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Alwin D R Huitema - One of the best experts on this subject based on the ideXlab platform.
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quantification of the hiv integrase inhibitor raltegravir and detection of its main metabolite in human plasma dried blood spots and peripheral blood mononuclear Cell Lysate by means of high performance liquid chromatography tandem mass spectrometry
Journal of Pharmaceutical and Biomedical Analysis, 2009Co-Authors: R Ter Heine, Michel J X Hillebrand, Hilde Rosing, E C M Van Gorp, J W Mulder, Jos H Beijnen, Alwin D R HuitemaAbstract:For the quantification of the HIV-integrase inhibitor raltegravir in human plasma, dried blood spots and peripheral blood mononuclear Cell (PBMC) Lysate, an assay was developed and validated, using liquid chromatography coupled with tandem mass spectrometry. The assay also allowed detection, but no quantification due to absence of reference substance, of the main metabolite, raltegravir-glucuronide. Raltegravir was extracted from plasma by means of protein precipitation with a mixture of methanol and acetonitrile using only 50microL plasma. Extraction from dried blood spots was performed with a simple one-step extraction with a mixture of methanol, acetonitrile and 0.2M zincsulphate in water (1:1:2, v/v/v) and extraction from Cell Lysate was performed in 50% methanol in water. Chromatographic separation was performed on a reversed phase C18 column (150mmx2.0mm, particle size 5microm) with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.25mL/min. The analytical run time was 10min. The triple quadrupole mass spectrometer was operated in the positive ion-mode and multiple reaction monitoring was used for drug quantification. The method was validated over a range of 50-10,000ng/mL in plasma and dried blood spots and a range of 1-500ng/mL in PBMC Lysate. Dibenzepine was used as the internal standard. The method was proven to be specific, accurate, precise and robust. Accuracies ranged from 104% to 105% in plasma, from 93% to 105% in dried blood spots and from 82% to 113% in PBMC Lysate. Precision over the complete concentration range was less than 6%, 11% and 13% in plasma, dried blood spots and PBMC Lysate, respectively. The method is now applied for therapeutic drug monitoring and pharmacological research in HIV-infected patients treated with raltegravir.
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quantification of etravirine tmc125 in plasma dried blood spots and peripheral blood mononuclear Cell Lysate by liquid chromatography tandem mass spectrometry
Journal of Pharmaceutical and Biomedical Analysis, 2009Co-Authors: R Ter Heine, Hilde Rosing, J W Mulder, Jos H Beijnen, E C M Van Gorp, Alwin D R HuitemaAbstract:Abstract For the quantification of the novel non-nucleoside reverse transcriptase inhibitor etravirine in human plasma, dried blood spots and peripheral blood mononuclear Cell (PBMC) Lysate, an assay was developed and validated, using liquid chromatography coupled with tandem mass spectrometry. Etravirine was extracted from plasma by means of protein precipitation with a mixture of methanol and acetonitrile using only 50 μL plasma. Extraction from dried blood spots was performed with a one-step extraction with a mixture of methanol, acetonitrile and 0.2 M zinc sulphate in water (1:1:2, v/v/v) and extraction from Cell Lysate was performed in 50% methanol in water. Chromatographic separation was performed on a reversed phase C18 column (150 mm × 2.0 mm, particle size 5 μm) with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.25 mL/min. 13 C 6 -efavirenz was used as an internal standard. The analytical run time was only 10 min. The triple quadrupole mass spectrometer was operated in the positive ion-mode and multiple reaction monitoring was used for drug quantification. The method was validated over a range of 25–5000 ng/mL in plasma, 50–10,000 ng/mL in dried blood spots and a range of 5–2500 ng/mL in PBMC Lysate. Accuracies ranged from 89% to 106% in plasma, from 94% to 109% in dried blood spots and from 91% to 105% in PBMC Lysate. Precisions at the all concentration levels ranged from 1.9% to 14% in plasma, 4.7% to 20% in dried blood spots and from 3.1% to 11% in PBMC Lysate. The bioanalytical assay was successfully incorporated with previously developed assays for the determination of all currently approved PIs and NNRTIs in plasma and dried blood spots and it is now applied for therapeutic drug monitoring and pharmacological research in HIV-infected patients treated with etravirine.
