Teresa Freitas

Variations on Fibrinogen-Erythrocyte Interactions during Cell Aging

Erythrocyte hyperaggregation, a cardiovascular risk factor, is considered to be caused by an increase in plasma adhesion proteins, particularly fibrinogen. We have recently reported a specific binding between fibrinogen and an erythrocyte integrin receptor with a β3 or β3-like subunit. In this study we evaluate the influence of erythrocyte aging on the fibrinogen binding. By atomic force microscopy-based force spectroscopy measurements we found that increasing erythrocyte age, there is a decrease of the binding to fibrinogen by decreasing the frequency of its occurrence but not its force. This observation is reinforced by zeta-potential and fluorescence spectroscopy measurements. We conclude that upon erythrocyte aging the number of fibrinogen molecules bound to each cell decreases significantly, due to the progressive impairment of the specific fibrinogen-erythrocyte receptor interaction. Knowing that younger erythrocytes bind more to fibrinogen, we could presume that this population is the main contributor to the cardiovascular diseases associated with increased fibrinogen content in blood, which could disturb the blood flow. Our data also show that the sialic acids exposed on the erythrocyte membrane contribute for the interaction with fibrinogen, possibly by facilitating its binding to the erythrocyte membrane receptor.

The role of blood cell membrane lipids on the mode of action of HIV-1 fusion inhibitor sifuvirtide

Sifuvirtide is a gp41 based peptide that inhibits HIV-1 fusion with the host cells and is currently under clinical trials. Previous studies showed that sifuvirtide partitions preferably to saturated phosphatidylcholine lipid membranes, instead of fluid-phase lipid vesicles. We extended the study to the interaction of the peptide with circulating blood cells, by using the dipole potential sensitive probe di-8-ANEPPS. Sifuvirtide decreased the dipole potential of erythrocyte and lymphocyte membranes in a concentration dependent manner, demonstrating its interaction. Also, the lipid selectivity of the peptide towards more rigid phosphatidylcholines was confirmed based on the dipole potential variations. Overall, the interaction of the peptide with the cell membranes is a contribution of different lipid preferences that presumably directs the peptide towards raft-like domains where the receptors are located, facilitating the reach of the peptide to its molecular target, the gp41 in its pre-fusion conformation.

Modulation of hemorheological parameters by the erythrocyte redox thiol status

BACKGROUND There is growing knowledge about the association between hemorheological blood disorders and compromised microcirculation in erythrocyte abnormalities. Effects of the non-neuronal cholinergic elements, especially acetylcholinesterase, on the erythrocyte hemorheological parameters were characterized in the past. However, alterations of these parameters have not been studied under the influence of the cellular redox thiol status. METHODS Aliquots of venous blood from ten healthy male subjects were incubated in vitro with increasing concentrations of a thiol reducer agent (dithiothreitol 1, 10, 50 microM final concentrations) in the presence and absence of acetylcholinesterase substrate (acetylcholine) or inhibitor (velnacrine maleate). The following parameters were determined in all blood samples aliquots: erythrocyte aggregation, erythrocyte deformability and lipid membrane fluidity. Blood smears were performed. RESULTS Dithiothreitol induces no significant changes on the hemorheological behaviour of human red cells. Upon intracellular thiol stimulation, the presence of AChE effectors (either acetylcholine or velnacrine) decreases erythrocyte aggregation and elongation indexes. CONCLUSION The addition of DTT to blood samples aliquots, contributing to the redox thiol status, is not directly involved in the modulation of erythrocyte rheological properties. However, upon acetylcholinesterase modulation by its substrate or inhibitor, changes on the hemorheological parameters are triggered by DTT. Associated pharmacological interest is considerable to address the hemorheology-hemostasis-microcirculation triad disorders.

Optimizing the use of linaclotide in patients with constipation-predominant irritable Bowel syndrome: an expert consensus report

