Vera Alves

Re-exploring the high-throughput potential of microextraction techniques, SPME and MEPS, as powerful strategies for medical diagnostic purposes. Innovative approaches, recent applications and future trends

AbstractThe human population continues to grow exponentially in the fast developing and most populated countries, whereas in Western Europe it is getting older and older each year. This inevitably raises the demand for better and more efficient medical services without increasing the economic burden in the same proportion. To meet these requirements, improvement of medical diagnosis is certainly a key aspect to consider. Therefore, we need powerful analytical methodologies able to go deeper and further in the characterization of human metabolism and identification of disease biomarkers and endogenous molecules in body fluids and tissues. The ultimate goal is to have a reliable and early medical diagnosis, mitigating the disease complications as much as possible. Microextraction techniques (METs) represent a key step in these analytical methodologies by providing samples in the suitable volumes and purification levels necessary for the characterization of the target analytes. In this aspect, solid-phase microextraction (SPME) and, more recently, microextraction by packed sorbent (MEPS), are powerful sample preparation techniques, characterized by their reduced time of analysis, low solvent consumption, and broad application. Moreover, as miniaturized techniques, they can be easily automatized to have a high-throughput performance in the clinical environment. In this review, we explore some of the most interesting MEPS and SPME applications, focusing on recent trends and applications to medical diagnostic, particularly the in vivo and near real time applications. FigureMETs as powerful strategies for medical diagnostic purposes

Combined effect of sodium selenite and docetaxel on PC3 metastatic prostate cancer cell line

Docetaxel and sodium selenite are well known for their anticancer properties. While resistance to docetaxel remains an obstacle in prostate cancer chemotherapy, sodium selenite, has been exploited as a new therapeutic approach. Currently, development of therapies affecting a multitude of cell targets, have been proposed as a strategy to overcome drug resistance. This association may reduce systemic toxicity counteracting a wide range of side effects. Here we report the effect of docetaxel and sodium selenite combination on the PC3 prostate cancer cell line, derived from bone metastasis. Therefore we evaluate cell growth, cell cycle progression, viability, mitochondria membrane potential, cytochrome C, Bax/Bcl2 ratio, caspase-3 expression and reactive oxygen species production. Our results suggest that sodium selenite and docetaxel combination have a synergistic effect on cell growth inhibition (67%) compared with docetaxel (22%) and sodium selenite (24%) alone. This combination also significantly induced cell death, mainly by late apoptosis vs necrosis, which is correlated with mitochondria membrane potential depletion. On the other hand, cytochrome C, Bax/Bcl2 ratio and caspase-3, known as proapoptotic factors, significantly increased in the presence of sodium selenite alone, but not in the presence of docetaxel in monotherapy or in combination with sodium selenite. These findings suggest that docetaxel and sodium selenite combination may be more effective on prostate cancer treatment than docetaxel alone warranting further evaluation of this combination in prostate cancer therapeutic approach.

Antileishmanial Activity of Semisynthetic Lupane Triterpenoids Betulin and Betulinic Acid Derivatives: Synergistic Effects with Miltefosine

