Biochemical study of the effect of stress conditions on the mandelonitrile-associated salicylic acid biosynthesis in peach.

Salicylic acid (SA) plays a central role in plant responses to environmental stresses. In a recent study, we suggested a third pathway for SA biosynthesis from mandelonitrile (MD) in peach plants. This pathway is an alternative to the phenylalanine ammonia-lyase pathway and links SA biosynthesis and cyanogenesis. In the present work, using biochemical approaches, we studied the effect of salt stress and Plum pox virus (PPV) infection on this proposed SA biosynthetic pathway from MD. Peach plants were submitted to salt stress and Plum pox virus (PPV) infection. We studied the levels of SA and its intermediates/precursors (phenylalanine, MD, amygdalin and benzoic acid) in in vitro shoots. Moreover, in peach seedlings, we analysed the content of H2 O2 -related enzymes, SA and the stress-related hormones abscisic acid and jasmonic acid. We showed that the contribution of this SA biosynthetic pathway from MD to the total SA pool does not seem to be important under the stress conditions assayed. Nevertheless, MD treatment not only affected the SA content, but also had a pleiotropic effect on abscisic acid and jasmonic acid levels. Furthermore, MD modulates the antioxidative metabolism via SA-dependent or -independent redox-related signalling pathways. Even though the proposed SA biosynthetic pathway seems to be functional under stress conditions, MD, and hence cyanogenic glycosides, may be operating more broadly than by influencing SA pathways and signalling. Thus, the physiological function of the proposed SA biosynthetic pathway remains to be elucidated.

The effect of mandelonitrile, a recently described salicylic acid precursor, on peach plant response against abiotic and biotic stresses.

In a previous work, we observed that mandelonitrile (MD), which controls cyanogenic glycoside turnover, is involved in salicylic acid (SA) biosynthesis in peach plants. In order to gain knowledge about the possible roles of this SA biosynthetic pathway, this current study looks at the effect of MD and phenylalanine (Phe; MD precursor) treatments on peach plant performance from an agronomic point of view. Abiotic (2 g·l-1 NaCl) and biotic (Plum pox virus, PPV) stresses were assayed. We recorded the following chlorophyll fluorescence parameters: quantum yield of photochemical energy conversion in PSII [Y(II)], photochemical quenching (qP) and quantum yield of regulated non-photochemical energy loss in PSII and its coefficient [Y(NPQ) and qN]. In addition, considering that environmental stresses lead to nutritional disorders, we determined the soluble K+ , Ca2+ , Na+ and Cl- concentrations in NaCl-stressed seedlings. In PPV-infected seedlings, we recorded the Ca2+ level, which has been suggested to play critical roles in regulating SA-related plant defence responses against pathogens. The MD treatment lessened the effect of both stresses on plant development. In addition, an increase in non-photochemical quenching parameters was observed in MD-treated seedlings, suggesting a safer dissipation of excess energy under stress conditions. In NaCl-stressed peach seedlings both treatments stimulated the accumulation of phytotoxic ions in roots, whereas in PPV-infected seedlings MD increased Ca2+ content. Our results suggest that MD and Phe influence the response of peach seedlings to the deleterious effects of salt and PPV infection stresses.

Salicylic acid negatively affects the response to salt stress in pea plants.

