Impact of endophyte inoculation on the morphological identity of cultivars of Lolium perenne (L) and Festuca arundinacea (Schreb.).

Grass endophytes have been shown to confer enhanced environmental resilience to symbiont cultivars with reports of modified growth. If inoculating with an endophyte (E+) made an accession morphologically distinct from its registered endophyte free (E-) accession, there could be protection and ownership issues for testing authorities and breeders. This study investigated if, in official Plant Breeders Rights (PBR) field trials, the morphological characteristics of E+and E- accessions of perennial ryegrass and tall fescue cultivars were sufficiently modified to designate them as mutually distinct and also distinct from their definitive accessions (Def), held by the testing authorities. Testing perennial ryegrass on 17 characters at 2 sites generated 48,960 observations and for tall fescue on 9 characters at 1 site, 12,960 observations (each for 3 accessions of 4 cultivars × 60 plants × 2 growing cycles). Distinctness required a p < 0.01 difference in a single character from the combined over years analysis (COYD). A few significant differences were recorded between E- and E+accessions. Cultivar Carn E+ was smaller than Carn E- for Infloresence Length (p < 0.01) in both years but COYD analysis (p < 0.05) was insufficient to declare distinctiveness. Overall, the number of observed differences between E-/E+ accessions was less or similar to the number expected purely by chance. In contrast, comparisons between Def and E- or E+ accessions showed a number of significant differences that were substantially more numerous than expected by chance. These results showed no conclusive evidence of endophyte inclusion creating false PBR distinctions but unexpectedly, several E- and E+ accessions were distinguished from their official definitive stock.

Lymphopoiesis in transgenic mice over-expressing Artemis.

Artemis is a factor of the non-homologous end joining pathway involved in DNA double-strand break repair that has a critical role in V(D)J recombination. Mutations in DCLRE1C/ARTEMIS gene result in radiosensitive severe combined immunodeficiency in humans owing to a lack of mature T and B cells. Given the known drawbacks of allogeneic hematopoietic stem cell transplantation (HSCT), gene therapy appears as a promising alternative for these patients. However, the safety of an unregulated expression of Artemis has to be established. We developed a transgenic mouse model expressing human Artemis under the control of the strong CMV early enhancer/chicken beta actin promoter through knock-in at the ROSA26 locus to analyze this issue. Transgenic mice present a normal development, maturation and function of T and B cells with no signs of lymphopoietic malignancies for up to 15 months. These results suggest that the over-expression of Artemis in mice (up to 40 times) has no deleterious effects in early and mature lymphoid cells and support the safety of gene therapy as a possible curative treatment for Artemis-deficient patients.

Quantification of lentiviral vector copy numbers in individual hematopoietic colony-forming cells shows vector dose-dependent effects on the frequency and level of transduction.

Lentiviral vectors are effective tools for gene transfer and integrate variable numbers of proviral DNA copies in variable proportions of cells. The levels of transduction of a cellular population may therefore depend upon experimental parameters affecting the frequency and/or the distribution of vector integration events in this population. Such analysis would require measuring vector copy numbers (VCN) in individual cells. To evaluate the transduction of hematopoietic progenitor cells at the single-cell level, we measured VCN in individual colony-forming cell (CFC) units, using an adapted quantitative PCR (Q-PCR) method. The feasibility, reproducibility and sensitivity of this approach were tested with characterized cell lines carrying known numbers of vector integration. The method was validated by correlating data in CFC with gene expression or with calculated values, and was found to slightly underestimate VCN. In spite of this, such Q-PCR on CFC was useful to compare transduction levels with different infection protocols and different vectors. Increasing the vector concentration and re-iterating the infection were two different strategies that improved transduction by increasing the frequency of transduced progenitor cells. Repeated infection also augmented the number of integrated copies and the magnitude of this effect seemed to depend on the vector preparation. Thus, the distribution of VCN in hematopoietic colonies may depend upon experimental conditions including features of vectors. This should be carefully evaluated in the context of ex vivo hematopoietic gene therapy studies.

