Characterization of [Ca2+]i responses in primary cultures of mouse cardiomyocytes induced by Trypanosoma cruzi trypomastigotes

Trypanosoma cruzi, the protozoan responsible for Chagas disease, employs distinct strategies to invade mammalian host cells. In the present work we investigated the participation of calcium ions on the invasion process using primary cultures of embryonic mice cardiomyocytes which exhibit spontaneous contraction in vitro. Using Fura 2-AM we found that T. cruzi was able to induce a sustained increase in basal intracellular Ca2+ level in heart muscle cells (HMC), the response being associated or not with Ca2+ transient peaks. Assays performed with both Y and CL strains indicated that the changes in intracellular Ca2+ started after parasites contacted with the cardiomyocytes and the evoked response was higher than the Ca2+ signal associated to the spontaneous contractions. The possible role of the extracellular and intracellular Ca2+ levels on T. cruzi invasion process was evaluated using the extracellular Ca2+ chelator EGTA alone or in association with the calcium ionophore A23187. Significant dose dependent inhibition of the invasion levels were found when intracellular calcium release was prevented by the association of EGTA +A23187 in calcium free medium. Dose response experiments indicated that EGTA 2.5 mM to 5 mM decreased the invasion level by 15.2 to 35.1% while A23187 (0.5 µM) alone did not induce significant effects (17%); treatment of the cultures with the protease inhibitor leupeptin did not affect the endocytic index, thus arguing against the involvement of leupeptin sensitive proteases in the invasion of HMC.

Basal and stimulated lactate fluxes in primary cultures of astrocytes are differentially controlled by distinct proteins.

Lactate release by astrocytes is postulated to be of importance for neuroenergetics but its regulation is poorly understood. Basigin, a chaperone protein for specific monocarboxylate transporters (MCTs), represents a putatively important regulatory element for lactate fluxes. Indeed, basigin knockdown by RNA interference in primary cultures of astrocytes partially reduced both proton-driven lactate influx and efflux. But more strikingly, enhancement of lactate efflux induced by glutamate was prevented while the effect of sodium azide was significantly reduced by treatment of cultured astrocytes with anti-basigin small interfering RNA. Enhancement of glucose utilization was unaffected under the same conditions. Basal lactate uptake and release were significantly reduced by MCT1 knockdown, even more so than with basigin knockdown, whereas glutamate-driven or sodium azide-induced enhancement of lactate release was not inhibited by either MCT1, 2, or 4 small interfering RNAs. In conclusion, MCT1 plays a pivotal role in the control of basal proton-driven lactate flux in astrocytes while basigin is only partly involved, most likely via its interaction with MCT1. In contrast, basigin appears to critically regulate the enhancement of lactate release caused by glutamate (or sodium azide) but via an effect on another unidentified transporter at least present in astrocytes in vitro.

Effect of angiotensin II and losartan on the phagocytic activity of peritoneal macrophages from Balb/C mice

Angiotensin II (AII), a product of rennin-angiotensin system, exerts an important role on the function of immune system cells. In this study, the effect of AII on the phagocytic activity of mouse peritoneal macrophages was assessed. Mice peritoneal macrophages were cultured for 48 h and the influence of different concentrations of AII (10-14 to 10-7 M) and/or losartan, 10-16 to 10-6 M), an AT1 angiotensin receptor antagonist, on phagocytic activity and superoxide anion production was determined. Dimethylthiazoldiphenyltetrazolium bromide reduction and the nucleic acid content were used to assess the cytotoxicity of losartan. A stimulatory effect on phagocytic activity (P < 0.05) was observed with 10-13 M and 10-12 M AII concentrations. The addition of losartan (up to10-14 M) to the cell cultures blocked (P < 0.001) the phagocytosis indicating the involvement of AT1 receptors. In contrast, superoxide anion production was not affected by AII or losartan. The existence of AT1 and AT2 receptors in peritoneal macrophages was demonstrated by immunofluorescence microscopy. These results support the hypothesis that AII receptors can modulate murine macrophage activity and phagocytosis, and suggest that AII may have a therapeutic role as an immunomodulatory agent in modifying the host resistance to infection.

