pre-emptive immunotherapy with DLT, administered on the basis of increasing mixed chimerism, is feasible and might prevent graft rejection.

Pitfalls in detection of contaminating neuroblastoma cells by tyrosine hydroxylase RT-PCR due to catecholamine-producing hematopoietic cells.

BACKGROUND: RT-PCR analysis of compounds of catecholamine metabolism (in particular tyrosine hydroxylase, TH) is widely used for the detection of contaminating neuroblastoma cells in hematopoietic stem cell preparations. Due to reports in the literature showing that hematopoietic cells are also able to produce catecholamines, we investigated whether TH-RT-PCR is really suitable for this purpose. MATERIALS AND METHODS: Besides neuroblastoma cells, mononuclear blood cells, apheresis preparations and hematopoietic stem cells were used for single and nested RT-PCR. In addition to TH, the expressions of dopamine-beta-hydroxylase and noradrenaline transporter were analyzed. RESULTS: Using single RT-PCR, a clear discrimination between neuroblastoma and hematopoietic cells was possible. However, by using nested RT-PCR, the "neuroblastoma markers" were also detected in a significant percentage of non-mobilized mononuclear blood cells, in mononuclear blood cells of healthy donors mobilized with G-CSF, and in highly purified CD34+ and CD133+ stem cells from healthy mobilized donors. CONCLUSION: Our results raise the question of whether the RT-PCR analysis of compounds of catecholamine metabolism is suitable and selective enough to detect the contamination of hematopoietic stem cells by a low number of neuroblastoma cells.

Receptor activator of nuclear factor kappaB ligand plays a nonredundant role in doxorubicin-induced apoptosis.

Doxorubicin induces apoptosis in a variety of cells. We investigated the expression and function of various tumor necrosis factor (TNF)alpha-homologues and their receptors. CEM cells did not differentially express any one of the TNFalpha-homologous receptors investigated nor TNF-related apoptosis-inducing ligand or TNF-related weakly apoptosis-inducing ligand (TWEAK) in the presence of doxorubicin. In addition to CD95 ligand, however, receptor activator of nuclear factor kappaB ligand (RANKL) was strongly up-regulated. Doxorubicin-induced apoptosis was greatly suppressed in the presence of either neutralizing antibody or RANK-Fc fusion protein. Moreover, neutralizing RANKL also prevented cytochrome c release from mitochondria. RANKL alone was unable to induce significant levels of apoptosis in CEM cells. However, doxorubicin-induced apoptosis was increased >2-fold when exogenous RANKL was added. Therefore, RANKL is necessary but not sufficient to account for early doxorubicin-induced apoptosis in CEM cells. This finding suggests improved chemotherapeutic efficiency of the anthracyclin against susceptible malignant cells in the presence with RANKL.

Consolidation treatment with chimeric anti-GD2-antibody ch14.18 in children older than 1 year with metastatic neuroblastoma.

PURPOSE: Antibody treatment is considered tolerable and potentially effective in the therapy of neuroblastoma. We have analyzed stage 4 neuroblastoma patients older than 1 year who underwent consolidation treatment with the chimeric monoclonal anti-GD2-antibody ch14.18. PATIENTS AND METHODS: Stage 4 patients older than 1 year who completed initial treatment without event were eligible. ch14.18 was scheduled in a dose of 20 mg/m2/d during 5 days in six cycles every 2 months. Patients who did not receive ch14.18 served as controls. RESULTS: Of 334 assessable patients, 166 received ch14.18, 99 received a 12-month low-dose maintenance chemotherapy (MT) instead, and 69 had no additional treatment. During 695 ch14.18 cycles, fever (55% of cycles), abnormal C-reactive protein without infection (35%), cough (24%), rash (22%), and pain (16%) were the main side effects. Univariate analysis found similar event-free survival (EFS) for the three groups (3-year EFS, 46.5% +/- 4.1%, 44.4% +/- 4.9%, 37.1% +/- 5.9% for patients treated with antibody ch14.18, MT, and no additional therapy, respectively; log-rank test, P =.314). For overall survival (OS), ch14.18 treatment (3-year OS, 68.5% +/- 3.9%) was superior to MT (3-year OS, 56.6% +/- 5.0%) or no additional therapy (3-year OS, 46.8% +/- 6.2%; log-rank test, P =.018). Separate univariate analysis of patients with autologous stem-cell transplantation revealed no difference between patients with ch14.18 treatment and no additional consolidation. Multivariate analysis failed to demonstrate an advantage of antibody treatment for EFS and OS. CONCLUSION: Consolidation treatment of stage 4 neuroblastoma with ch14.18 was associated with considerable but manageable side effects. Compared with oral maintenance chemotherapy and no consolidation treatment, ch14.18 had no clear impact on the outcome of patients.

