Impact of Olive Oil Supplement Intake on Dendritic Cell Maturation after Strenuous Physical Exercise: A Preliminary Study

Physical exercise is known to have a dose-dependent effect on the immune system and can result in an inflammatory process in athletes that is proportional to the intensity and duration of exertion. This inflammatory process can be measured by cell markers such as dendritic cells (DCs), which, in humans, consist of the myeloid DC (mDCs) and plasmacytoid DC (pDCs) subpopulations. The aim of this study was to measure DC differentiation to determine the possible anti-inflammatory effects, after intense aerobic effort, of the intake of a 25 mL extra-virgin olive oil supplement. Three healthy sports-trained subjects went through resistance exercise loads on two days separated by a week: on one day after active supplement intake and on the other day after placebo supplement intake. The results show that the highest increase (77%) in the percentage of mDCs as a proportion of pDCs was immediately after testing. Independently of the supplement taken, mature mDCs showed a decreasing trend between the test one hour after and 24 h after testing ended. Nevertheless, measured in terms of the coefficient of variation, only the decrease (46%) for extra-virgin olive oil supplementation was statistically significant (95% CI: 30-62%; p = 0.05). In conclusion, an extra-virgin olive oil supplement could reduce the inflammatory impact of intense aerobic effort and improve recovery at 24 h

Mycophenolic acid interferes the transcriptional regulation and protein trafficking of maturation surface markers in dendritic cells

Background: The ability of dendritic cells (DCs) to regulate adaptive immunity makes them interesting cells to be used as therapeutic targets modulating alloimmune responses. Mycophenolic acid (MPA) is an immunosuppressor commonly used in transplantation, and its effect on DCs has not been fully investigated. Methods: Monocyte-derived DCs were obtained from healthy volunteers and cultured for 7 days. Cells were treated with MPA on day 2 and matured by lipopolysaccharide (LPS) stimulation. Functionality of mature DC (mDCs) was evaluated by allogeneic mixed lymphocytes reaction. Surface expression of maturation markers (CD40, CD83, CD86, and ICAM-1) was analysed in both immature DCs (iDCs) and mDCs by flow cytometry. To assess transcriptional regulation and protein subcellular location, RT-PCR and confocal microscopy were used, respectively. Results: MPA decreased surface expression of all maturation markers in mDCs and significantly abrogated DCs-induced allogeneic T-cell proliferation after MPA pre-treatment. In iDCs, the reduced surface protein expression after MPA paralleled with mRNA downregulation of their genes. In mDCs, the mRNA levels of ICAM-1, CD40 and CD83 were enhanced in MPA-treated mDCs with an increase in the expression of CD83 and ICAM-1 near the Golgi compared to non-treated mDCs. In contrast, mRNA levels of CD86 were diminished after MPA treatment. Conclusions: The reduced surface markers expression in mDCs exerted by MPA produced a decline in their capacity to activate immune responses. Moreover, the inhibition of guanosine-derived nucleotide biosynthesis by MPA treatment leads to DC maturation interference by two mechanisms depending on the marker, transcriptional downregulation or disrupted intracellular protein trafficking

Online haemodiafiltration improves inflammatory state in dialysis patients: A longitudinal study

Background: Patients undergoing conventiona l hemodialysis (C-HD) present a greater immuno-inflam- matory state probably related to uremia, sympathetic nervous system (SNS) activation and /or membrane bioincompat ibility, which could improve with a technique-switch ing to online hemodiafiltration (OL-HD). The antigen-indep endent pathway activation of this modified immunologic state turns dendritic cells (DC) into an accurate cell model to study these patients. The aim of this study is to further evaluate the immune-inflammat ory state of patients in C-HD assessed by DC maturation. Methods: 31 patients were submitted to C-HD and after 4 months switched to the OL-HD technique. Monocytes-derive d DCs from HD patients were cultured in the presence of IL-4/GM-CSF. DC-maturation was evaluated by assessing the maturation phenotype by flow cytometry (FACs). DCs-functiona l capacity to elicit T-cell alloresponse was studied by mixed leuco- cyte reaction. Cytokine release was assessed by FACs and SNS was evaluated measuring renalase levels by ELISA. Results: An up-regulation of maturation markers was observed in C-HD DCs which induced two fold more T cells proliferation than OL-HD DCs. Also, C-HD-mDCs presented with over-produc- tion of pro-inflammatory cytokines (IL-6, IL-1 β , IL-8, IL-10 and TNF- α ) compared with OL- HD-mDC (P < 0·05). Results were correlated with clinical data. When SNS was evaluated, hypotension events and blood pressure were significantly lower and renalase levels were significantly higher after conversion to OL-HD. Diabetes mellitus type 2 patients also found beneficial reduction of mDC when converted to OL-HD compared to non-diabetics. Conclusions: OL-HD could interfere with immuno-inflammatory state in HD patients with an improvement of renalase levels as potential key mediators in the mechanistic pathway of down-regulation of DC maturatio