Norman J Dovichi - One of the best experts on this subject based on the ideXlab platform.
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capillary zone electrophoresis multiple reaction monitoring from 100 pg of raw 264 7 Cell Lysate digest
Analyst, 2013Co-Authors: Yihan Li, Matthew M Champion, Roza Wojcik, Norman J DovichiAbstract:Capillary zone electrophoresis-multiple/single reaction monitoring (CZE-MRM/SRM), which employed an electrokinetically driven sheath-flow electrospray interface, was used for the rapid and highly sensitive detection of protein analytes in complex tryptic digests. MRM channels were developed against a commercial exponential mixture of bovine proteins. Five proteins spanning four orders of magnitude concentration range were confidently detected from only 2.5 ng of the digest mixture; the mass detection limits (S/N = 3) of two detected proteins, alpha-casein and glutamate dehydrogenase were about 600 zmol and 30 amol, respectively. This technique was then applied to a RAW 264.7 Cell Lysate digest. Three proteins were confidently and reproducibly detected from 100 pg of this digest. The sample amount corresponds to the approximate protein content from a single Cell, which suggests that CZE-MRM may be a useful analytical tool in chemical cytometry. In addition to providing highly sensitive detection of proteins in complex mixtures, this system is highly rapid; migration time of the protein digests was less than 10 min.
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integrated capillary zone electrophoresis electrospray ionization tandem mass spectrometry system with an immobilized trypsin microreactor for online digestion and analysis of picogram amounts of raw 264 7 Cell Lysate
Analytical Chemistry, 2013Co-Authors: Liangliang Sun, Guijie Zhu, Norman J DovichiAbstract:A capillary zone electrophoresis (CZE) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) system was integrated with an immobilized trypsin microreactor. The system was evaluated and then applied for online digestion and analysis of picogram loadings of RAW 264.7 Cell Lysate. Protein samples were dissolved in a buffer containing 50% (v/v) acetonitrile (ACN), and then directly loaded into the capillary for digestion, followed by CZE separation and MS/MS identification. The organic solvent (50% (v/v) ACN) assisted the immobilized trypsin digestion and simplified the protein sample preparation protocol. Neither protein reduction nor alkylation steps were employed, which minimized sample loss and contamination. The integrated CZE–ESI-MS/MS system generated confident identification of bovine serum albumin (BSA) with 19% sequence coverage and 14 peptide identifications (IDs) when 20 fmol was loaded. When only 1 fmol of BSA was injected, one BSA peptide was consistently detected. For the analysis of ...
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comparison of the ltq orbitrap velos and the q exactive for proteomic analysis of 1 1000 ng raw 264 7 Cell Lysate digests
Rapid Communications in Mass Spectrometry, 2013Co-Authors: Liangliang Sun, Guijie Zhu, Norman J DovichiAbstract:Rational There is interest in extending bottom-up proteomics to the smallest possible sample size. We investigated the performance of two modern instruments for the analysis of samples ranging from 1 ng to 1 µg of RAW 264.7 Cell Lysate digests.
Matthias Trost - One of the best experts on this subject based on the ideXlab platform.
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hydrophilic strong anion exchange hsax chromatography for highly orthogonal peptide separation of complex proteomes
Journal of Proteome Research, 2013Co-Authors: Maria Stella Ritorto, Ken Cook, Kshitiz Tyagi, Patrick G A Pedrioli, Matthias TrostAbstract:Due to its compatibility and orthogonality to reversed phase (RP) liquid chromatography (LC) separation, ion exchange chromatography, and mainly strong cation exchange (SCX), has often been the first choice in multidimensional LC experiments in proteomics. Here, we have tested the ability of three strong anion exchanger (SAX) columns differing in their hydrophobicity to fractionate RAW264.7 macrophage Cell Lysate. IonPac AS24, a strong anion exchange material with ultralow hydrophobicity, demonstrated to be superior to other materials by fractionation and separation of tryptic peptides from both a mixture of 6 proteins as well as mouse Cell Lysate. The chromatography displayed very high orthogonality and high robustness depending on the hydrophilicity of column chemistry, which we termed hydrophilic strong anion exchange (hSAX). Mass spectrometry analysis of 34 SAX fractions from RAW264.7 macrophage Cell Lysate digest resulted in an identification of 9469 unique proteins and 126318 distinct peptides in on...
