An efficient and reproducible plant regeneration system, initiated in somatic tissues, has been devised for cassava (Manihot esculenta Crantz). Somatic embryogenesis has been induced from shoot tips and immature leaves of in vitro shoot cultures of 15 cassava genotypes. Somatic embryos developed directly on the explants when cultured on a medium containing 4-16 mg/l 2,4-D. Differences were observed with respect to the embryogenic capacity of the explants of different varieties. Secondary embryogenesis has been induced by subculture on solid or liquid induction medium. Long term cultures were established and maintained for up to 18 months by repeated subculture of the proliferating somatic embryos. Plantlets developed from primary and secondary embryos in the presence of 0.1 mg/l BAP, 1mg/l GA3, and 0.01 mg/l 2,4-D. Regenerated plants were transferred to the field, and were grown to maturity.

BackgroundExperimental evidence and clinical studies in breast cancer suggest that some anti-tumor therapy regimens generate stimulation of the immune system that accounts for tumor clinical responses, however, demonstration of the immunostimulatory power of these therapies on cancer patients continues to be a formidable challenge. Here we present experimental evidence from a breast cancer patient with complete clinical response after 7 years, associated with responsiveness of tumor specific T cells.MethodsT cells were obtained before and after anti-tumor therapy from peripheral blood of a 63-years old woman diagnosed with ductal breast cancer (HER2/neu+++, ER-, PR-, HLA-A*02:01) treated with surgery, followed by paclitaxel, trastuzumab (suspended due to cardiac toxicity), and radiotherapy. We obtained a leukapheresis before surgery and after 8 months of treatment. Using in vitro cell cultures stimulated with autologous monocyte-derived dendritic cells (DCs) that produce high levels of IL-12, we characterize by flow cytometry the phenotype of tumor associated antigens (TAAs) HER2/neu and NY-ESO 1 specific T cells. The ex vivo analysis of the TCR-Vβ repertoire of TAA specific T cells in blood and Tumor Infiltrating Lymphocytes (TILs) were performed in order to correlate both repertoires prior and after therapy.ResultsWe evidence a functional recovery of T cell responsiveness to polyclonal stimuli and expansion of TAAs specific CD8+ T cells using peptide pulsed DCs, with an increase of CTLA-4 and memory effector phenotype after anti-tumor therapy. The ex vivo analysis of the TCR-Vβ repertoire of TAA specific T cells in blood and TILs showed that whereas the TCR-Vβ04-02 clonotype is highly expressed in TILs the HER2/neu specific T cells are expressed mainly in blood after therapy, suggesting that this particular TCR was selectively enriched in blood after anti-tumor therapy.ConclusionsOur results show the benefits of anti-tumor therapy in a breast cancer patient with clinical complete response in two ways, by restoring the responsiveness of T cells by increasing the frequency and activation in peripheral blood of tumor specific T cells present in the tumor before therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2625-2) contains supplementary material, which is available to authorized users.

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BackgroundVaccination of mice with tumors treated with Doxorubicin promotes a T cell immunity that relies on dendritic cell (DC) activation and is responsible for tumor control in vaccinated animals. Despite Doxorubicin in combination with Cyclophosphamide (A/C) is widely used to treat breast cancer patients, the stimulating effect of A/C on T and APC compartments and its correlation with patient’s clinical response remains to be proved.MethodsIn this prospective study, we designed an in vitro system to monitor various immunological readouts in PBMCs obtained from a total of 17 breast cancer patients before, and after neoadjuvant anti-tumor therapy with A/C.ResultsThe results show that before treatment, T cells and DCs, exhibit a marked unresponsiveness to in vitro stimulus: whereas T cells exhibit poor TCR internalization and limited expression of CD154 in response to anti-CD3/CD28/CD2 stimulation, DCs secrete low levels of IL-12p70 and limited CD83 expression in response to pro-inflammatory cytokines. Notably, after treatment the responsiveness of T and APC compartments was recovered, and furthermore, this recovery correlated with patients’ residual cancer burden stage.ConclusionsOur results let us to argue that the model used here to monitor the T and APC compartments is suitable to survey the recovery of immune surveillance and to predict tumor response during A/C chemotherapy.Electronic supplementary materialThe online version of this article (10.1186/s12885-017-3982-1) contains supplementary material, which is available to authorized users.

Neem (Azadirachta indica) produces several of compounds known as limonoids, which have an antifeedant effect on insects. These compounds are extremely sensitive to some environmental factors that cause their degradation. Despite this, they are widely used in many formulations of commercial bioinsecticides. We evaluated the photodegradation of the crude extract from A. indica cell culture and designed the formulation of a botanical active substance for controlling insects. The crude extract was subjected to 368 nm UV light for 24 h, and its degradation was examined. Limonoids present in the crude extract were analyzed via high-performance liquid chromatography (HPLC). The composition of some compounds in the extract decreased by 55% after 214 min and 83% after 1440 min. For the insect bioassay, we prepared six formulations containing ethanolic extracts from A. indica cell culture as the active ingredient. The formulation also contained a photoprotector and two stabilizers, emulsified with water, castor oil, and Tween 80. Formulations were subjected to stability tests, and the relative phase separation was assessed. To evaluate their biological activity, the antifeedant index and the affected leaf area on corn infested with Spodoptera frugiperda were determined using laboratory-and field-scale bioassays. Three formulations showed good stability, and two presented the highest antifeedant indices (98.5 and 99.7%) in laboratory-scale bioassays. They provided the greatest field-level protection (leaf areas affected were 0.6 and 1.9%, respectively). Therefore, the emulsion containing 0.76% p/p ethanolic extract, 0.72% 8-hydroxyquinoline, 1.00% anthraquinone and epichlorohydrin, 0.20% Tween 80, and 50/50 aqueous phase/oil phase was selected as the best formulation for the insect biocontroller. This thus addresses the problem of metabolite degradation in the field. To our knowledge, this is the first effective formulation of a botanical active substance for controlling insects using A. indica cell culture extract.