Increasing mixed chimerism is an important prognostic factor for unfavorable outcome in children with acute lymphoblastic leukemia after allogeneic stem-cell transplantation: possible role for pre-emptive immunotherapy?

PURPOSE: We recently reported that children with acute leukemias who show increasing mixed chimerism (MC) after allogeneic stem-cell transplantation have a significantly enhanced risk of relapse. Here we present the results of a prospective multicenter study to investigate (1) whether relapse of acute lymphoblastic leukemia (ALL) can be determined in advance by serial analysis of chimerism, and (2) if outcome can be influenced by withdrawal of immunosuppression and/or by low-dose donor lymphocyte infusion when increasing MC is detected. PATIENTS AND METHODS: Serial and quantitative analysis of chimerism was performed using a fluorescent-based short-tandem-repeat-polymerase chain reaction in 163 children with ALL. RESULTS: One hundred one patients revealed complete chimerism (CC) or low-level MC (CC/low-level MC); increasing MC was found in 46 patients; and decreasing MC, in 16 patients. Relapse was significantly more frequent in patients with increasing MC (26 of 46) than in patients with CC/low-level MC (eight of 101) or in patients with decreasing MC (0 of 16; P <.0001). The probability of 3-year event-free survival (EFS) was 54% for all patients, 66% for patients with CC/low-level MC (n = 101), 66% for patients with decreasing MC (n = 16), and 23% for patients with increasing MC (n = 46; P <.0001). Of the 46 patients with increasing MC, 31 received immunotherapy. This group had a significantly higher 3-year EFS estimate (37%) than the 15 patients who did not receive immunotherapy (0%; P <.001). CONCLUSION: Serial analysis of chimerism reliably identifies patients at highest risk to relapse. The 3-year EFS of patients with increasing MC without immunotherapy was 0%, by which overt relapse could be prevented in a considerable group of patients.

Isolation and expansion of human adenovirus-specific CD4+ and CD8+ T cells according to IFN-gamma secretion for adjuvant immunotherapy.

OBJECTIVE: In patients with lymphopenia following allogeneic stem cell transplantation adenovirus (ADV) infection is associated with high morbidity and mortality despite aggressive antiviral drug therapy. Virus-specific T cells seem to be essential for virus elimination. The aim of this study was to isolate and expand donor-derived human ADV-specific T lymphocytes for adoptive transfer of sufficient cell numbers to restore protective immunity after allogeneic stem cell transplantation. MATERIALS AND METHODS: A clinical-grade strategy to generate ADV-specific T cells using the interferon-gamma (IFN-gamma) secretion assay, followed by expansion to numbers sufficient for clinical application with interleukin-2 (IL-2) and feeder cell stimulation, is described. RESULTS: A mean number of 3.4 x 10(6) (+/-3 SD) ADV antigen-specific T lymphocytes were isolated from 0.1 to 2 x 10(9) mononuclear cells from peripheral blood (n=5) or leukapheresis products (n=6). Characterization of ADV-specific T cells after isolation revealed a mean purity of 85.1% (+/-12% SD) using antigen-specific intracellular cytokine staining. Isolated cells were expanded ex vivo for a median of 18 days (range 7-29 days; n=5) to greater than 10(8) total cells using IL-2 and autologous feeder cell stimulation. ADV-specific response to adenovirus antigen was confirmed in the generated T cell lines, using intracellular cytokine staining, IFN-gamma Elispot assay, and (3)H-thymidine uptake. Generated T-cell lines showed specific killing of ADV-infected B-LCL (n=4). Alloreactive proliferation of generated T-cell lines in mixed lymphocyte cultures was significantly reduced when compared to unmanipulated PBMCs. CONCLUSION: Generation of adenovirus-specific T cells in a simple and rapid clinical-grade protocol was established, using IFN-gamma secretion assay with short expansion times, leading to sufficient numbers of ADV-specific T cells that can be used for adoptive immunotherapy.