We studied the effect of salicylic acid (SA) treatment on the response of pea plants to salinity. Sodium chloride (NaCl)-induced damage to leaves was increased by SA, which was correlated with a reduction in plant growth. The content of reduced ascorbate and glutathione in leaves of salt-treated plants increased in response to SA, although accumulation of the respective oxidised forms occurred. An increase in hydrogen peroxide also occurred in leaves of salt-exposed plants treated with SA. In the absence of NaCl, SA increased ascorbate peroxidase (APX; 100 µm) and glutathione-S transferase (GST; 50 µm) activities and increased catalase (CAT) activity in a concentration-dependent manner. Salinity decreased glutathione reductase (GR) activity, but increased GST and CAT activity. In salt-stressed plants, SA also produced changes in antioxidative enzymes: 100 µm SA decreased APX but increased GST. Finally, a concentration-dependent increase in superoxide dismutase (SOD) activity was induced by SA treatment in salt-stressed plants. Induction of PR-1b was observed in NaCl-stressed plants treated with SA. The treatment with SA, as well as the interaction between salinity and SA treatment, had a significant effect on PsMAPK3 expression. The expression of PsMAPK3 was not altered by 70 mm NaCl, but was statistically higher in the absence than in the presence of SA. Overall, the results show that SA treatment negatively affected the response of pea plants to NaCl, and this response correlated with an imbalance in antioxidant metabolism. The data also show that SA treatment could enhance the resistance of salt-stressed plants to possible opportunistic pathogen attack, as suggested by increased PR-1b gene expression.

Benzothiadiazole and l-2-oxothiazolidine-4-carboxylic acid reduce the severity of Sharka symptoms in pea leaves: effect on antioxidative metabolism at the subcellular level.

The effect of treatment with benzothiadiazole (BTH) or l-2-oxothiazolidine-4-carboxylic acid (OTC), and their interaction with Plum pox virus (PPV) infection, on antioxidative metabolism of pea plants was studied at the subcellular level. PPV infection produced a 20% reduction in plant growth. Pre-treatment of pea plants with OTC or BTH afforded partial protection against PPV infection, measured as the percentage of leaves showing symptoms, but neither BTH nor OTC significantly reduced the virus content. PPV infection caused oxidative stress, as monitored by increases in lipid peroxidation and protein oxidation in soluble and chloroplastic fractions. In leaves of non-infected plants, OTC increased the content of reduced glutathione (GSH) and total glutathione; accordingly, an increase in the redox state of glutathione was observed. An increase in oxidized glutathione (GSSG) was found in symptomatic leaves from infected plants. A similar increase in GSSG was also observed in asymptomatic leaves from infected, untreated plants. However, no changes in GSSG occurred in asymptomatic leaves from infected plants treated with BTH and OTC and, accordingly, a higher redox state of GSH was recorded in those leaves, which could have had a role in the reduction of symptoms, as observed in asymptomatic leaves from infected plants treated with BTH or OTC. Treatment with BTH or OTC had some effect on antioxidant enzymes in soluble and chloroplastic fractions from infected pea leaves. An increase in antioxidative mechanisms, such as GSH-related enzymes (DHAR, GR and G6PDH), as well as APX and POX, at the subcellular level was observed, which could play a role in reducing the severity of cellular damage induced by Sharka in pea leaves.

Interaction between hydrogen peroxide and plant hormones during germination and the early growth of pea seedlings.

Hydrogen peroxide (H(2)O(2)) increased the germination percentage of pea seeds, as well as the growth of seedlings in a concentration-dependent manner. The effect of H(2)O(2) on seedling growth was removed by incubation with 10 microm ABA. The H(2)O(2)-pretreatment produced an increase in ascorbate peroxidase (APX), peroxidase (POX) and ascorbate oxidase (AAO). The increases in these ascorbate-oxidizing enzymes correlated with the increase in the growth of the pea seedlings as well as with the decrease in the redox state of ascorbate. Moreover, the increase in APX activity was due to increases in the transcript levels of cytosolic and stromal APX (cytAPX, stAPX). The proteomic analysis showed that H(2)O(2) induced proteins related to plant signalling and development, cell elongation and division, and cell cycle control. A strong correlation between the effect of H(2)O(2) on plant growth and the decreases in ABA and zeatin riboside (ZR) was observed. The results suggest an interaction among the redox state and plant hormones, orchestrated by H(2)O(2), in the induction of proteins related to plant signalling and development during the early growth of pea seedlings.

The apoplastic antioxidant system in Prunus: response to long-term plum pox virus infection.