Validation of a mutated PRE sequence allowing high and sustained transgene expression while abrogating WHV-X protein synthesis: application to the gene therapy of WAS.

The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) is widely used in retroviral gene transfer vectors. However, this element contains an open-reading frame (ORF) encoding a truncated peptide of the woodchuck hepatitis virus X protein (WHX). Because we are developing a lentiviral vector for the gene therapy of Wiskott-Aldrich syndrome (WAS), we evaluated whether the WPRE was needed in the gene transfer cassette and tested the possibility of replacing it with a mutated derivative. The transcriptional activity of the WPRE was undetectable in the context of the lentiviral vector but the element was capable of translating a polypeptide. This capability was abrogated by mutating the WHX ORF translation start. The WPRE was required to express high levels of the transgene and for that, the native form or mutated derivatives functioned equivalently. The vector using a WAS gene promoter and the mut6 WPRE induced long-term expression of the WAS transgene in vivo, correcting cytoskeletal defects, thymocyte and B-cell numbers and improved the colitis of WAS-null mice. By providing additional evidence of efficacy of this WAS lentiviral vector with improved safety features, our results validate a mutated WPRE, which should be useful in future gene therapy applications.

Lentiviral vectors targeting WASp expression to hematopoietic cells, efficiently transduce and correct cells from WAS patients.

Gene therapy has been proposed as a potential treatment for Wiskott-Aldrich syndrome (WAS), a severe primary immune deficiency characterized by multiple hematopoietic-specific cellular defects. In order to develop an optimal lentiviral gene transfer cassette for this application, we compared the performance of several internal promoters in a variety of cell lineages from human WAS patients. Vectors using endogenous promoters derived from short (0.5 kb) or long (1.6 kb) 5' flanking sequences of the WAS gene, expressed the transgene in T, B, dendritic cells as well as CD34(+) progenitor cells, but functioned poorly in non-hematopoietic cells. Defects of T-cell proliferation and interleukin-2 production, and the cytoskeletal anomalies in WAS dendritic cells were also corrected. The levels of reconstitution were comparable to those obtained following transduction with similar lentiviral vectors incorporating constitutive PGK-1, EF1-alpha promoters or the spleen focus forming virus gammaretroviral LTR. Thus, native regulatory sequences target the expression of the therapeutic WAS transgene to the hematopoietic system, as is naturally the case for WAS, and are effective for correction of multiple cellular defects. These vectors may have significant advantages for clinical application in terms of natural gene regulation, and reduction in the potential for adverse mutagenic events.

A lentiviral vector encoding the human Wiskott-Aldrich syndrome protein corrects immune and cytoskeletal defects in WASP knockout mice.

Wiskott-Aldrich syndrome (WAS) is an immune deficiency with thrombopenia resulting from mutations in the WASP gene. This gene normally encodes the Wiskott-Aldrich syndrome protein (WASP), a major cytoskeletal regulator expressed in hematopoietic cells. Gene therapy is a promising option for the treatment of WAS, requiring that clinically applicable WASP gene transfer vectors demonstrate efficacy in preclinical studies. Here, we describe a self-inactivating HIV-1-derived lentiviral vector encoding human WASP and show that it effectively transduced bone marrow progenitor cells of WASP knockout (WKO) mice. Transplantation of these transduced cells into lethally irradiated WKO recipients led to stable expression of WASP and correction of immune, inflammatory and cytoskeletal defects. Splenic T-cell proliferation was restored, podosomes were reinstated on bone-marrow-derived dendritic cells and colon inflammation was reduced. This shows for the first time (a) that cytoskeletal defects can be corrected in WKO mice, (b) that human WASP is biologically active in mice and (c) that a lentiviral vector is effective to express human WASP in vivo over several months. These data support further development of such lentiviral vectors for the gene therapy of WAS.

Hematological recovery and peripheral blood progenitor cell mobilization after induction chemotherapy and GM-CSF plus G-CSF in breast cancer.