In order to assess the antifungal activity of methanolic extracts from neem tree (Azadirachta indica A. Juss.), several bioassays were conducted following M38-A2 broth microdilution method on 14 isolates of the dermatophytes Trichophyton mentagrophytes, Trichophyton rubrum, Microsporum canis and Epidermophyton floccosum. Neem extracts were obtained through methanol-hexane partitioning of mature green leaves and seed oil. Furthermore, high performance liquid chromatography (HPLC) analyses were carried out to relate the chemical profile with their content of terpenoids, of widely known antifungal activity. The antimycotic Terbinafine served as a positive control. Results showed that there was total growth inhibition of the dermatophytes isolates at minimal inhibitory concentrations (MIC) between 50 μg/mL and 200 μg/mL for leaves extract, and between 625 μg/mL and 2500 μg/mL for seed oil extract. The MIC of positive control (Terbinafine) ranged between 0.0019 μg/mL and 0.0313 μg/mL. Both neem leaves and seed oil methanol extracts exhibited different chromatographic profiles by HPLC, which could explain the differences observed in their antifungal activity. This analysis revealed the possible presence of terpenoids in both extracts, which are known to have biological activity. The results of this research are a new report on the therapeutic potential of neem to the control of dermatophytosis.Keywords: HPLC, neem, microbial sensitivity tests, minimum inhibitory concentration, terpenoids. RESUMENSe determinó la actividad antifúngica de extractos metanólicos de la especie Azadirachta indica A. Juss. (Meliaceae), conocida comúnmente como neem, empleando el método de microdilución en caldo M38-A2 de referencia para hongos filamentosos y dermatofitos. Se evaluaron 14 aislamientos de los dermatofitos Trichophyton mentagrophytes, Trichophyton rubrum, Microsporum canis y Epidermophyton floccosum. Los extractos de neem fueron obtenidos mediante partición metanol-hexano a partir de aceite de semillas y hojas verdes maduras. Adicionalmente, se analizaron por cromatografía líquida de alta resolución (CLAR) con el fin de relacionar su perfil químico con el contenido de terpenoides, de conocida actividad antifúngica. Se empleó como control positivo el antimicótico Terbinafina. Los resultados mostraron inhibición total del crecimiento de los aislamientos de dermatofitos a concentraciones mínimas inhibitorias (CMI) entre 50 μg/mL y 200 μg/mL para el extracto de hojas y entre 625 μg/mL y 2500 μg/mL para el extracto de aceite de semillas. La CMI encontrada para el control positivo (Terbinafina) fluctuó entre 0,0078 μg/mL y 0,0313 μg/mL. Los extractos metanólicos de hojas y aceite de semillas de neem exhibieron diferentes perfiles cromatográficos en CLAR, lo cual podría explicar las diferencias observadas en su actividad antifúngica. Éste análisis químico reveló la posible presencia de compuestos INTRODUCTIONThe research about neem has been focused not only on its known antifeedant activity on insects, but also on its antifung...

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IntroductionAnimal studies and preclinical studies in cancer patients suggest that the induction of immunogenic cell death (ICD) by neoadjuvant chemotherapy with doxorubicin and cyclophosphamide (NAC-AC) recovers the functional performance of the immune system. This could favor immunotherapy schemes such as the administration of antigen-free autologous dendritic cells (DCs) in combination with NAC-AC to profit as cryptic vaccine immunogenicity of treated tumors.ObjectiveTo explore the safety and immunogenicity of autologous antigen-free DCs administered to breast cancer patients (BCPs) in combination with NAC-AC.Materials and MethodsA phase I/II cohort clinical trial was performed with 20 BCPs treated with NAC-AC [nine who received DCs and 11 who did not (control group)]. The occurrence of adverse effects and the functional performance of lymphocytes from BCPs before and after four cycles of NAC-AC receiving DCs or not were assessed using flow cytometry and compared with that from healthy donors (HDs). Flow cytometry analysis using manual and automated algorithms led us to examine functional performance and frequency of different lymphocyte compartments in response to a stimulus in vitro. This study was registered at clinicaltrials.gov (NCT03450044).ResultsNo grade II or higher adverse effects were observed associated with the transfer of DCs to patients during NAC-AC. Interestingly, in response to the in vitro stimulation, deficient phosphorylation of Zap70 and AKT proteins observed before chemotherapy in most patients’ CD4 T cells significantly recovered after NAC-AC only in patients who received DCs.ConclusionsThe transfer of autologous DCs in combination with NAC-AC in BCPs is a safe procedure. That, in BCPs, the administration of DCs in combination with NAC-AC favors the recovery of the functional capacity of T cells suggests that this combination may potentiate the adjuvant effect of ICD induced by NAC-AC on T cells and, hence, potentiate the immunogenicity of tumors as cryptic vaccines.