Different Storing and Processing Conditions of Human Lymphocytes do not Alter P-Glycoprotein Rhodamine 123 Efflux

P-glycoprotein (Pgp), a protein codified by Multi Drug Resistance (MDR1) gene, has a detoxifying function and might influence the toxicity and pharmacokinetics and pharmacodynamics of drugs. Sampling strategies to improve Pgp studies could be useful to optimize the sensitivity and the reproducibility of efflux assays. This study aimed to compare Pgp expression and efflux activity by measuring Rhodamine123 (Rh123) retention in lymphocytes stored under different conditions, in order to evaluate the potential utility of any of the storing conditions in Pgp functionality. Our results show no change in protein expression of Pgp by confocal studies and Western blotting, nor changes at the mRNA level (qRT-PCR). No differences in Rh123 efflux by Pgp activity assays were found between fresh and frozen lymphocytes after 24 hours of blood extraction, using either of the two Pgp specific inhibitors (VP and PSC833). Different working conditions in the 24 hours post blood extraction do not affect Rh123 efflux. These results allow standardization of Pgp activity measurement in different individuals with different timing of blood sampling and in different geographic areas. ______________

P-Glycoprotein functional activity in peripheral blood lymphocytes. Role of immunosuppressants, pharmacogenomics and alloimmune response