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hydrophilic strong anion exchange hsax chromatography for highly orthogonal peptide separation of complex proteomes
Journal of Proteome Research, 2013Co-Authors: Maria Stella Ritorto, Ken Cook, Kshitiz Tyagi, Patrick G A Pedrioli, Matthias TrostAbstract:Due to its compatibility and orthogonality to reversed phase (RP) liquid chromatography (LC) separation, ion exchange chromatography, and mainly strong cation exchange (SCX), has often been the first choice in multidimensional LC experiments in proteomics. Here, we have tested the ability of three strong anion exchanger (SAX) columns differing in their hydrophobicity to fractionate RAW264.7 macrophage Cell Lysate. IonPac AS24, a strong anion exchange material with ultralow hydrophobicity, demonstrated to be superior to other materials by fractionation and separation of tryptic peptides from both a mixture of 6 proteins as well as mouse Cell Lysate. The chromatography displayed very high orthogonality and high robustness depending on the hydrophilicity of column chemistry, which we termed hydrophilic strong anion exchange (hSAX). Mass spectrometry analysis of 34 SAX fractions from RAW264.7 macrophage Cell Lysate digest resulted in an identification of 9469 unique proteins and 126318 distinct peptides in one week of instrument time. Moreover, when compared to an optimized high pH/low pH RP separation approach, the method presented here raised the identification of proteins and peptides by 10 and 28%, respectively. This novel hSAX approach provides robust, reproducible, and highly orthogonal separation of complex protein digest samples for deep coverage proteome analysis.
Cristian Pereda - One of the best experts on this subject based on the ideXlab platform.
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a heat shocked melanoma Cell Lysate vaccine enhances tumor infiltration by prototypic effector t Cells inhibiting tumor growth
Journal for ImmunoTherapy of Cancer, 2020Co-Authors: Maria Alejandra Gleisner, Cristian Pereda, Andres Tittarelli, Mariela Navarrete, Camila Fuentes, Ignacio AvalosAbstract:Background Immune checkpoint blocker (ICB) therapy has shown survival benefits for some patients with cancer. Nevertheless, many individuals remain refractory or acquire resistance to treatment, motivating the exploration of complementary immunotherapies. Accordingly, cancer vaccines offer an attractive alternative. Optimal delivery of multiple tumor-associated antigens combined with potent adjuvants seems to be crucial for vaccine effectiveness. Methods Here, a prototype for a generic melanoma vaccine, named TRIMELVax, was tested using B16F10 mouse melanoma model. This vaccine is made of heat shock-treated tumor Cell Lysates combined with the Concholepas concholepas hemocyanin as adjuvant. Results While B16F10 Lysate provides appropriate melanoma-associated antigens, both a generic human melanoma Cell Lysate and hemocyanin adjuvant contributes with danger signals promoting conventional dendritic type 1 Cells (cDC1), activation, phagocytosis and effective antigen cross-presentation. TRIMELVax inhibited tumor growth and increased mice survival, inducing Cellular and humoral immune responses. Furthermore, this vaccine generated an increased frequency of intratumor cDC1s but not conventional type 2 dendritic Cells (cDC2s). Augmented infiltration of CD3+, CD4+ and CD8+ T Cells was also observed, compared with anti-programmed Cell death protein 1 (PD-1) monotherapy, while TRIMELVax/anti-PD-1 combination generated higher tumor infiltration of CD4+ T Cells. Moreover, TRIMELVax promoted an augmented proportion of PD-1lo CD8+ T Cells in tumors, a phenotype associated with prototypic effector Cells required for tumor growth control, preventing dysfunctional T-Cell accumulation. Conclusions The therapeutic vaccine TRIMELVax efficiently controls the weakly immunogenic and aggressive B16F10 melanoma tumor growth, prolonging tumor-bearing mice survival even in the absence of ICB. The strong immunogenicity shown by TRIMELVax encourages clinical studies in patients with melanoma.