Haploidentical transplantation for acute lymphoblastic leukemia in childhood.

Haploidentical transplantation in childhood acute lymphoblastic leukemia (ALL) is a promising option for children lacking a suitable donor. We have updated our series of patients with ALL and report the results. Additionally, we reviewed the literature and try to embed our own experiences in the published results. We performed HLA-mismatched stem cell transplantations with megadoses of purified positively selected mobilized peripheral blood CD34+ progenitor cells (PBPC) from adult donors in 27 children with acute lymphoblastic leukemia (ALL) in first (CR1 n = 7), second (CR2 n = 10), or third (CR3 n = 4) complete remission, and in refractory state (NR n = 6). The patients received a mean number of 19.1+/-11.3 x 10(6)/kg purified CD34+ and a mean number of 15.5+/-24.2 x 10(3)/kg CD3+ T-cells. No additional graft-versus-host disease (GVHD) prophylaxis was used, except as short-term CSA in the first 3 patients. The myeloablative treatment was based on busulfan in 12 and on TBI in 14 patients. One patient was grafted with a non-myeloablative approach. Engraftment was rapid in 26 patients, with two patients suffering from a rejection. These two and one patient with initial non-engraftment had been successfully regrafted. The probability of survival of the total group is 0.34+/-0.09; the 12 patients transplanted in remission showed a probability of survival of 0.44+/-0.11. None of the patients transplanted in non-remission survived. There was no statistical difference in survival for patients with a 1, 2 or 3 antigen mismatched donor (out of 6 HLA antigens) or for patients in 1st, 2nd or 3rd remission. Causes of death were relapses in 10 patients, veno-occlusive disease (VOD) in 1, multi-organ failure (MOF) in 2 and infections in 4 patients. 3/24 evaluable patients without any additional GVHD-prophylaxis developed grade 1 or 2 GVHD. Ten patients were treated with additional donor lymphocyte infusion (DLI), from which 4 developed a maximum grade 3 GVHD. We conclude that the HLA barrier can be overcome by transplantation of megadoses of highly purified CD34+ PBPC and GVHD can effectively be prevented. This approach offers a promising treatment option for patients with acute lymphoblastic leukemia needing urgently transplantation but lacking a suitable donor.

Organ-specific T cell receptor repertoire in target organs of murine graft-versus-host after transplantation across minor histocompatibility antigen barriers.

BACKGROUND: Minor histocompatibility antigens (miHags) are recognized by alloreactive cytotoxic donor T lymphocytes and trigger potent immune reactions such as graft-versus-host disease (GvHD) after major histocompatibility complex-matched transplantation. Our study focuses on tissue-specific T-cell responses to miHag-encoded peptides in GvHD target organs during the first 30 days in a murine transplant model. METHODS: Complementarity determining region (CDR)3-size spectratyping was used to study T cell receptor (TCR) repertoires in recipient skin, liver, ileum, colon, spleen, and heart. RESULTS: GvHD occurred as early as day 14 and was proven by histology in skin, liver, ileum, and colon. The heart was histologically not affected by GvHD but showed endomyocardial "quilty lesions." Two distinct patterns of TCR diversities could be identified. In skin, a restricted V beta usage in combination with all J beta segments contrasted with a complete V beta repertoire in intestinal organs combined with a restricted J beta usage. Interestingly, TCR repertoire in the heart was almost identical with intestinal CDR3-size patterns. Persisting clones were found in skin from day 9 to 30. In intestine and heart, identical sequences were obtained from several organs on day 14 and 21, but no persistence of CDR3 sequences could be observed. CONCLUSIONS: These results suggest that in the skin a limited number of persisting T cell clones maintains GvHD, whereas in the intestine, temporary expansions of different clones may fuel the process of GvHD. Strategies that eliminate tissue-specific T cells on the basis of their activational status rather than their V beta expression but at the same time preserve a broad, overall TCR repertoire will help to increase the efficacy and safety of allogeneic stem cell transplantation.