In order to determine the effect of GM-CSF plus G-CSF in combination in breast cancer patients receiving an effective induction regimen, we compared hematological recovery and peripheral blood progenitor cell (PBPC) mobilization according to colony-stimulating factor (CSF) support. Forty-three breast cancer patients were treated by TNCF (THP-doxorubicin, vinorelbine, cyclophosphamide, fluorouracil, D1 to D4) with CSF support: 11 patients received GM-CSF (D5 to D14); 16 patients G-CSF (D5 to D14) and 16 patients GM-CSF (D5-D14) plus G-CSF (D10-D14). Between two subsequent cycles, progenitor cells were assessed daily, from D13 to D17. The WBC count was similar for patients receiving G-CSF alone or GM-CSF plus G-CSF, but significantly greater than that of patients receiving GM-CSF alone (P<0.001). The GM-CSF plus G-CSF combination led to better PBPC mobilization, with significantly different kinetics (P<0.001) and optimal mean values of CFU-GM, CD34+ cells and cells in cycle, at D15 compared to those obtained with G-CSF or GM-CSF alone. The significantly greater PBPC mobilization obtained with a CSF combination by D15 could be of value for PBPC collection and therapeutic reinjection after high-dose chemotherapies.

Effect of ultrasound emulsification on cheese aroma encapsulation by carbohydrates.

The encapsulation of liquid cheese aroma (20%) in different carbohydrate matrices by spray-drying was investigated. Carbohydrate stabilized emulsions have been prepared by two emulsifying methods, ultrasonic or Ultra-Turrax treatments, and have been compared in terms of emulsion stability and encapsulation efficiency. The use of ultrasound is particularly effective to obtain a stable emulsion with maltodextrin as support, which is known for poor emulsification properties. In the same way, the spray-dried maltodextrin microcapsules were more effective for retaining cheese aroma when ultrasound (12.7 g/100 g of dry powder) was used for the emulsification step rather than Ultra-Turrax (10.7 g/100 g of dry powder). In terms of encapsulation efficiency, the best system of cheese aroma encapsulation is obtained using ultrasound for the emulsification step and modified starch as support (94.3%). With this support, the positive effect of ultrasound resulted in a lower microcaspsule size and in a higher aroma retention than when Ultra-Turrax was used (83.3%). Studies on the aroma profiles showed changes after encapsulation that depend not only on the nature of the support and the emulsification method but also on the interactions between the aroma compound and the matrix. In terms of flavor quality, the best system of cheese aroma encapsulation is obtained using ultrasound and maltodextrin as support.

[Neoadjuvant chemotherapy of breast cancer. Role of surgery in cases of complete clinical response]. / Chimiothérapie néo-adjuvante des cancers du sein. Place de la chirurgie en cas de réponse clinique complète.

OBJECTIVES: Assess the importance of systematic surgical exploration of the tumor focus in case of complete clinical response to neoadjuvant chemotherapy for operable breast cancer. PATIENTS AND METHODS: Between 1985 and 1997, first intention chemotherapy (4 to 6 cures) was given to 433 breast cancer patients with resectable tumors measuring > or = 3 cm but who required mammectomy. Complete clinical response was observed in 112 patients (26%); 31 of them had normal mammography and ultrasound examinations (7%). Local treatment in this favorable context varied: 82 patients underwent surgery (71 conservative procedures, 11 radical procedures) and 30 patients received radiation therapy alone. RESULTS: Complete histological response was obtained in 22 cases accounting for 6% of the entire series, 27% of the complete clinical responses, and 45% of the complete clinical and imaging responses. The incidence of local recurrence at a mean 107 month follow-up was compared between patients given complementary surgical or radiation therapy after complete clinical response. Recurrence was observed in 10 of the 82 operated patients versus 8 of the 30 nonoperated patients (12% versus 27%, NS). CONCLUSION: Complete clinical response after neoadjuvant chemotherapy does not rule out the need for surgical resection of the tumor focus because the risk of neoplastic reliquats remains high (3 out of 4 cases) and because the surgical specimen provides important histological information for prognosis (in vivo chemosensitivity test). lumpectomy also contributes to reducing the risk of local recurrence.

Mobilization of peripheral blood progenitor cells after induction chemotherapy (THP-doxorubicin-vinorelbine-cyclophosphamide-fluorouracil) and granulocyte colony-stimulating factor in breast cancer.