Leishmaniasis is a neglected tropical disease (NTDs), endemic in 88 countries, affecting more than 12 million people. The treatment consists in pentavalent antimony compounds, amphotericin B, pentamidine and miltefosine, among others. However, these current drugs are limited due to their toxicity, development of biological resistance, length of treatment and high cost. Thus, it is important to continue the search for new effective and less toxic treatments. The anti-Leishmania activity of sixteen semisynthetic lupane triterpenoids derivatives of betulin (BT01 to BT09) and betulinic acid (AB10 to AB16) were evaluated. Drug interactions between the active compounds and one current antileishmanial drug, miltefosine, were assessed using the fixed ratio isobologram method. In addition, effects on the cell cycle, apoptosis/necrosis events, morphology and DNA integrity were studied. The derivatives BT06 (3β-Hydroxy-(20R)-lupan-29-oxo-28-yl-1H-imidazole-1-carboxylate) and AB13 (28-(1H-imidazole-1-yl)-3,28-dioxo-lup-1,20(29)-dien-2-yl-1H-imidazole-1-carboxylate) were found to be the most active, with IC50 values of 50.8 µM and 25.8 µM, respectively. Interactions between these two compounds and miltefosine were classified as synergistic, with the most effective association being between AB13 and miltefosine, where decreases of IC50 values to 6 µM were observed, similar to the miltefosine activity alone. AB13 induced significant morphological changes, while both derivatives produced anti-proliferative activity through cell cycle arrest at the G0/G1 phase. Neither of these derivatives induced significant apoptosis/necrosis, as indicated by phosphatidylserine externalization and DNA fragmentation assays. In addition, neither of the derivatives induced death in macrophage cell lines. Thus, they do not present any potential risk of toxicity for the host cells. This study has identified the betulin derivative BT06 and the betulinic acid derivative AB13 as promising molecules in the development of new alternative therapies for leishmaniasis, including those involving combined-therapy with miltefosine.

Oxidative stress adaptation in aggressive prostate cancer may be counteracted by the reduction of glutathione reductase

PC3 shows higher ROS production but also the highest GSH levels and Gl‐Red activity, possibly contributing to oxidative stress resistance. This is also associated with higher mitochondrial membrane potential, TAS and lower lipid peroxidation. On the other hand, we identified Gl‐Red activity reduction as a new strategy in overcoming oxidative stress resistance, by inducing H2O2 cytotoxicity. Therefore these results suggest Gl‐Red activity reduction as a new potential therapeutic approach, in prostate cancer.

Membrane progesterone receptors in human regulatory T cells: a reality in pregnancy

To provide evidence of the existence of membrane progesterone receptor alpha (mPRα) on regulatory T cells (Treg) in peripheral blood during pregnancy, postulating a possible explanation for the effect of progesterone on preterm birth.

Leukocyte, Platelet and Endothelial Activation in Patients with Acute Renal Failure Treated by Intermittent Hemodialysis