Both immunological and non immunological factors are involved in chronic allograft nephropathy such as donor specific alloreactivity, chronic inflammation and nephrotoxicity induced by anticalcineurinics. Different degrees of nephrotoxicity are related to immunosuppression and to the level of inhibition of the different transporter proteins such as MDR1, MRP1 and MRP2. Those proteins could be subjected to genetic inter-individual variability thus modifying pharmacokinetics parameters. Furthermore, optimal immunosuppressive dose could be predicted by genotype characterization of MDR1, MRP1 or MRP2 genes. Polymorphisms in these genes have been related to immunosuppressive exposure, nephrotoxicity and renal allograft rejection. Those proteins have also been associated to immunological factors. Both MDR1 and MRP1 have been involved in T cell activation and also in dendritic cells (DCs) differentiation, migration and maturation. It has been hypothesized that ABC transporter proteins play a major role in drug efflux and also may be an underlying factor as an immunomodulator. Considering that many immunosuppressants are substrates and also inhibitors of this ABC transporter proteins, the hypothesis of this doctoral thesis is to evaluate if Pgp activity would participate in lymphocyte activation and if the polymorphisms of this protein in renal transplant recipients with different immunosuppressive regimen could have a functional role in the pharmacokinetics of the immunosuppressants. The main objective is to study the role of P-glycoprotein as an efflux pump and its contribution in the pharmacokinetics and pharmacogenetics of immunosuppressants. On the other hand, far from the well known role in drug exposition and nephrotoxicity, to improve the knowledge on its function in the alloimmunity responses. In the first study we compared Pgp expression and efflux activity by measuring Rho123 retention in lymphocytes stored under different conditions to evaluate the potential influence of any of the storing conditions on Pgp expression and functionality. To improve Pgp studies, especially multicentric ones, sampling strategies should be considered in order to optimize the sensitivity and reproducibility of efflux assays. There is no data which compares Pgp activity measured from fresh lymphocytes with cryo-preserved lymphocytes. Moreover, most authors do not specify under which conditions cells are preserved and stored before measuring Pgp activity. Here, we isolated lymphocytes from fresh venous blood from 12 healthy volunteers and four storage conditions of lymphocytes were used. 1) Fresh lymphocytes (Fresh/Non-frozen) (F/NFr); 2) Lymphocytes frozen immediately after the extraction (Fresh/Frozen) (F/Fr); 3) Lymphocytes isolated within 24 hours after the extraction (Non-fresh/Non-frozen) (NF/NFr); 4) And lymphocytes isolated within 24 hours after the extraction and immediately frozen (Non-fresh/Frozen) (NF/Fr). The objective of the second study was to investigate the association of different ABCB1 polymorphisms (C3435T, G2677T, C1236T and T129C) with Pgp activity and exposure of different immunosuppressive drugs in renal transplant patients from eight different centers. This substudy of pharmacogenomics was part of the Symphony study. The main objective of the Symphony trail was to establish the differences on MPA and its metabolites exposure among the four immunosuppressive groups: MMF in combination with normal or low doses of CsA, low doses of Tac and low doses of SRL. The study was evaluated in seventy patients: CsA (n=30), tacrolimus (n=13) and sirolimus (n=23). For the pharmacokinetic analysis, the AUC0-12 of all immunosuppressants was compared at each time along the follow-up. For this purpose, 11 blood samples were collected for each point: before the first MMF administration of the day and up to 12h post-dose. To perform the study, patients were genotyped for SNPs in ABCB1 gene and moreover, Pgp activity was evaluated in PBMCs using the Rho123 efflux assay. Considering earlier studies in our group which describes the in vivo impact of the association of Rapa (mTORi) with two calcineurin inhibitors (CsA and Tac) on Pgp function and the involvement of Pgp in this immunosupressor-related renal toxicity, in the next study, we analyzed the activity of Pgp on different T-cell subsets and we studied the effects of the same immunosuppressants in monotherapy and associated with rapamycin. Rho123 uptake, efflux and kinetic of extrusion in CD4+ and CD8+ subsets of peripheral blood mononuclear cells isolated from eight healthy volunteers were measured. We also studied the antigen-specific memory T-cell responses by measuring T-cell proliferation and cytokine secretion using an allogeneic mixed lymphocyte reaction.En la nefropatía crónica del aloinjerto renal (NCT) se implican factores aloinmunes y no aloinmunes tales como la aloreactividad donante específica, inflamación crónica y nefrotoxicidad inducida por los anticalcineurínicos. La nefrotoxicidad varía en función de la combinación de los inmunosupresores. Así mismo, el diferente grado de nefrotoxicidad podría estar relacionado con el grado de inhibición de las diferentes proteínas transportadoras (MDR1, MRP1 y MRP2) que son las responsables de afluir el fármaco hacia el exterior de las células. Considerando la importancia del papel de éstas proteínas, las diferentes variantes genéticas de los genes que las codifican y su función, pueden afectar a la variabilidad interindividual con sus consecuencias en la farmacocinética de muchos fármacos. La monitorización del genotipo de los genes MDR1, MRP1 y MRP2, así como también su función, podría predecir la dosis óptima de los inmunosupresores en receptores de trasplante renal y la dosis inicial que necesita cada paciente individual para obtener la inmunosupresión adecuada. Los polimorfismos de estos genes están relacionados con la exposición al fármaco, la nefrotoxicidad y el rechazo. Estas proteínas también se asocian a factores inmunológicos. Tanto MDR1 como MRP1 juegan un papel importante en la activación de la célula T y también en la diferenciación y maduración de la célula dendrítica. El papel de estas proteínas transportadoras va más allá de una bomba de extrusión ya que se conoce que participan en la respuesta inmune siendo una nueva diana terapéutica para la inmunosupresión. El principal objetivo fue estudiar el rol de la P-Glicoproteína (MDR1/Pgp) tanto como una bomba transportadora de fármacos como su contribución en la farmacocinética y farmacogenética de los inmunosupresores. También se estudió el rol de Pgp en la respuesta aloinmune. En un primer estudio, se comparó la expresión de Pgp y la actividad de eflujo mediante el ensayo de Rodamina (Rho123) en linfocitos de voluntarios sanos guardados en diferentes condiciones para evaluar la influencia que podían tener estas condiciones de almacenaje sobre la expresión y funcionalidad de Pgp. Se puso a punto un método para la medida de actividad de Pgp en linfocitos T, para poder ser aplicada en muestras de linfocitos de pacientes trasplantados renales. Se aislaron linfocitos de la sangre de 12 voluntarios sanos y se evaluó las diferencias de expresión de Pgp a nivel de RNA y proteína, entre linfocitos procesados y guardados en diferentes condiciones de almacenaje. En un segundo objetivo se analizó la asociación de diferentes polimorfismos (SNPs) de Pgp/ABCB1 (C3435T, C1236T, G2677T i T129C) con la actividad de Pgp y la exposición a diferentes fármacos inmunosupresores en 70 pacientes de trasplante renal de diferentes hospitales. En un estudio in vivo donde se describe un modelo de nefrotoxicidad en ratas, se estudió el papel de Pgp en la asociación de Rapa (mTORi) con dos inhibidores de calcineurina (CsA y Tac). A partir de este trabajo, se realizó el siguiente estudio donde se analizó la actividad de Pgp in vitro en diferentes sub-poblaciones de célula T y los efectos de los mismos inmunosupresores solos y asociados con Rapamicina