In order to evaluate the mobilization of peripheral blood progenitor cells (PBPC) after an effective induction regimen in breast cancer, we performed a study on 15 breast cancer patients. Between January 1995 and June 1996, these patients received TNCF (THP-doxorubicin. vinorelbine, cyclophosphamide, fluorouracil for four days, every 21 days) with G-CSF support (5 microg/kg for 10 days after chemotherapy) to reduce aplasia. This regimen is known to result in a complete pathological response in 30% of patients. Between two cycles of TNCF treatment, hematological recovery was observed. Progenitor cells (CFU-GM and CD34+ cells) and mononuclear cells in DNA synthesis (MCDS) counts were performed daily, between the 12th and 17th post-chemotherapy days (81 samples). The results showed a similarity for hematological recovery and PBPC mobilization kinetics depending on the number of treatment cycles. The three methods used for PBPC evaluation were well correlated (P < 0.01) with an optimal mean PBPC recruitment by the last day of G-CSF administration: respectively, 11 520 (1729-26539) CFU-GM/ml of blood, 249 (14-1160) CD34+ cells/microl of blood and 211 (21-554) MCDS/microl of blood. These results suggested that a daily injection of G-CSF after one or two TNCF cycles will produce an effective PBPC mobilization in comparison with currently used regimens.

Threonine and methionine are limiting amino acids for protein synthesis in patients with AIDS.

This study was conducted to identify the most rate-limiting amino acids for whole-body protein synthesis in acquired immunodeficiency syndrome (AIDS) patients. We postulated that an essential amino acid that would be rate limiting in AIDS should have a low basal plasma concentration and should remain at a low level during amino acid infusion. Seven male AIDS patients (median age 37 y, CD4 cell count: 76 mm-3) without any clinically active opportunistic infection during the month before the experiment were infused intravenously with a complete amino acid-glucose mixture for 2.5 h. Eight healthy volunteers were used as controls. Before the infusion, the concentrations of most free essential amino acids (methionine, threonine, histidine, isoleucine, leucine and tryptophan) were significantly lower (P < 0.05) in AIDS patients than in controls. Most plasma free essential amino acids increased significantly during infusion. However, the absolute increase above basal levels for threonine, valine, lysine, (P < 0.05) and methionine (P < 0.073) was smaller in AIDS patients than in control subjects. Thus, threonine and possibly methionine may be rate limiting for whole-body protein synthesis in AIDS patients, suggesting that there are selective amino acid requirements in patients with AIDS.

Primary chemotherapy in breast cancer: correlation between tumor response and patient outcome.

This study focused on the correlation between tumor response and patient outcome in 329 breast cancers treated with primary chemotherapy. There were 141 stage IIIB tumors, including 109 inflammatory carcinomas. Other malignancies (34 IIIA, 99 IIB, 55 IIA) were operable but considered to be too large (> 3 cm) for conservative surgery and received primary chemotherapy to avoid mastectomy. All received the AVCF regimen, comprising 4-week cycles of doxorubicin (30 mg/m2) day 1, vincristine (1 mg/m2) day 1, 5-fluorouracil (5-FU; 400 mg/m2) days 2 through 5, cyclophosphamide (300 mg/m2) days 2 through 5. In 189 cases, methotrexate (15 mg/m2) was added at day 2 and day 3. Patients received 6 cycles, then underwent locoregional treatment (surgery, radiotherapy, or both) according to tumor regression. The response rate was assessed by clinical, mammographic, and echographic examinations: a 50% rate of objective responses were noted, of which 15% were complete responses (tumor shrinkage allowed breast conservation in 68% of patients who had stages II or IIIA). For the whole population studied, median follow-up was 111 months (range, 60- 196). One hundred fifty-seven patients had disease relapse (48 local, 14 contralateral, 95 distant). Kaplan-Meier estimates showed an increased 10-year overall survival for patients in complete response, as compared with noncomplete response: 70% versus 50% (p < 0.03). Complete response to neoadjuvant chemotherapy seems a good prognostic factor.