Down-regulation of protein kinase Ceta by antisense oligonucleotides sensitises A549 lung cancer cells to vincristine and paclitaxel.

Previous studies point to protein kinase C (PKC) isozyme eta as a resistance factor in cancer cells. Therefore, we investigated whether down-regulation of PKCeta with second generation antisense oligonucleotides (ODNs) would sensitise A549 human lung carcinoma cells to cytostatics. The effects were compared to the outcome of Bcl-xL down-regulation. Upon treatment with antisense ODNs, PKCeta and Bcl-xL were both significantly reduced on mRNA and protein level. Down-regulation of either PKCeta or Bcl-xL in combination with vincristine or paclitaxel resulted in a significant increase in caspase-3 activity compared to that in the control oligonucleotide treated cells. In addition, PKCeta down-regulation augmented vincristine-induced dissipation of mitochondrial transmembrane potential. In conclusion, these results confirm that PKCeta might represent a considerable resistance factor and an interesting target to improve anticancer chemotherapy.

Impaired T-cell activation and cytokine productivity after transplantation of positively selected CD34+ allogeneic hematopoietic stem cells.

Transplantation of positively selected, CD34(+) peripheral blood stem cells from alternative donors frequently results in delayed immune reconstitution. A shift towards a type 2 cytokine production might be a major contributing factor. We therefore decided to measure IFN-gamma, IL-2, IL-4, and IL-10 after stimulation of peripheral mononuclear cells with PMA/ionomycin and on a single cell level by intracellular cytokine staining during different stages of immune reconstitution. Immediately after transplantation, secretion of all selected cytokines was substantially diminished, and remained subnormal compared to controls until the end of the first year despite normalizing T-cell levels. IL-2 was predominantly produced by CD4(+)CD45RA(+) naïve, whereas IFN-gamma originated mainly from CD8(+)CD45RO(+) memory T cells. Secretion of IL-2 was correlated with the numbers of naive CD4(+) T cells, whereas IFN-gamma secretion correlated with total CD3(+) T-cell counts. IL-4 and IL-10 were produced by CD4(+) and CD8(+) memory T cells; secretion of these cytokines was low, however, and did not increase during follow-up. Therefore, a shift towards a preferential production of type 2 cytokines could not be observed. Analysis of CD69 upregulation upon stimulation revealed a deficiency in patient T-cell activation, which unexpectedly comprised both naïve and memory T-cell subpopulations. Therefore, we suggest that a defect in T-cell activation intrinsic to the host and not graft-versus-host disease, post-transplant immunosuppression or a shift towards a type 2 cytokine pattern contributes to the impaired production of cytokines post-transplant. Further studies will focus on the elimination of host factors that may adversely affect T-cell function after transplantation.

Biology and plasticity of CD133+ hematopoietic stem cells.