Impact of small molecules immunosuppressants on P-glycoprotein activity and T-cell function

Purpose. P-glycoprotein (Pgp) is a member of the ABC-transporter family that transports substances across cellular membranes acting as an efflux pump extruding drugs out of the cells. Pgp plays a key role on the pharmacokinetics of several dr ugs. Herein, we have studied the effects of immunosuppressants on Pgp function, assessing rhodamine-123 (Rho123) uptake and efflux in different T- cell subsets. Methods. Different immunosuppressants such as Cyclosporine (CsA), Rapamycin (Rapa) and Tacrolimus (Tac) were used to assess the in vitro effect on Pgp function of main T-cell subsets among healthy volunteers. We measured Rho123 upta ke, efflux and kinetic of extrusion in CD4 + and CD8 + subsets by flow cytometry. Antigen-specific memory T-ce ll responses were assessed by measuring T-cell proliferation and cytokine secretion using an allogeneic mixed lymphocyte reaction. Results. Rho123 uptake in groups treated with CsA and CsA+Rapa was signif icantly decreased compared to non-treated group and the other immunosupressants in both T cells subsets. Pgp activity was also reduced in CsA and CsA+Rapa compared to the other immunosupressants but it was only significant in the CsA group for CD8 + subset. Kinetic extrusion of Rho123 by Pgp in all groups was faster in CD8 + T cells. All immunosuppressants and the specific Pgp inhibitor PSC833 diminished antigen-primed T-cell proliferation, especially CD8 + T-cell subset. Conclusions. Our data indicate that small molecules immunosuppressants, especially CsA, inhibit Pgp activity and T-cell function being the CD8 + T cells more susceptible to this effect. These findings support the importance of Pgp when designing combined immunosuppressive regimens

Online haemodiafiltration improves inflammatory state in dialysis patients: A longitudinal study

Background: Patients undergoing conventiona l hemodialysis (C-HD) present a greater immuno-inflam- matory state probably related to uremia, sympathetic nervous system (SNS) activation and /or membrane bioincompat ibility, which could improve with a technique-switch ing to online hemodiafiltration (OL-HD). The antigen-indep endent pathway activation of this modified immunologic state turns dendritic cells (DC) into an accurate cell model to study these patients. The aim of this study is to further evaluate the immune-inflammat ory state of patients in C-HD assessed by DC maturation. Methods: 31 patients were submitted to C-HD and after 4 months switched to the OL-HD technique. Monocytes-derive d DCs from HD patients were cultured in the presence of IL-4/GM-CSF. DC-maturation was evaluated by assessing the maturation phenotype by flow cytometry (FACs). DCs-functiona l capacity to elicit T-cell alloresponse was studied by mixed leuco- cyte reaction. Cytokine release was assessed by FACs and SNS was evaluated measuring renalase levels by ELISA. Results: An up-regulation of maturation markers was observed in C-HD DCs which induced two fold more T cells proliferation than OL-HD DCs. Also, C-HD-mDCs presented with over-produc- tion of pro-inflammatory cytokines (IL-6, IL-1 β , IL-8, IL-10 and TNF- α ) compared with OL- HD-mDC (P < 0·05). Results were correlated with clinical data. When SNS was evaluated, hypotension events and blood pressure were significantly lower and renalase levels were significantly higher after conversion to OL-HD. Diabetes mellitus type 2 patients also found beneficial reduction of mDC when converted to OL-HD compared to non-diabetics. Conclusions: OL-HD could interfere with immuno-inflammatory state in HD patients with an improvement of renalase levels as potential key mediators in the mechanistic pathway of down-regulation of DC maturatio

Online haemodiafiltration improves inflammatory state in dialysis patients: A longitudinal study