This work describes, for the first time, the changes taking place in the antioxidative system of the leaf apoplast in response to plum pox virus (PPV) in different Prunus species showing different susceptibilities to PPV. The presence of p-hydroxymercuribenzoic acid (pHMB)-sensitive ascorbate peroxidase (APX) (class I APX) and pHMB-insensitive APX (class III APX), superoxide dismutase (SOD), peroxidase (POX), NADH-POX, and polyphenoloxidase (PPO) was described in the apoplast from both peach and apricot leaves. PPV infection produced different changes in the antioxidant system of the leaf apoplast from the Prunus species, depending on their susceptibility to the virus. In leaves of the very susceptible peach cultivar GF305, PPV brought about an increase in class I APX, POX, NADH-POX, and PPO activities. In the susceptible apricot cultivar Real Fino, PPV infection produced a decrease in apoplastic POX and SOD activities, whereas a strong increase in PPO was observed. However, in the resistant apricot cultivar Stark Early Orange, a rise in class I APX as well as a strong increase in POX and SOD activities was noticed in the apoplastic compartment. Long-term PPV infection produced an oxidative stress in the apoplastic space from apricot and peach plants, as observed by the increase in H2O2 contents in this compartment. However, this increase was much higher in the PPV-susceptible plants than in the resistant apricot cultivar. Only in the PPV-susceptible apricot and peach plants was the increase in apoplastic H2O2 levels accompanied by an increase in electrolyte leakage. No changes in the electrolyte leakage were observed in the PPV-inoculated resistant apricot leaves, although a 42% increase in the apoplastic H2O2 levels was produced. Two-dimensional electrophoresis analyses revealed that the majority of the polypeptides in the apoplastic fluid had isoelectric points in the range of pI 4-6. The identification of proteins using MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) and peptide mass fingerprinting analyses showed the induction of a thaumatin-like protein as well as the decrease of mandelonitrile lyase in peach apoplast due to PPV infection. However, most of the selected polypeptides showed no homology with known proteins. This fact emphasizes that, at least in Prunus, most of the functions of the apoplastic space remain unknown. It is concluded that long-term PPV infection produced an oxidative stress in the leaf apoplast, contributing to the deleterious effects produced by PPV infection in leaves of inoculated, susceptible Prunus plants.

Prognostic value of minimal residual disease (MRD) in acute myeloid leukemia (AML) with favorable cytogenetics [t(8;21) and inv(16)].

Most patients with acute myeloid leukemia (AML) and t(8;21) or inv(16) have a good prognosis with current anthracycline- and cytarabine-based protocols. Tandem analysis with flow cytometry (FC) and real-time RT-PCR (RQ-PCR) was applied to 55 patients, 28 harboring a t(8;21) and 27 an inv(16), including one case with a novel CBFbeta/MYH11 transcript. A total of 31% (n=17) of CR patients relapsed: seven with t(8;21) and 10 with inv(16). The mean amount of minimal residual disease (MRD) detected by FC in relapsed and nonrelapsed patients was markedly different: 0.3 vs 0.08% (P=0.002) at the end of treatment. The mean number of fusion transcript copies/ ABL x 10(4) also differed between relapsed and non-relapsed patients: 2385 vs 122 (P=0.001) after induction, 56 vs 7.6 after intensification (P=0.0001) and 75 vs 3.3 (P=0.0001) at the end of chemotherapy. Relapses were more common in patients with FC MRD level >0.1% at the end of treatment than in patients with < or = 0.1%: cumulative incidence of relapse (CIR) was 67 and 21% (P=0.03), respectively. Likewise, using RQ-PCR, a cutoff level of >10 copies at the end of treatment correlated with a high risk of relapse: CIR was 75% for patients with RQ-PCR >10 compared to 21% for patients with RQ-PCR levels < or = 10 (P=0.04). Combined use of FC and RQ-PCR may improve MRD detection, and provide useful clinical information on relapse kinetics in AML patients.

Adverse prognostic impact of CD36 and CD2 expression in adult de novo acute myeloid leukemia patients.