Maladies inflammatoires de l'intestin [Main therapeutic advances in IBD in 2008]

A coimmunosuppression with azathioprine or methotrexate in addition to infliximab does not improve the therapeutic efficacy in Crohn's disease but increase the risks of infectious complications and neoplasia.

Therapeutic Drug Monitoring of the new targeted anticancer agents imatinib, nilotinib, dasatinib, sunitinib, sorafenib and lapatinib by LC tandem mass spectrometry.

The treatment of some cancer patients has shifted from traditional, non-specific cytotoxic chemotherapy to chronic treatment with molecular targeted therapies. Imatinib mesylate, a selective inhibitor of tyrosine kinases (TKIs) is the most prominent example of this new era and has opened the way to the development of several additional TKIs, including sunitinib, nilotinib, dasatinib, sorafenib and lapatinib, in the treatment of various hematological malignancies and solid tumors. All these agents are characterized by an important inter-individual pharmacokinetic variability, are at risk for drug interactions, and are not devoid of toxicity. Additionally, they are administered for prolonged periods, anticipating the careful monitoring of their plasma exposure via Therapeutic Drug Monitoring (TDM) to be an important component of patients' follow-up. We have developed a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) requiring 100 microL of plasma for the simultaneous determination of the six major TKIs currently in use. Plasma is purified by protein precipitation and the supernatant is diluted in ammonium formate 20 mM (pH 4.0) 1:2. Reverse-phase chromatographic separation of TKIs is obtained using a gradient elution of 20 mM ammonium formate pH 2.2 and acetonitrile containing 1% formic acid, followed by rinsing and re-equilibration to the initial solvent composition up to 20 min. Analyte quantification, using matrix-matched calibration samples, is performed by electro-spray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effects variability (<9.6%), overall process efficiency (87.1-104.2%), as well as TKIs short- and long-term stability in plasma. The method is precise (inter-day CV%: 1.3-9.4%), accurate (-9.2 to +9.9%) and sensitive (lower limits of quantification comprised between 1 and 10 ng/mL). This is the first broad-range LC-MS/MS assay covering the major currently in-use TKIs. It is an improvement over previous methods in terms of convenience (a single extraction procedure for six major TKIs, reducing significantly the analytical time), sensitivity, selectivity and throughput. It may contribute to filling the current knowledge gaps in the pharmacokinetics/pharmacodynamics relationships of the latest TKIs developed after imatinib and better define their therapeutic ranges in different patient populations in order to evaluate whether a systematic TDM-guided dose adjustment of these anticancer drugs could contribute to minimize the risk of major adverse reactions and to increase the probability of efficient, long lasting, therapeutic response.

Differential expression of notch signaling-related transcripts accompanies Pro-thymocyte proliferation and phenotype transition induced by epidermal growth factor plus insulin in fetal thymus organ cultures

Thymus regression upon stressing stimuli, such as infectious diseases, is followed by organ reconstitution, paralleling its development in ontogeny. A narrow window of thymus development was here studied, encompassing the pro-T lymphoid precursor expansion during specification stages, by the use of epidermal growth factor plus insulin (INS) in murine fetal thymus organ cultures. Aiming to disclose signaling pathways related to these stages, cultured thymus lobes had their RNA extracted, for the search of transcripts differentially expressed using RNAse protection assays and reverse transcriptase-polymerase chain reactions. We found no difference that could explain INS-driven thymocyte growth, in the pattern of transcripts for death/proliferation mediators, or for a series of growth factor receptors and transcriptional regulators known as essential for thymus development. Thymocyte suspensions from cultured lobes, stained for phenotype analysis by fluorescence activated cell sorting, showed a decreased staining for Notch1 protein at cell surfaces upon INS addition. We analyzed the expression of Notch-related elements, and observed the recruitment of a specific set of transcripts simultaneous and compatible with INS-driven thymocyte growth, namely, transcripts for Notch3, for its ligand Jagged2, and for Deltex1, a mediator of a poorly characterized alternative pathway downstream of the Notch receptor.