IntroductionIrritable bowel syndrome (IBS) is a functional bowel disorder characterized by chronic or recurrent abdominal pain in association with defecation or a change in bowel habits. A predominant disorder of bowel habits, IBS is classified into three main subtypes: constipation-predominant IBS (IBS-C), diarrhea-predominant IBS (IBS-D) and IBS alternating between constipation and diarrhea (IBS-M). Linaclotide is a first-in-class, oral, once-daily guanylate cyclase-C receptor agonist (GC-CA) that is licensed for the symptomatic treatment of moderate-to-severe IBS-C in adults. This review aims to facilitate and optimize clinical practices, establishing common guidelines to monitor patients with IBS-C that are treated with linaclotide.MethodsA group of experts in functional digestive disorders was convened to review the efficacy and safety of linaclotide and to develop an updated consensus report for the treatment of patients with IBS-C. A search was performed for English, French and Spanish language articles in PubMed. On the basis of the articles identified, an initial document was drafted addressing different issues frequently raised by general practitioners and GI specialists that are related to the prescription, efficacy and safety of linaclotide. This document was then reviewed and modified by the expert panel until a final text was agreed upon and validated.ResultsBased on the evidence, the panel addressed the following recommendations: (1) Linaclotide is indicated for the treatment of moderate to severe IBS-C in adults; (2) it is recommended that patients take linaclotide continuously and not sporadically; (3) patients should be warned about the risk of diarrhea and given choices concerning how to deal with this possible side effect; (4) the absence of tachyphylaxis or potential risks implies that linaclotide treatment can be maintained for long periods of time.ConclusionsThis document seeks to lay down a set of recommendations and to identify key issues that may be useful for the clinical management of IBS-C patients treated with linaclotide.

Impact of Histological and Endoscopic Remissions on Clinical Recurrence and Recurrence-free Time in Ulcerative Colitis

Background: Clinical and endoscopic remissions constitute the therapeutic goals in ulcerative colitis (UC). Histological healing is currently not a target in UC. This study aims to determine the impact of the definition of endoscopic remission (Mayo endoscopic subscore [MSe] 0–1) and histological activity in the recurrence of UC and recurrence-free survival time. Methods: Patients with UC in clinical remission (partial Mayo score ⩽ 1) and endoscopic remission (MSe ⩽ 1) who underwent colonoscopy with biopsies between March 2010 and December 2013 were included. The validated Nancy score was used to evaluate histological activity, which considers inactivity if 0 to 1 and activity if 2 to 4. The recurrence-free time was evaluated and recurrence was defined as partial Mayo score ≥ 2, therapy to induce remission, hospitalization, or colectomy. Predictive factors associated with recurrence and time to recurrence were determined. Results: Sixty patients were included; 58.3% (n = 35) were women, with a mean age of 52.7 years. MSe = 1 was observed in 46.7% (n = 28) and histological activity in 38.3% (n = 23). Clinical recurrence occurred in 31.7% (n = 19) of patients, with a cumulative risk of 17.1%/24.5%/26.7%/40.1% at 12/24/36/48 months, respectively. MSe = 1 (P = 0.02) and histological activity (P = 0.007) were significantly associated with recurrence. Of these, only histological activity (P = 0.03) was an independent predictive factor of recurrence. Patients with MSe = 1 (P = 0.02) and with histological activity (P = 0.01) had a significantly shorter recurrence-free time in univariate analysis. In multivariate analysis, only histological activity (P = 0.02) was an independent predictive factor of lower recurrence-free time. Conclusion: The presence of histological activity represents an independent predictive factor of recurrence and time to recurrence, which was not verified with MSe 0 to 1.

Erythrocyte nitric oxide in glaucoma patients : ex vivo study

BACKGROUND Glaucoma is an optic neuropathy associated with vascular dysregulation and increased intra-ocular pressure (IOP). Timolol is used as treatment for reducing IOP, by limiting aqueous humour production. Increased NOS expression as well as decreased levels of nitric oxide (NO) metabolites, and high activity of erythrocyte acetylcholinesterase (AChE) were observed in primary open angle glaucoma patients. OBJECTIVE This ex vivo study aims to evaluate timolol effect in NO efflux and its derivatives in glaucoma patients erythrocytes. METHODS Venous blood from 15 glaucoma patients was collected. Erythrocyte suspensions were incubated with the AChE modulators acetylcholine (ACh) and timolol at 10 μM. Erythrocyte NO efflux and S-nitrosoglutathione (GSNO) concentration were measured. RESULTS No significant differences were obtained in erythrocyte NO efflux and GSNO concentration in response to ACh or timolol when compared with the untreated erythrocytes of glaucoma patients. When comparing the same incubation conditions for erythrocyte suspensions between glaucoma patients and healthy subjects, those from glaucoma patients showed higher NO efflux in presence and absence of timolol, and higher values of GSNO in the presence of timolol. CONCLUSIONS We demonstrated that erythrocytes from glaucoma patients have similar availability to release NO both in absence and presence of timolol, and have higher GSNO values in presence of timolol.

Identification of erythrocyte biomarkers in amyotrophic lateral sclerosis.