AC133 (CD133) is a highly conserved antigen expressed on hematopoietic stem cells with unknown function. In order to further characterize CD133(+) progenitor cells, we purified CD133(+) stem cells using the method of magnetic activated cell sorting (MACS) from healthy adult volunteers mobilized with granulocyte colony-stimulating growth factor (G-CSF) to a mean purity of 94%. The purified CD133(+) cells highly engrafted NOD/SCID mice. In addition, unseparated mononuclear cells or CD133(+) stem cells isolated from the bone marrow of transplanted NOD/SCID mice gave rise to engraftment of secondary recipients. Upon ex vivo culture of purified CD133(+) cells with FLT3/Flk2 ligand (FL) and interleukin-6 (IL-6), a plastic-adherent cell population could be observed after 6 weeks in culture. These adherent cells did not express CD34 or CD133 antigens on their surface, nor did they express markers for endothelial, mesenchymal, or dendritic cells. After incubation of these adherent cells with stem cell factor (SCF), non-adherent cells were observed which partially co-expressed CD133, but were negative for CD34. These nonadherent CD34(-) cells showed a high engraftment capacity in NOD/SCID mice. From our results, we conclude that CD133 might be a marker of early progenitors with a high NOD/SCID engraftment potential. The fact that CD133(+) hematopoietic progenitors can give rise to an adherent population which is CD133(-) and CD34(-) and that these cells can again give rise to a CD133(+)CD34(-) stem cell population with high NOD/SCID engraftment potential indicates the plasticity of hematopoietic precursors. CD133(+) stem cells might be useful for research and for clinical application.

Expression and regulation of B7 family molecules on macrophages (MPhi) in preterm and term neonatal cord blood and peripheral blood of adults.

BACKGROUND: Macrophage (MPhi) receptors of the B7 family (CD80, CD86) play a crucial role in T cell activation: the lack of costimulation leads to anergy or apoptosis of reactive T cells. MPhi may differentiate into different subsets, the balance of which defines MPhi-dependent T cell reactions. The aim of this study was to examine neonatal and adult T cell response with respect to the costimulatory MPhi-potential in order to identify molecular predictors for the neonatal immune defense. METHODS: MPhi from peripheral blood (PBMPhi) or cord blood (CBMPhi) were stimulated with interferon-gamma (IFN-gamma), cyclic adenosine monophosphate (cAMP), CD40 ligand (CD40L), or alphaCD3. RESULTS: As compared to PBMPhi, CBMPhi showed a significantly decreased upregulation of CD80 and/or CD86 after stimulation with IFN-gamma, cAMP, CD40L, and alphaCD3. Accordingly, the proliferative T cell response was impaired in the presence of CBMPhi. The fraction of T cells that underwent cell death was higher, and blast formation was significantly lower than that observed in the presence of PBMPhi. CONCLUSIONS: CBMPhi, as compared to PBMPhi, delivered fewer costimulatory but more cytotoxic signals to T cells. These observations suggest that MPhi are one factor explaining the suboptimal immune defense of neonates and their increased susceptibility to infection. Using the costimulatory MPhi-potential as a predictor for immune responses requires a separate reference value system in neonatology.

Induction of drug resistance and protein kinase C genes in A2780 ovarian cancer cells after incubation with antineoplastic agents at sublethal concentrations.

We examined the inducibility of drug resistance (MDR1, MRP1, LRP) and protein kinase C (PKC) isozyme (alpha, epsilon, eta, theta, tau, zeta) corresponding genes in A2780 ovarian cancer cells after a 24-hour treatment with adriamycin (ADR), camptothecin (CAM), etoposide (ETO) or vincristine (VCR). Sublethal concentrations of drugs were used to exclude short-term effects caused by selection. Cell cycle analysis was performed to identify possible correlation between resistance factors, PKC isozymes and proliferation. We found a mostly combined induction of MDR1, LRP, PKC tau and PKC zeta by CAM, ETO and VCR. PKC alpha, epsilon, eta and theta gene expression altered variably. Cell cycle analysis showed that A2780 cells responded with a marked G2/M arrest after a 24-hour treatment with CAM, ETO and VCR but an association between the induction of PKC isozymes corresponding genes and proliferation was not seen. Our analysis points to a possible link between atypical PKC tau/PKC zeta and MDR1/LRP in cytostatic stress response of cancer cells.

Bone marrow-derived factors support growth of N-type, but not of melanocytic neuroblastoma cells.