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melanoma Cell Lysate induces ccr7 expression and in vivo migration to draining lymph nodes of therapeutic human dendritic Cells
Immunology, 2014Co-Authors: Fermin E Gonzalez, Cristian Pereda, Mercedes N Lopez, Maria Alejandra Gleisner, Carolina Ortiz, Montserrat Reyes, Nicolas Dutzan, Vyomesh Patel, Silvio J Gutkind, Flavio SalazaronfrayAbstract:We have previously reported a novel method for the production of tumour-antigen-presenting Cells (referred to as TAPCells) that are currently being used in cancer therapy, using an allogeneic melanoma-derived Cell Lysate (referred to as TRIMEL) as an antigen provider and activation factor. It was recently demonstrated that TAPCell-based immunotherapy induces T-Cell-mediated immune responses resulting in improved long-term survival of stage IV melanoma patients. Clinically, dendritic Cell (DC) migration from injected sites to lymph nodes is an important requirement for an effective anti-tumour immunization. This mobilization of DCs is mainly driven by the C-C chemokine receptor type 7 (CCR7), which is up-regulated on mature DCs. Using flow cytometry and immunohistochemistry, we investigated if TRIMEL was capable of inducing the expression of the CCR7 on TAPCells and enhancing their migration in vitro, as well as their in vivo relocation to lymph nodes in an ectopic xenograft animal model. Our results confirmed that TRIMEL induces a phenotypic maturation and increases the expression of surface CCR7 on melanoma patient-derived DCs, and also on the monocytic/macrophage Cell line THP-1. Moreover, in vitro assays showed that TRIMEL-stimulated DCs and THP-1 Cells were capable of migrating specifically in the presence of the CCR7 ligand CCL19. Finally, we demonstrated that TAPCells could migrate in vivo from the injection site into the draining lymph nodes. This work contributes to an increased understanding of the biology of DCs produced ex vivo allowing the design of new strategies for effective DC-based vaccines for treating aggressive melanomas.
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tumour Cell Lysate loaded dendritic Cell vaccine induces biochemical and memory immune response in castration resistant prostate cancer patients
British Journal of Cancer, 2013Co-Authors: Diego Reyes, Lorena Salazar, E Espinoza, Cristian Pereda, Enrique A Castellon, R Valdevenito, C Huidobro, Ines M Becker, Alvaro Lladser, Mercedes N LopezAbstract:Background: Recently, we produced a tumour antigen-presenting Cells (TAPCells) vaccine using a melanoma Cell Lysate, called TRIMEL, as an antigen source and an activation factor. Tumour antigen-presenting Cells induced immunological responses and increased melanoma patient survival. Herein, we investigated the effect of TAPCells loaded with prostate cancer Cell Lysates (PCCL) as an antigen source, and TRIMEL as a dendritic Cell (DC) activation factor; which were co-injected with the Concholepas concholepas haemocyanin (CCH) as an adjuvant on castration-resistant prostate cancer (CRPC) patients. Methods: The Lysate mix capacity, for inducing T-Cell activation, was analysed by flow cytometry and Elispot. Delayed-type hypersensitivity (DTH) reaction against PCCL, frequency of CD8 þ memory T Cells (Tm) in blood and prostate-specific antigen (PSA) levels in serum were measured in treated patients. Results: The Lysate mix induced functional mature DCs that were capable of activating PCCL-specific T Cells. No relevant adverse reactions were observed. Six out of 14 patients showed a significant decrease in levels of PSA. DTH þ patients showed a prolonged PSA doubling-time after treatment. Expansion of functional central and effector CD8 þ Tm were detected.
Hilde Rosing - One of the best experts on this subject based on the ideXlab platform.
