Flávia Isaura de Santi Ferreira

Mesenchymal stromal cells up-regulate CD39 and increase adenosine production to suppress activated T-lymphocytes.

Mesenchymal stromal cells (MSCs) suppress T cell responses through mechanisms not completely understood. Adenosine is a strong immunosuppressant that acts mainly through its receptor A(2a) (ADORA2A). Extracellular adenosine levels are a net result of its production (mediated by CD39 and CD73), and of its conversion into inosine by Adenosine Deaminase (ADA). Here we investigated the involvement of ADO in the immunomodulation promoted by MSCs. Human T lymphocytes were activated and cultured with or without MSCs. Compared to lymphocytes cultured without MSCs, co-cultured lymphocytes were suppressed and expressed higher levels of ADORA2A and lower levels of ADA. In co-cultures, the percentage of MSCs expressing CD39, and of T lymphocytes expressing CD73, increased significantly and adenosine levels were higher. Incubation of MSCs with media conditioned by activated T lymphocytes induced the production of adenosine to levels similar to those observed in co-cultures, indicating that adenosine production was mainly derived from MSCs. Finally, blocking ADORA2A signaling raised lymphocyte proliferation significantly. Our results suggest that some of the immunomodulatory properties of MSCs may, in part, be mediated through the modulation of components related to adenosine signaling. These findings may open new avenues for the development of new treatments for GVHD and other inflammatory diseases.

Low expression of CD39 on regulatory T cells as a biomarker for resistance to methotrexate therapy in rheumatoid arthritis

Significance Methotrexate (MTX) is the first-line therapy for rheumatoid arthritis (RA). However, about 40% of patients are resistant to MTX. Furthermore, MTX resistance is only apparent after a prolonged continuous MTX treatment (>3 mo), by which time the disease of the nonresponders would have aggravated. Thus, there is a considerable unmet need for a biomarker to select MTX-resistant patients and place them immediately on alternative therapy. We found here that the low density of CD39 on peripheral regulatory T cells in RA patients is a rapid, convenient, and reliable (P < 0.01) biomarker for MTX resistance. Our findings also provide previously unrecognized information on aspects of immune regulation in RA and the mechanism of action of MTX. Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by joint destruction and severe morbidity. Methotrexate (MTX) is the standard first-line therapy of RA. However, about 40% of RA patients are unresponsive to MTX treatment. Regulatory T cells (Tregs, CD4+CD25+FoxP3+) are thought to play an important role in attenuating RA. To investigate the role of Tregs in MTX resistance, we recruited 122 RA patients (53 responsive, R-MTX; 69 unresponsive, UR-MTX) and 33 healthy controls. Three months after MTX treatment, R-MTX but not UR-MTX showed higher frequency of peripheral blood CD39+CD4+CD25+FoxP3+ Tregs than the healthy controls. Tregs produce adenosine (ADO) through ATP degradation by sequential actions of two cell surface ectonucleotidases: CD39 and CD73. Tregs from UR-MTX expressed a lower density of CD39, produced less ADO, and had reduced suppressive activity than Tregs from R-MTX. In a prospective study, before MTX treatment, UR-MTX expressed a lower density of CD39 on Tregs than those of R-MTX or control (P < 0.01). In a murine model of arthritis, CD39 blockade reversed the antiarthritic effects of MTX treatment. Our results demonstrate that MTX unresponsiveness in RA is associated with low expression of CD39 on Tregs and the decreased suppressive activity of these cells through reduced ADO production. Our findings thus provide hitherto unrecognized mechanism of immune regulation in RA and on mode of action of MTX. Furthermore, our data suggest that low expression of CD39 on Tregs could be a noninvasive biomarker for identifying MTX-resistant RA patients.

Hydroxycarbamide modulates components involved in the regulation of adenosine levels in blood cells from sickle-cell anemia patients

Recent studies have demonstrated the role of adenosine (ADO) in sickle-cell anemia (SCA). ADO is produced by CD39 and CD73 and converted to inosine by adenosine deaminase (ADA). We evaluated the effects of hydroxycarbamide (HU) treatment on the modulation of adenosine levels in SCA patients. The expressions of CD39, CD73, and CD26 were evaluated by flow cytometry on blood cells in 15 HU-treated and 17 untreated patients and 10 healthy individuals. RNA was extracted from monocytes, and ADA gene expression was quantified by real-time PCR. ADA activity was also evaluated. We found that ADA transcripts were two times higher in monocytes of HU-treated patients, compared with untreated (P = 0.039). Monocytes of HU-treated patients expressed CD26, while monocytes of controls and untreated patients did not (P = 0.023). In treated patients, a lower percentage of T lymphocytes expressed CD39 compared with untreated (P = 0.003), and the percentage of T regulatory (Treg) cells was reduced in the treated group compared with untreated (P = 0.017) and controls (P = 0.0009). Besides, HU-treated patients displayed increased ADA activity, compared with untreated. Our results indicate a novel mechanism of action of HU mediated by the reduction of adenosine levels and its effects on pathophysiological processes in SCA.

Olanzapine, weight change and metabolic effects: a naturalistic 12-month follow up.