BACKGROUND: Neuroblastoma and melanoma cells have a common embryonal origin. In contrast to melanoma, most neuroblastoma tumours preferentially metastasize into bone marrow. Previously, we described that bone marrow-conditioned medium (BM-CM) supports the proliferation of catecholamine-producing (N-type) neuroblastoma (SK-N-SH, IMR-32, Kelly)-, but not of melanoma cells. Both neuroblastoma and melanoma produce DOPA (3,4 dihydroxyphenylalanine); while melanoma cells use tyrosinase for DOPA synthesis, neuroblastoma cells usually utilize tyrosine hydroxylase. RESULTS: Certain neuroblastoma cells (in our study: SK-N-LO, LS, SH-EP) express tyrosinase instead of tyrosine hydroxylase for synthesis of DOPA, and do not synthesize catecholamines, as shown by HPLC and RT-PCR analysis. Strikingly and in contrast to catecholamine-producing N-type cells, the proliferation of these melanocytic neuroblastoma cells is not supported by BM-CM. CONCLUSION: With respect to proliferation in the presence of BM-CM, melanocytic neuroblastoma cells behave more like melanoma cells and may represent the subfraction of neuroblastoma cells with a minor tendency to metastasize into bone marrow.

Low-density lipoprotein modification by normal, myeloperoxidase-deficient and NADPH oxidase-deficient granulocytes and the impact of redox active transition metal ions.

The modification of low-density lipoprotein (LDL) by normal, myeloperoxidase (MPO)-deficient and NADPH oxidase-deficient granulocytes was investigated using the monoclonal antibody (mAb) OB/04, which was originally generated against copper-oxidized LDL. Incubation of LDL with normal granulocytes increased the reactivity of LDL with mAb OB/04. These effects were even more pronounced using MPO-deficient granulocytes. Inhibitors of oxidative reactions (the NADPH oxidase inhibitor diphenyleneiodonium chloride [DPI], catalase, superoxide dismutase [SOD]) did not significantly reduce LDL oxidation by normal granulocytes. Furthermore, granulocytes of a patient with NADPH oxidase deficiency were almost equally effective as normal granulocytes, indicating that oxidative burst-derived reactive oxygen species are of only minor importance in the generation of mAb OB/04-detectable new epitopes on LDL in vitro. In contrast, incubation of LDL with iron and copper prior to and during incubation with normal granulocytes markedly enhanced the generation of OB/04-detectable epitopes. It is supposed that, besides superoxide (in normal and MPO-deficient granulocytes) or instead of superoxide (in NADPH oxidase-deficient granulocytes), lytic enzymes released by activated granulocytes may enhance the availability of transition metals for oxidation of LDL. Our results support the concept that transition-metal-dependent pathways of LDL oxidation in combination with degranulation products of granulocytes are important.

Differences in the accumulation of ascorbic acid in normal, myeloperoxidase deficient and NADPH-oxidase deficient granulocytes.

Granulocytes contain large quantities of ascorbic acid (AA). The uptake mechanism is mainly restricted to the accumulation of the oxidized form, dehydroascorbate (DHA). We investigated the uptake of ascorbic acid and dehydroascorbate of normal, myeloperoxidase (MPO)-deficient, and NADPH-oxidase-deficient granulocytes. The accumulation of ascorbic acid was increased in all types of granulocytes after stimulation with phorbol-myristate-acetate, whereas the NADPH-oxidase-deficient cells showed a decreased uptake compared to normal and MPO-deficient cells. The intracellular concentration of ascorbic acid was further enhanced after incubation of granulocytes with DHA, most prominently in NADPH-oxidase-deficient granulocytes. MPO-deficient granulocytes are not able to produce HOCl after activation. The granulocytes of one individual with total MPO deficiency accumulated ascorbate in higher concentrations than did cells with partial MPO deficiency, indicating that HOCl is of minor importance for the oxidation of ascorbate. Since the ability of MPO-deficient cells to kill microorganisms is pronounced in contrast to NADPH-oxidase-deficient cells, effective mechanisms of compensating for the absence of HOCl must exist. We hypothesize that the enhanced uptake of ascorbic acid combined with an enhanced superoxide anion production may favor the generation of OH radicals via the Fenton reaction.