INTRODUCTION Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease of the motor system. It has been hypothesised that red blood cells (RBCs) may be involved in the disease process by the release of damaging molecules. OBJECTIVE The aim of this ex vivo study is to compare RBCs biochemical and hemorheological parameters between ALS patients and healthy donors to identify novel biomarkers of the ALS disease. METHODS We included 82 ALS patients and 40 gender age-matched healthy donors. We performed quantification of erythrocyte aggregation and deformability, nitric oxide (NO) efflux from RBCs, acetylcholinesterase (AChE) enzyme activity and intraerythrocytic concentration of nitrite, nitrate and S-nitrosogluthatione (GSNO). RESULTS Erythrocyte deformability and AChE activity were increased in patients with ALS in comparison to healthy donors. NO efflux from RBCs and concentration of intraerythrocytic nitrite were lower in ALS patients. In patients, we found that for higher NO range of values the respiratory function is worse and that for higher AChE range of values the RBCs nitrite content increase. CONCLUSION The results of the present study indicate that NO efflux from RBCs and RBCs AChE should be further explored as potential biomarkers for ALS.

An ex vivo study of nitric oxide efflux from human erythrocytes in both genders.

INTRODUCTION Acetylcholinesterase (AChE) is located on outer surface of erythrocyte membrane. Gender-related differences in erythrocyte AChE enzyme activity had been verified in young adults. It is also known that binding of acetylcholine (ACh) with AChE on erythrocyte membrane initiates a signal transduction mechanism that stimulates nitric oxide (NO) efflux. AIMS This ex vivo study was done to compare the amount of NO efflux obtained from erythrocytes of healthy donors in males and females. METHODS We included 66 gender age-matched healthy donors (40-60 years old). We performed quantification of erythrocyte NO efflux from erythrocytes and of the membrane AChE enzyme activity. RESULTS There are no significant differences in NO efflux from erythrocytes between men and women. Regarding AChE enzyme activity values, in this range of age, no differences between genders were obtained. However, the values of AChE enzyme activity in the third quartile of NO efflux values were significantly higher (p < 0.05) in women than in men. CONCLUSIONS The efflux of NO from erythrocyte of healthy humans did not change with gender. For the same range of values of NO efflux from erythrocytes, in both gender, it was verified higher values of AChE enzyme activity in women.

Taking nanomedicine teaching into practice with atomic force microscopy and force spectroscopy

Atomic force microscopy (AFM) is a useful and powerful tool to study molecular interactions applied to nanomedicine. The aim of the present study was to implement a hands-on atomic AFM course for graduated biosciences and medical students. The course comprises two distinct practical sessions, where students get in touch with the use of an atomic force microscope by performing AFM scanning images of human blood cells and force spectroscopy measurements of the fibrinogen-platelet interaction. Since the beginning of this course, in 2008, the overall rating by the students was 4.7 (out of 5), meaning a good to excellent evaluation. Students were very enthusiastic and produced high-quality AFM images and force spectroscopy data. The implementation of the hands-on AFM course was a success, giving to the students the opportunity of contact with a technique that has a wide variety of applications on the nanomedicine field. In the near future, nanomedicine will have remarkable implications in medicine regarding the definition, diagnosis, and treatment of different diseases. AFM enables students to observe single molecule interactions, enabling the understanding of molecular mechanisms of different physiological and pathological processes at the nanoscale level. Therefore, the introduction of nanomedicine courses in bioscience and medical school curricula is essential.

Blood cell membrane fluidity and intracellular Ca2+ changes in antiretroviral-naïve and -treated HIV-1-infected patients

We previously showed that lymphocytes and erythrocytes of HIV-1–infected patients, prior to antiretroviral therapy, presented significant changes in intracellular calcium concentration ([Ca2+]int) and membrane fluidity. The present study evaluates the same parameters after response to highly active antiretroviral therapy (HAART). Blood samples were collected from patients prior to and after antiretroviral therapy, and from control subjects. Membrane fluidity and [Ca2+]int were assessed by fluorescence spectroscopy measurements, using three different probes: TMA-DPH and DPH for membrane fluidity, and fura-2 for Ca2+. When compared with the control group, both untreated and treated patients presented increased lymphocyte [Ca2+]int and decreased lymphocyte membrane fluidity, without significant differences between the two groups of patients. On the contrary, the therapy reversed the membrane fluidity variations observed in erythrocytes. The decreased erythrocyte [Ca2+]int of untreated patients was not reversed by HAART. AIDS patients present changes in lymphocyte (mostly noninfected) and erythrocyte properties, partially reversed by HAART, consistent with a process of facilitated propagation of the infection to new cells, stimulation of virion production, and maintenance of a reservoir of erythrocyte-bound infectious virus. These observations can be related with the action of the HIV Nef protein in the cells proteins and lipid composition, as well as with the recently observed cell infection by HIV-1 via endocytosis.