Objective: Olanzapine is an atypical antipsychotic drug used to treat schizophrenia. Some of the adverse effects related to its use are obesity, hyperlipidemia, type 2 diabetes and hypertension, which may result in development of metabolic syndrome. This study aimed to investigate a possible increase in some anthropometric and biochemical parameters, and the existence of any correlation between them in Brazilian patients with schizophrenia treated with olanzapine in the mid term. Methods: Thirty subjects with schizophrenia were evaluated, 16 women and 14 men, aged between 18 and 47 years. All patients underwent blood collection and anthropometric measurements at four different times during 12 months of follow up; thus each patient was his or her own control. Results: Evaluation of some anthropometric measurements showed significant differences when comparing the mean values obtained in each of the different data collection times (p < 0.05). However, the biochemical indicators of development of metabolic syndrome measured in our study did not show the same rate of increment, with only the total cholesterol and glucose levels presenting statistically significant changes (p < 0.05), but without the same magnitude of weight change. Conclusion: We conclude that medium-term treatment with olanzapine promoted a substantial weight gain and increased visceral fat, while the metabolic profile did not show the same magnitude of change, suggesting a dissociation between weight gain and blood parameters, despite the severe weight gain observed among subjects evaluated.

An investigation of the influence of patient-related factors and comedications on lamotrigine clearance in patients with epilepsy

Lamotrigine (LTG) is one of the most widely used antiepileptic drugs. Confusion still exists in the literature as to the relative influence of age, body weight, and concomitant drug therapy on LTG pharmacokinetics. So, the objective of this study is to evaluate the influence of patient‐related factors and comedication on LTG apparent oral clearance (CL/F). A therapeutic drug‐monitoring database was used to identify steady‐state plasma LTG concentrations in 210 patients. LTG CL/F values were calculated for each patient according to the equation CL/F (L/h per kg) = LTG daily dose (mg/kg)/Css (steady state concentration) (mg/L) × 24 h. A linear‐regression model was used to assess the influence of gender, dose, age, and body weight in LTG CL/F. The influence of comedication on LTG CL/F was investigated by applying the Bonferroni post‐test. The lowest LTG CL/F was found in patients comedicated with valproate (VPA) (mean, 0.0183 L/h per kg), followed by patients receiving VPA + enzyme inducers (0.0271 L/h per kg), patients on LTG monotherapy (0.0298 L/h per kg) and patients comedicated with enzyme inducers (0.056 L/h per kg) LTG CL/F correlated significantly with LTG dose (P < 0.01), but showed no significant relationship with gender, weight, and age. LTG CL/F is influenced by the type of antiepileptic comedication. The correlation with dose may be a spurious finding related to the fact that physicians, in adjusting dosage according to clinical response, are more likely to use larger doses in patients with high clearance values.

The social context and the need of information from patients with epilepsy: evaluating a tertiary referral service

OBJECTIVE Characterize the social profile and the need of information from patients with refractory epilepsy. METHOD A semi-structured questionnaire was applied to 103 patients to investigate sociodemographic aspects, pharmacotherapy and any doubts about epilepsy. RESULTS Patients were highly dependent on having a free and accessible supply of antiepileptic drugs. Sixty-eight percent of the population was unemployed, and 26% confirmed receiving social security benefits due to epilepsy. Twenty-nine percent of the population reached high school. Eighty-five percent of the patients had at least one doubt about epilepsy; treatment and epilepsy aspects in general were the main topics. CONCLUSION As observed in developed countries, patients with refractory epilepsy from a developing country also have high rates of unemployment and low educational levels. The results raise a concern about the need of information about epilepsy by patients and their families, urging the necessity to invest in strategies to solve this deficiency in knowledge.

Stir bar-sorptive extraction, solid phase extraction and liquid-liquid extraction for levetiracetam determination in human plasma: comparing recovery rates

Levetiracetam (LEV), an antiepileptic drug (AED) with favorable pharmacokinetic profile, is increasingly being used in clinical practice, although information on its metabolism and disposition are still being generated. Therefore a simple, robust and fast liquid-liquid extraction (LLE) followed by high-performance liquid chromatography method is described that could be used for both pharmacokinetic and therapeutic drug monitoring (TDM) purposes. Moreover, recovery rates of LEV in plasma were compared among LLE, stir bar-sorptive extraction (SBSE), and solid-phase extraction (SPE). Solvent extraction with dichloromethane yielded a plasma residue free from usual interferences such as commonly co-prescribed AEDs, and recoveries around 90% (LLE), 60% (SPE) and 10% (SBSE). Separation was obtained using reverse phase Select B column with ultraviolet detection (235 nm). Mobile phase consisted of methanol:sodium acetate buffer 0.125 M pH 4.4 (20:80, v/v). The method was linear over a range of 2.8-220.0 µg mL-1. The intra- and inter-assay precision and accuracy were studied at three concentrations; relative standard deviation was less than 10%. The limit of quantification was 2.8 µg mL-1. This robust method was successfully applied to analyze plasma samples from patients with epilepsy and therefore might be used for pharmacokinetic and TDM purposes.