Background: Using different types of dialysis membranes to treat acute renal failure (ARF), clinical and experimental studies have demonstrated discordant results relatively to the effect of membrane bioincompatibility on patient outcome. Nevertheless, there are few data on the biocompatibility indices in patients with ARF who need to be treated by hemodialysis. Objective: To characterize the impact of intermittent hemodialysis (IHD) on indices of leukocyte, platelet and endothelial activation in patients with ARF. Patients and Methods: We prospectively studied 27 patients with severe ARF treated by IHD. They were characterized relatively to gender, age, ARF etiology, urinary output per 24 h, Simplified Acute Physiology Score (SAPS), number of dialysis sessions and outcome. We evaluated the effect of hemodialysis with two types of low-flux dialyzers (cuprophane and polysulfone dialyzers) on plasma concentrations of soluble TNF-RI (TNF-sR55), TNF-RII (TNF-sR75), interleukin (IL)-6, soluble CD23 molecule, soluble P-selectin and von Willebrand factor (vWF). Results: There were no significant differences between the two groups of patients dialyzed with cellulose-based and synthetic membranes in terms of age, sex, urinary output per 24 h, SAPS, number of dialysis sessions and mortality. We verified high plasma concentrations of TNF-sR55, TNF-sR75, IL-6, sCD23, sP-selectin and vWF in the global population studied. Patients dialyzed with cuprophane membranes showed significantly lower preand postdialysis concentrations of sP-selectin than patients dialyzed with polysulfone membranes. After a hemodialysis session, with correction for differences of blood hematocrit, we did not observe any significant modification in the concentrations of TNF-sR55, TNF-sR75, IL-6, sP-selectin and vWF of the plasma. On the other hand, a significant increase of sCD23 molecule was found in the group dialyzed with polysulfone membranes (p = 0.009). Conclusions: The group of 27 patients with ARF who needed IHD, showed increased plasma concentrations of some leukocyte, platelet and endothelial activation markers (TNF-sR55, TNF-sR75, IL-6, sCD23, sP-selectin and vWF). These mediators characterize the inflammatory and procoagulant state associated with this pathologic condition. After a hemodialysis session with these low-flux membranesBACKGROUND Using different types of dialysis membranes to treat acute renal failure (ARF), clinical and experimental studies have demonstrated discordant results relatively to the effect of membrane bioincompatibility on patient outcome. Nevertheless, there are few data on the biocompatibility indices in patients with ARF who need to be treated by hemodialysis. OBJECTIVE To characterize the impact of intermittent hemodialysis (IHD) on indices of leukocyte, platelet and endothelial activation in patients with ARF. PATIENTS AND METHODS We prospectively studied 27 patients with severe ARF treated by IHD. They were characterized relatively to gender, age, ARF etiology, urinary output per 24 h, Simplified Acute Physiology Score (SAPS), number of dialysis sessions and outcome. We evaluated the effect of hemodialysis with two types of low-flux dialyzers (cuprophane and polysulfone dialyzers) on plasma concentrations of soluble TNF-RI (TNF-sR55), TNF-RII (TNF-sR75), interleukin (IL)-6, soluble CD23 molecule, soluble P-selectin and von Willebrand factor (vWF). RESULTS There were no significant differences between the two groups of patients dialyzed with cellulose-based and synthetic membranes in terms of age, sex, urinary output per 24 h, SAPS, number of dialysis sessions and mortality. We verified high plasma concentrations of TNF-sR55, TNF-sR75, IL-6, sCD23, sP-selectin and vWF in the global population studied. Patients dialyzed with cuprophane membranes showed significantly lower pre- and postdialysis concentrations of sP-selectin than patients dialyzed with polysulfone membranes. After a hemodialysis session, with correction for differences of blood hematocrit, we did not observe any significant modification in the concentrations of TNF-sR55, TNF-sR75, IL-6, sP-selectin and vWF of the plasma. On the other hand, a significant increase of sCD23 molecule was found in the group dialyzed with polysulfone membranes (p = 0.009). CONCLUSIONS The group of 27 patients with ARF who needed IHD, showed increased plasma concentrations of some leukocyte, platelet and endothelial activation markers (TNF-sR55, TNF-sR75, IL-6, sCD23, sP-selectin and vWF). These mediators characterize the inflammatory and procoagulant state associated with this pathologic condition. After a hemodialysis session with these low-flux membranes (cellulose-based and polysulfone membranes), we did not observe any significant variation in the concentrations of TNF-sR55, TNF-sR75, IL-6, sP-selectin and vWF of the plasma. Patients dialyzed with polysulfone membranes presented higher basal plasma concentrations of sP-selectin and significant postdialysis increase of plasma concentrations of CD23 molecule compared to patients dialyzed with cuprophane dialyzers.

An improved analytical strategy combining microextraction by packed sorbent combined with ultra high pressure liquid chromatography for the determination of fluoxetine, clomipramine and their active metabolites in human urine

A powerful and sensitive method, by microextraction packed sorbent (MEPS), and ultra-high performance liquid chromatography (UHPLC) with a photodiode array (PDA) detection, is described for the determination of fluoxetine, clomipramine and their active metabolites in human urine samples. The MEPS variables, such as sample volume, pH, number of extraction cycles (draw-eject), and desorption conditions (solvent and solvent volume of elution) were optimized. The analysis were carried out using small sample volumes (500μL) and in a short time period (5min for the entire sample preparation step). Good linearity was obtained for all antidepressants with the correlation coefficients (R(2)) above 0.9965. The limits of detection (LOD) ranged from 0.068 to 0.087μgmL(-1). The recoveries were from 93% to 98%, with relative standard deviations less than 6%. The inter-day precision, expressed as the relative standard deviation, varied between 3.8% and 8.5% while the intra-day precision between 3.0% and 7.1%. In order to evaluate the proposed method for clinical use, the MEPS/UHPLC-PDA method was applied to analysis of urine samples from depressed patients.