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quantification of the hiv integrase inhibitor raltegravir and detection of its main metabolite in human plasma dried blood spots and peripheral blood mononuclear Cell Lysate by means of high performance liquid chromatography tandem mass spectrometry
Journal of Pharmaceutical and Biomedical Analysis, 2009Co-Authors: R Ter Heine, Michel J X Hillebrand, Hilde Rosing, E C M Van Gorp, J W Mulder, Jos H Beijnen, Alwin D R HuitemaAbstract:For the quantification of the HIV-integrase inhibitor raltegravir in human plasma, dried blood spots and peripheral blood mononuclear Cell (PBMC) Lysate, an assay was developed and validated, using liquid chromatography coupled with tandem mass spectrometry. The assay also allowed detection, but no quantification due to absence of reference substance, of the main metabolite, raltegravir-glucuronide. Raltegravir was extracted from plasma by means of protein precipitation with a mixture of methanol and acetonitrile using only 50microL plasma. Extraction from dried blood spots was performed with a simple one-step extraction with a mixture of methanol, acetonitrile and 0.2M zincsulphate in water (1:1:2, v/v/v) and extraction from Cell Lysate was performed in 50% methanol in water. Chromatographic separation was performed on a reversed phase C18 column (150mmx2.0mm, particle size 5microm) with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.25mL/min. The analytical run time was 10min. The triple quadrupole mass spectrometer was operated in the positive ion-mode and multiple reaction monitoring was used for drug quantification. The method was validated over a range of 50-10,000ng/mL in plasma and dried blood spots and a range of 1-500ng/mL in PBMC Lysate. Dibenzepine was used as the internal standard. The method was proven to be specific, accurate, precise and robust. Accuracies ranged from 104% to 105% in plasma, from 93% to 105% in dried blood spots and from 82% to 113% in PBMC Lysate. Precision over the complete concentration range was less than 6%, 11% and 13% in plasma, dried blood spots and PBMC Lysate, respectively. The method is now applied for therapeutic drug monitoring and pharmacological research in HIV-infected patients treated with raltegravir.
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quantification of etravirine tmc125 in plasma dried blood spots and peripheral blood mononuclear Cell Lysate by liquid chromatography tandem mass spectrometry
Journal of Pharmaceutical and Biomedical Analysis, 2009Co-Authors: R Ter Heine, Hilde Rosing, J W Mulder, Jos H Beijnen, E C M Van Gorp, Alwin D R HuitemaAbstract:Abstract For the quantification of the novel non-nucleoside reverse transcriptase inhibitor etravirine in human plasma, dried blood spots and peripheral blood mononuclear Cell (PBMC) Lysate, an assay was developed and validated, using liquid chromatography coupled with tandem mass spectrometry. Etravirine was extracted from plasma by means of protein precipitation with a mixture of methanol and acetonitrile using only 50 μL plasma. Extraction from dried blood spots was performed with a one-step extraction with a mixture of methanol, acetonitrile and 0.2 M zinc sulphate in water (1:1:2, v/v/v) and extraction from Cell Lysate was performed in 50% methanol in water. Chromatographic separation was performed on a reversed phase C18 column (150 mm × 2.0 mm, particle size 5 μm) with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.25 mL/min. 13 C 6 -efavirenz was used as an internal standard. The analytical run time was only 10 min. The triple quadrupole mass spectrometer was operated in the positive ion-mode and multiple reaction monitoring was used for drug quantification. The method was validated over a range of 25–5000 ng/mL in plasma, 50–10,000 ng/mL in dried blood spots and a range of 5–2500 ng/mL in PBMC Lysate. Accuracies ranged from 89% to 106% in plasma, from 94% to 109% in dried blood spots and from 91% to 105% in PBMC Lysate. Precisions at the all concentration levels ranged from 1.9% to 14% in plasma, 4.7% to 20% in dried blood spots and from 3.1% to 11% in PBMC Lysate. The bioanalytical assay was successfully incorporated with previously developed assays for the determination of all currently approved PIs and NNRTIs in plasma and dried blood spots and it is now applied for therapeutic drug monitoring and pharmacological research in HIV-infected patients treated with etravirine.