BACKGROUND AND OBJECTIVES: A consecutive series of acute myeloid leukemias (AML) patients was analyzed in conditions which reduce the inter-assay variations (the same flow cytometer, the same observers and the same panel of monoclonal antibodies) in order to investigate the prognostic information provided by flow cytometry. DESIGN AND METHODS: Two hundred and sixty-six bone marrow (BM) samples from 326 patients enrolled in the LMA-99 protocol from the CETLAM group were studied by multiparametric flow cytometry. Immunophenotyping studies were performed on erythrocyte-lysed BM samples. Antigen expression of leukemic cells was analyzed using triple stainings with fluorochrome-conjugated combinations of monoclonal antibodies. RESULTS: CD2 was positive in 21 cases (8%); an associated inv(16) was detected in eight CD2+ cases (38%). Two-year overall survival (OS) rate for CD2+/inv(16)+ patients was 75%, whereas it was 0% for CD2+/inv(16)- patients and 47% for CD2- patients (p=0.0001). CD36 was expressed in 37% of patients (n=98). Two-year leukemia-free survival (LFS) rate was 34% for CD36+ patients and 55% for CD36- patients (p=0.001). In the multivariate analysis, CD2+ (RR=8.4; p=0.0001) and adverse karyotype (RR=10.2; p=0.0001) were associated with a lower CR rate, CD36+ (RR=1.5; p=0.03), CD2+ (RR=2; p=0.04) and adverse karyotype (RR=4; p=0.0001) were associated with a lower OS and CD36+ (RR=2; p=0.002) and adverse karyotype (RR=3.5; p=0.005) predicted a lower LFS. CONCLUSIONS: CD2+ patients had a very poor OS when CD2/inv(16)+ cases were excluded. CD36 and CD2 expression at diagnosis can provide prognostically important information in adult de novo AML.

Favourable effect of the combination of acute and chronic graft-versus-host disease on the outcome of allogeneic peripheral blood stem cell transplantation for advanced haematological malignancies.

To assess the influence of graft-versus-host disease (GVHD) on the outcome of patients with advanced haematological malignancies (AHM) who received a primary, unmodified allogeneic peripheral blood progenitor cells transplant (allo-PBT) from a human leucocyte antigen (HLA) identical sibling donor, we analysed 136 patients with myeloid neoplasms (n = 70) or lymphoproliferative disorders (n = 66), transplanted at 19 Spanish institutions. Median age was 35 years (range 1-61). The cumulative incidence of relapse for all patients was 34% (95% CI, 26-42%), 41% (95% CI, 33-49) for patients without GVHD and 14% (95% CI, 3-25) (P = 0.001) for patients with acute and chronic GVHD. After a median follow-up of 11 months (range 2-49), 60 (44%) patients remained alive with an actuarial probability of overall survival and disease-free survival (DFS) at 30 months of 31% (95% CI, 21-41%) and 28% (95% CI, 17-39%) respectively. In patients surviving > 100 d, the low incidence of relapse in those with acute and chronic GVHD led to a DFS of 57% (95% CI, 38-76%) compared with a DFS of 34% (95% CI, 17-51%) in the remaining patients (P = 0.03). Our results indicate a reduced incidence of relapse for patients with AHM receiving an unmodified allo-PBT and developing acute and chronic GVHD, which results in an improved DFS.

Rituximab can be useful as treatment for minimal residual disease in bcr-abl-positive acute lymphoblastic leukemia.

We report the results of administering CD20 monoclonal antibody (MoAb) in a 32-year-old man with bcr-abl-positive acute lymphoblastic leukemia. Morphological complete remission was achieved after two lines of chemotherapy with persistence of blast cells (2%) in flow cytometric analysis of marrow cells. Since no HLA-matched donor for allogeneic bone marrow transplantation (BMT) was found, anti-CD20 MoAb therapy was administered for in vivo marrow purging, prior to autologous peripheral blood stem cell (PBSC) harvest and transplantation. After MoAb therapy <0.1% of blast cells were observed and the molecular abnormality (bcr-abl gene rearrangement) disappeared.

Treatment with interleukin-2 (IL-2) and interferon (IFN(alpha 2b)) after autologous bone marrow or peripheral blood stem cell transplantation in onco-hematological malignancies with a high risk of relapse.