Oxidative stress and mitochondrial dysfunction play a role in myelodysplastic syndrome development, diagnosis, and prognosis: A pilot study

Abstract The imbalance between reactive oxygen species (ROS) production and their elimination by antioxidants leads to oxidative stress. Depending on their concentration, ROS can trigger apoptosis or stimulate cell proliferation. We hypothesized that oxidative stress and mitochondrial dysfunction may participate not only in apoptosis detected in some myelodysplastic syndrome (MDS) patients, but also in increasing proliferation in other patients. We investigated the involvement of oxidative stress and mitochondrial dysfunction in MDS pathogenesis, as well as assessed their diagnostic and prognostic values. Intracellular peroxides, superoxide, superoxide/peroxides ratio, reduced glutathione (GSH), and mitochondrial membrane potential (Δψmit) levels were analyzed in bone marrow cells from 27 MDS patients and 12 controls, by flow cytometry. We observed that all bone marrow cell types from MDS patients had increased intracellular peroxide levels and decreased GSH content, compared with control cells. Moreover, oxidative stress levels were MDS subtype— and risk group—dependent. Low-risk patients had the highest ROS levels, which can be related with their high apoptosis; and intermediate-2-risk patients had high Δψmit that may be associated with their proliferative potential. GSH levels were negatively correlated with transfusion dependency, and peroxide levels were positively correlated with serum ferritin level. GSH content proved to be an accurate parameter to discriminate patients from controls. Finally, patients with high ROS or low GSH levels, as well as high superoxide/peroxides ratio had lower overall survival. Our results suggest that oxidative stress and mitochondrial dysfunction are involved in MDS development, and that oxidative stress parameters may constitute novel diagnosis and/or prognosis biomarkers for MDS.

The role of immune system exhaustion on cancer cell escape and anti-tumor immune induction after irradiation.

Immune surveillance seems to represent an effective tumor suppressor mechanism. However, some cancer cells survive and become variants, being poorly immunogenic and able to enter a steady-state phase. These cells become functionally dormant or remain hidden clinically throughout. Neoplastic cells seem to be able to instruct immune cells to undergo changes promoting malignancy. Radiotherapy may act as a trigger of the immune response. After radiotherapy a sequence of reactions occurs, starting in the damage of oncogenic cells by multiple mechanisms, leading to the immune system positive feedback against the tumor. The link between radiotherapy and the immune system is evident. T cells, macrophages, Natural Killer cells and other immune cells seem to have a key role in controlling the tumor. T cells may be dysfunctional and remain in a state of T cell exhaustion, nonetheless, they often retain a high potential for successful defense against cancer, being able to be mobilized to become highly functional. The lack of clinical trials on a large scale makes data a little robust, in spite of promising information, there are still many variables in the studies relating to radiation and immune system. The clarification of the mechanisms underlying immune response to radiation exposure may contribute to treatment improvement, gain of life quality and span of patients.

Functional and molecular characterization of cancer stem-like cells in bladder cancer: a potential signature for muscle-invasive tumors

Striking evidence associates cancer stem cells (CSCs) to the high recurrence rates and poor survival of patients with muscle-invasive bladder cancer (BC). However, the prognostic implication of those cells in risk stratification is not firmly established, mainly due to the functional and phenotypic heterogeneity of CSCs populations, as well as, to the conflicting data regarding their identification based on a single specific marker. This emphasizes the need to exploit putative CSC-related molecular markers with potential prognostic significance in BC patients. This study aimed to isolate and characterize bladder CSCs making use of different functional and molecular approaches. The data obtained provide strong evidence that muscle-invasive BC is enriched with a heterogeneous stem-like population characterized by enhanced chemoresistance and tumor initiating properties, able to recapitulate the heterogeneity of the original tumor. Additionally, a logistic regression analysis identified a 2-gene stem-like signature (SOX2 and ALDH2) that allows a 93% accurate discrimination between non-muscle-invasive and invasive tumors. Our findings suggest that a stemness-related gene signature, combined with a cluster of markers to more narrowly refine the CSC phenotype, could better identify BC patients that would benefit from a more aggressive therapeutic intervention targeting CSCs population.