Adjuvant chemotherapy with doxorubicin-containing regimen for 326 stage II breast cancers: 15-year results.

Between 1975 and 1986, 326 patients with stage II breast cancer were treated with an adjuvant combination of doxorubicin, vincristine, cyclophosphamide, and 5-fluorouracil (AVCF) following regional therapy (232 modified radical mastectomy, 94 lumpectomies, 304 irradiations). The AVCF regimen consisted of 4-week cycles of doxorubicin (30 mg/m2 day 1, modified radical mastectomy), vincristine (1 mg/m2 day 2), 5-fluorouracil 400 (mg/m2), and cyclophosphamide (300 mg/m2) days 3-6. Two hundred twenty-four patients (pts) had six cycles and 102 pts 12 cycles; 90 pts also received 30 mg daily tamoxifen for 1 year after chemotherapy. As of March 1994, the median follow-up was 130 months (range 86-221). One hundred eighteen pts developed recurrences (7 local, 19 controlateral, 92 metastatic) and 104 died. Estimated disease-free survival (DFS) was 5 years, 76 +/- 5%; 10 years, 64 +/- 5%; 15 years, 54 +/- 9%. Overall survival (OS) was 5 years, 85 +/- 4%; 10 years, 70 +/- 5%; 15 years, 58 +/- 10%. Survival was affected by the number of involved lymph nodes (258 pts were N+), menopausal status (OS at 15 years: 53% for MP+ and 65% for MP-) and Scarff-Bloom-Richardson grading, but not by hormonal receptors, number of courses, or associated hormonotherapy. Minimal cardiac toxicity was induced by doxorubicin either during or subsequent to treatment completion.

Clinical and pathological response to primary chemotherapy in operable breast cancer.

Neoadjuvant chemotherapy is used to improve patients' survival in locally-advanced and inflammatory breast cancer and to increase conservative surgical procedures in bulky tumours. Pathological complete responses are unusual. The aim of this pilot study was to assess the clinical and pathological response rates and to evaluate toxicity with a new protocol of primary chemotherapy in 50 high-risk breast cancer patients. All tumours were > 3 cm and had at least one other adverse prognostic factor: lymph node involvement (32 N1, 6 N2), SBR grade III (20), aneuploidy (29), negative hormonal receptors (19). Patients were treated by 3-week cycles of THP-doxorubicin 20 mg/m2 D1 to 3, vinorelbine 25 mg/m2 D1 and 4, cyclophosphamide 300 mg/m2 and 5-fluorouracil 400 mg/m2 D1 to 4 (TNCF). 38 patients received G-CSF or GM-CSF support. After 4-6 cycles, all underwent surgery (39 conservative, 11 modified radical). Tumour response was assessed clinically, by mammography and echography and on pathological specimens. An objective clinical response was observed for 43 patients: 26 complete (51%) and 18 partial (37%). After pathological review, 11 patients (22%) were devoid of any tumour cells, 4 others (8%) had only in situ carcinoma. From 253 evaluated cycles, grade III-IV toxicity occurred, 81% with neutropenia, 25% with anaemia, and 20% with thrombocytopenia. All patients recovered. This regimen induced a severe but not life-threatening haematological toxicity and resulted in a high pathological response rate (30%).

[First-line chemotherapy in operable breast neoplasms: results of 3 protocols]. / Chimiothérapie première dans les cancers du sein opérables supérieurs à 3 cm: résultats de 3 protocoles.