Nine patients with onco-hematological malignancies with a poor prognosis due to high risk of relapse received immunotherapy with interleukin-2 (IL-2) and interferon (IFN(alpha 2b)) s.c. as maintenance therapy after receiving autologous bone marrow or peripheral blood stem cell transplantation (ABMT/PBSCT). All the patients were considered at very high risk of relapse. We attempted to assess the efficiency, toxicity and clinical effects of these cytokines in these patients. Five patients were treated with high-dose of IL-2 and the other four patients with escalating doses every month. Side-effects in the first group of patients consisted of fever, chills, weakness, nausea, anorexia, loss of weight and local dermatitis in the injection site. Toxicity on the WHO scale was grade II in three patients and grade IV in the other two patients. In the second group of patients, the same clinical signs of toxicity appeared, but these were grade I on the WHO scale in all patients. None of the patients had infections or died in relation to administration of IL-2. Four patients died of relapse or progression of their hematological malignancies. The other five patients are alive, one in chronic phase of CML and the other four patients are in complete remission of their malignancies.

Immunomagnetic bone marrow purging in children with acute lymphoblastic leukemia.

Autologous bone marrow transplantation (ABMT) offers a therapeutic alternative for children with poor prognosis acute lymphoblastic leukemia (ALL) who lack an HLA-matched sibling donor. The most common cause of treatment failure after ABMT in these patients is leukemia relapse. We have developed an ex vivo autologous marrow purging program for children with ALL using an immunomagnetic method. BM purging has been performed in 37 children with ALL (31 B-lineage ALL and 6 T-lineage ALL) following an indirect method, using panels of mouse monoclonal antibodies (MAbs) directed against B or T cell antigens, Dynabeads M-450 (Dynal) coated with sheep antimouse (SAM) antibodies, and the MaxSep Magnetic Cell Separator (Baxter). Purging efficiency has been assessed by flow cytometry. Considering the limit of detection of target cells 0.1%, the median depletion was 2.0 log (range 0.8- > 2.8 log) for the B-lineage ALL and 2.7 (range 2.2- > 2, 9 log) for the T-lineage ALL patients. Twenty-seven patients have been autografted (6 in first complete remission, CR, 13 in second CR, and 8 in third or subsequent CR). Engraftment has been satisfactory in all of them, reaching levels of 500 neutrophils/mm3 and 20,000 platelets/mm3 after a median of 17 (range 12-39) and 30 (range 13-96) days post-ABMT, respectively. In summary, our results show that this immunomagnetic procedure achieves high levels of target cell depletion and can be safely applied to bone marrow purging in childhood ALL patients.

Allogeneic peripheral blood progenitor cell transplantation: analysis of short-term engraftment and acute GVHD incidence in 33 cases. allo-PBPCT Spanish Group.

The results of 33 allogeneic peripheral blood progenitor cells transplants (allo-PBPCT) in adult patients with hematologic malignancies were analyzed in a retrospective and multicenter study. In 21 of 33 cases (63%) the disease was refractory or in advanced stage and eight of the 33 cases (24%) were second transplants after relapse. Donors were treated with a median of 10 (4-16) micrograms/kg/day of rhG-CSF subcutaneously for 5-7 days. Three required a central venous line for harvesting. Peripheral blood leukapheresis product contained a median of 5.9 (1.8-13) 10(6)/kg CD34+ cells and a median of 309.5 (153-690) 10(6)/kg CD3+ cells. After a myeloablative regimen, all patients received PBPC from HLA-identical donors as the sole source of progenitor cells. Cyclosporin A (CsA) alone (n = 2), CsA and steroids (n = 9), and CsA and methotrexate (MTX) (n = 22) were used for GVHD prophylaxis. Growth factors post-transplant were given to 11 patients (33%). The median follow-up of the patients was 3 months. Actuarial median day for hemopoietic recovery was: neutrophils to >0.5 (>1) x 10(9)/l, day 14 (15); platelets to >20 (>50) x 10(9)/l, day 14 (21). The quantity of CD34+ cells infused did not significantly affect the engraftment kinetics, from a starting cutoff of 2.5 x 10(6)/kg. The speed of neutrophil recovery seemed to be influenced strongly by using rhG-CSF post-transplant and marginally by the type of GVHD prophylaxis. Actuarial probability for grade II-IV acute GVHD of the whole group was 37% (95% Cl, 20-54%).