Background: Patients undergoing conventiona l hemodialysis (C-HD) present a greater immuno-inflam- matory state probably related to uremia, sympathetic nervous system (SNS) activation and /or membrane bioincompat ibility, which could improve with a technique-switch ing to online hemodiafiltration (OL-HD). The antigen-indep endent pathway activation of this modified immunologic state turns dendritic cells (DC) into an accurate cell model to study these patients. The aim of this study is to further evaluate the immune-inflammat ory state of patients in C-HD assessed by DC maturation. Methods: 31 patients were submitted to C-HD and after 4 months switched to the OL-HD technique. Monocytes-derive d DCs from HD patients were cultured in the presence of IL-4/GM-CSF. DC-maturation was evaluated by assessing the maturation phenotype by flow cytometry (FACs). DCs-functiona l capacity to elicit T-cell alloresponse was studied by mixed leuco- cyte reaction. Cytokine release was assessed by FACs and SNS was evaluated measuring renalase levels by ELISA. Results: An up-regulation of maturation markers was observed in C-HD DCs which induced two fold more T cells proliferation than OL-HD DCs. Also, C-HD-mDCs presented with over-produc- tion of pro-inflammatory cytokines (IL-6, IL-1 β , IL-8, IL-10 and TNF- α ) compared with OL- HD-mDC (P < 0·05). Results were correlated with clinical data. When SNS was evaluated, hypotension events and blood pressure were significantly lower and renalase levels were significantly higher after conversion to OL-HD. Diabetes mellitus type 2 patients also found beneficial reduction of mDC when converted to OL-HD compared to non-diabetics. Conclusions: OL-HD could interfere with immuno-inflammatory state in HD patients with an improvement of renalase levels as potential key mediators in the mechanistic pathway of down-regulation of DC maturatio

Different Storing and Processing Conditions of Human Lymphocytes do not Alter P-Glycoprotein Rhodamine 123 Efflux

P-glycoprotein (Pgp), a protein codified by Multi Drug Resistance (MDR1) gene, has a detoxifying function and might influence the toxicity and pharmacokinetics and pharmacodynamics of drugs. Sampling strategies to improve Pgp studies could be useful to optimize the sensitivity and the reproducibility of efflux assays. This study aimed to compare Pgp expression and efflux activity by measuring Rhodamine123 (Rh123) retention in lymphocytes stored under different conditions, in order to evaluate the potential utility of any of the storing conditions in Pgp functionality. Our results show no change in protein expression of Pgp by confocal studies and Western blotting, nor changes at the mRNA level (qRT-PCR). No differences in Rh123 efflux by Pgp activity assays were found between fresh and frozen lymphocytes after 24 hours of blood extraction, using either of the two Pgp specific inhibitors (VP and PSC833). Different working conditions in the 24 hours post blood extraction do not affect Rh123 efflux. These results allow standardization of Pgp activity measurement in different individuals with different timing of blood sampling and in different geographic areas. ______________

Impact of small molecules immunosuppressants on P-glycoprotein activity and T-cell function

Purpose. P-glycoprotein (Pgp) is a member of the ABC-transporter family that transports substances across cellular membranes acting as an efflux pump extruding drugs out of the cells. Pgp plays a key role on the pharmacokinetics of several dr ugs. Herein, we have studied the effects of immunosuppressants on Pgp function, assessing rhodamine-123 (Rho123) uptake and efflux in different T- cell subsets. Methods. Different immunosuppressants such as Cyclosporine (CsA), Rapamycin (Rapa) and Tacrolimus (Tac) were used to assess the in vitro effect on Pgp function of main T-cell subsets among healthy volunteers. We measured Rho123 upta ke, efflux and kinetic of extrusion in CD4 + and CD8 + subsets by flow cytometry. Antigen-specific memory T-ce ll responses were assessed by measuring T-cell proliferation and cytokine secretion using an allogeneic mixed lymphocyte reaction. Results. Rho123 uptake in groups treated with CsA and CsA+Rapa was signif icantly decreased compared to non-treated group and the other immunosupressants in both T cells subsets. Pgp activity was also reduced in CsA and CsA+Rapa compared to the other immunosupressants but it was only significant in the CsA group for CD8 + subset. Kinetic extrusion of Rho123 by Pgp in all groups was faster in CD8 + T cells. All immunosuppressants and the specific Pgp inhibitor PSC833 diminished antigen-primed T-cell proliferation, especially CD8 + T-cell subset. Conclusions. Our data indicate that small molecules immunosuppressants, especially CsA, inhibit Pgp activity and T-cell function being the CD8 + T cells more susceptible to this effect. These findings support the importance of Pgp when designing combined immunosuppressive regimens