In order to avoid modified radical mastectomy, a neoadjuvant approach was adopted in our institute for operable bulky breast cancers. From January, 1982, to December, 1995, 288 patients received primary chemotherapy with 3 different regimens (all doses mg/m2): (1) AVCF/AVCFM, 167 patients (adriamycin 30, vincristine 1 d1, cyclophosphamide 300, fluorouracil 400 d2-d5 and methotrexate 20 d2 and d4, every 28 days); (2) NEM, 78 patients (vinorelbine 25, epirubicin 35, methotrexate 20 d1 and d8, every 28 days); and (3) TNCF, 43 patients (THP-adria 20, d1-d3, vinorelbine 25 d1 and d4, cyclophosphamide 300, fluorouracil 400 d1-d4, every 21 days). Evaluation of the response comprised 3 methods: clinical (C), echographic (E), mammographic (M). The overall objective response rate (C: 63/90/93; E: 49/61/85; M: 53/65/83%) is higher with regimens (2) and (3). The complete response rate was increased 2-fold with TNCF but the hematologic toxicity was very superior with this combination. Patients were all operated for (2) and (3), only several for (1), and the breast conservation rate (68/83/79%) was quite similar in the 3 regimens. The pathological complete response rate reached 23% with TNCF. However the impact on patient survival has to be confirmed.

Technetium-99m-sestamibi uptake in breast tumor and associated lymph nodes.

UNLABELLED: The aim of this study was to measure the accumulation of 99mTc-sestamibi in breast tumors and their axillary lymph nodes in patients undergoing scintimammography. METHODS: Eighteen patients who were scheduled for breast surgery underwent scintimammography with 740 MBq of 99mTc-sestamibi on the day before the operation. The next morning, reinjection with 370 MBq was performed. Immediately after the surgical procedure, the 99mTc activity of the tumor samples and, when available, the related lymph nodes was measured in a gamma counter. The samples were weighed and prepared for histological analysis. The activity of each sample was normalized to the mean activity of normal tissue samples obtained from the same patient. RESULTS: Among the 198 samples analyzed, the relative uptake of sestamibi was increased in 111 containing normal lymph nodes (1.80+/-0.79 vs 1.00+/-0.22, p<0.05), as well as in the seven containing invaded lymph nodes (2.01+/-0.83, p<0.01) and more dramatically, in the 22 with a carcinoma (5.64+/-3.06, p<0.001). In two patients with a benign lesion, both scintigraphy and counting demonstrated increased activity in the tumor. Four patients had negative scan results despite the presence of malignant tumor and a more than fourfold increase of sestamibi concentration in two of them. CONCLUSION: Technetium-99m-sestamibi concentrates strongly in breast carcinoma, sometimes even when the scan results appear normal, and mildly in lymph nodes, especially when invaded; it also concentrates in some benign tumors, possibly in relation to the presence of epithelial hyperplasia.

Response of leucine metabolism to hyperinsulinemia under amino acid replacement in experimental hyperthyroidism.

We investigated the responsiveness of protein metabolism to insulin as a mediator of the protein catabolic response to hyperthyroidism in humans. Six healthy volunteers were studied in a postabsorptive state before and after oral intake of thyroid hormones (2 micrograms.kg-1.day-1 L-thyroxine for 6 wk along with 1 microgram.kg-1.day-1 triiodothyronine for the last 2 wk). Insulin was infused at 7.14 nmol.kg-1.min-1 for 140 min under euglycemic and eukalemic clamps. An appropriate amino acid infusion was used to blunt insulin-induced hypoaminoacidemia. Leucine kinetics were assessed using a primed continuous infusion of L-[1-13C]leucine. Hyperthyroidism induced a significant increase (P < 0.05) in leucine endogenous appearance rate (a reflection of proteolysis; 2.15 +/- 0.06 vs. 1.76 +/- 0.03 mumol.kg-1.min-1 in the control state), oxidation (0.54 +/- 0.04 vs. 0.47 +/- 0.07), and nonoxidative disposal (a reflection of protein synthesis; 1.80 +/- 0.06 vs. 1.45 +/- 0.06). Insulin lowered proteolysis. Further hyperthyroidism improved the ability of insulin to inhibit proteolysis, whether considered as an absolute decrease (-0.57 +/- 0.02 vs. -0.45 +/- 0.05 mumol.kg-1.min-1, P < 0.05) or related to insulinemia [1.59 +/- 0.11 vs. 1.01 +/- 0.08 mumol leucine.kg-1.min-1/(nmol insulin/l), P < 0.05]. Insulin also moderately (but significantly P < 0.05) lowered protein synthesis in both control and hyperthyroid states. These changes in insulin action may provide a mechanism to save body protein during hyperthyroidism.