Feasibility and results of bone marrow transplantation after remission induction and intensification chemotherapy in de novo acute myeloid leukemia. Catalan Group for Bone Marrow Transplantation.

PURPOSE: To evaluate prospectively the feasibility and results of bone marrow transplantation (BMT) after induction and intensification chemotherapy (CT) in patients with de novo acute myeloid leukemia (AML). PATIENTS AND METHODS: A total of 159 patients less than 51 years of age were treated. Induction CT consisted of daunorubicin 60 mg/m2 for 3 days, cytarabine (ARA-C) 100mg/m2 for 7 days, and etoposide 100 mg/m2 for 3 days. The first intensification therapy included mitoxantrone 10 mg/m2 for 3 days and ARA-C 1.2 g/m2 every 12 hours for 4 days. Amsacrine (100 or 150 mg/m2 for 3 days) and ARA-C (1.2 g/m2 every 12 hours for 2 or 4 days) were given as the second intensification therapy. Depending on the availability of a human leukocyte antigen (HLA)-identical sibling, the intention of treatment after CT was allogeneic BMT (allo-BMT) or autologous BMT (ABMT). RESULTS: Complete remission (CR) was obtained in 120 patients (75%) and partial remission (PR) in 11 (7%), while 15 patients (10%) were refractory and 13 (8%) died during induction. There was a trend for better leukemia-free survival (LFS) at 4 years for patients assigned to the ABMT group (50% +/- 6%) compared with the allo-BMT group (31% +/- 7%) (P = .08). This difference in LFS reached statistical significance when considering only transplanted patients (63% +/- 3% at 4 years after ABMT and 38% +/- 11% after allo-BMT, P = .02). The favorable results in patients who received ABMT (no toxic deaths and 37% +/- 7% probability of relapse at 4 years) contrast with the poor outcome of allografted patients (11 patients with transplant-related mortality). CONCLUSION: Our study reflects the difficulties in the completion of a therapeutic strategy that include BMT and suggests that intensification before BMT may be useful in the setting of ABMT, but this approach was associated with a high mortality rate in allo-BMT patients.

Kaposi's sarcoma after autologous bone marrow transplantation for multiple myeloma.

We report a case of Kaposi's sarcoma in a patient who underwent autologous bone marrow transplantation (ABMT) for multiple myeloma. Four months after ABMT he presented with numerous asymptomatic, dark blue and purplish macules and nodules on the trunk and lower extremities. Biopsy revealed the typical histologic pattern of Kaposi's sarcoma. The patient died due to disseminated Kaposi's sarcoma while in complete remission of his hematologic malignancy.

Outpatient total body irradiation for bone marrow transplantation.

Five selected patients entering a BMT program were included in a prospective feasibility study to evaluate the tolerance to total body irradiation (TBI) on an outpatient basis. Four fractions of 3 Gy in 4 consecutive days (8 Gy lung total dose) were given. Ondansetron 8 mg/8 h orally was used without sedation as anti-emetic regimen. After each treatment dose, patients went home where they remained in close telephone contact with the BMT team. After the last TBI fraction, patients were hospitalized and treated with cyclophosphamide 60 mg/g/day for 2 consecutive days. The outpatient TBI regimen was well tolerated in four cases. Only one patient presented with nausea and vomiting after the second treatment day. She was admitted to the hospital and treated with chlorpromazine. During the conditioning and hematological recovery period, no complications related to the outpatient TBI could be identified. We conclude that TBI can be given on an outpatient basis with safety. Additionally, it represents a cost saving of US$ 1160 per patient.