Therapeutic drug monitoring of imatinib: Bayesian and alternative methods to predict trough levels

Background: The imatinib trough plasma concentration (C(min)) correlates with clinical response in cancer patients. Therapeutic drug monitoring (TDM) of plasma C(min) is therefore suggested. In practice, however, blood sampling for TDM is often not performed at trough. The corresponding measurement is thus only remotely informative about C(min) exposure. Objectives: The objectives of this study were to improve the interpretation of randomly measured concentrations by using a Bayesian approach for the prediction of C(min), incorporating correlation between pharmacokinetic parameters, and to compare the predictive performance of this method with alternative approaches, by comparing predictions with actual measured trough levels, and with predictions obtained by a reference method, respectively. Methods: A Bayesian maximum a posteriori (MAP) estimation method accounting for correlation (MAP-ρ) between pharmacokinetic parameters was developed on the basis of a population pharmacokinetic model, which was validated on external data. Thirty-one paired random and trough levels, observed in gastrointestinal stromal tumour patients, were then used for the evaluation of the Bayesian MAP-ρ method: individual C(min) predictions, derived from single random observations, were compared with actual measured trough levels for assessment of predictive performance (accuracy and precision). The method was also compared with alternative approaches: classical Bayesian MAP estimation assuming uncorrelated pharmacokinetic parameters, linear extrapolation along the typical elimination constant of imatinib, and non-linear mixed-effects modelling (NONMEM) first-order conditional estimation (FOCE) with interaction. Predictions of all methods were finally compared with 'best-possible' predictions obtained by a reference method (NONMEM FOCE, using both random and trough observations for individual C(min) prediction). Results: The developed Bayesian MAP-ρ method accounting for correlation between pharmacokinetic parameters allowed non-biased prediction of imatinib C(min) with a precision of ±30.7%. This predictive performance was similar for the alternative methods that were applied. The range of relative prediction errors was, however, smallest for the Bayesian MAP-ρ method and largest for the linear extrapolation method. When compared with the reference method, predictive performance was comparable for all methods. The time interval between random and trough sampling did not influence the precision of Bayesian MAP-ρ predictions. Conclusion: Clinical interpretation of randomly measured imatinib plasma concentrations can be assisted by Bayesian TDM. Classical Bayesian MAP estimation can be applied even without consideration of the correlation between pharmacokinetic parameters. Individual C(min) predictions are expected to vary less through Bayesian TDM than linear extrapolation. Bayesian TDM could be developed in the future for other targeted anticancer drugs and for the prediction of other pharmacokinetic parameters that have been correlated with clinical outcomes.

Differences in the stability of the plasmids of Yersinia pestis cultures in vitro: impact on virulence

Plasmid and chromosomal genes encode determinants of virulence for Yersinia pestis, the causative agent of plague. However, in vitro, Y. pestis genome is very plastic and several changes have been described. To evaluate the alterations in the plasmid content of the cultures in vitro and the impact of the alterations to their pathogenicity, three Y. pestis isolates were submitted to serial subculture, analysis of the plasmid content, and testing for the presence of characteristic genes in each plasmid of colonies selected after subculture. Different results were obtained with each strain. The plasmid content of one of them was shown to be stable; no apparent alteration was produced through 32 subcultures. In the other two strains, several alterations were observed. LD50 in mice of the parental strains and the derived cultures with different plasmid content were compared. No changes in the virulence plasmid content could be specifically correlated with changes in the LD50.

Presence of JC virus-specific CTL in the cerebrospinal fluid of PML patients: rationale for immune-based therapeutic strategies.

OBJECTIVES: There is urgent need of a treatment for progressive multifocal leukoencephalopathy (PML), caused by the polyomavirus JC (JCV). To evaluate the rationale for immunotherapy of PML, we explored whether JCV-specific cytotoxic T lymphocytes (CTL) can penetrate the central nervous system (CNS). In addition, we studied the breadth of their T-cell receptor (TCR) repertoire, and sought to establish a reliable method to expand these cells in vitro. DESIGN AND METHODS: We enrolled 18 patients in this study, including 16 with proven or possible PML (15 HIV-positive and one HIV-negative), and two HIV-positive patients with other neurological diseases. Detection of JCV-specific CTL in the blood and the cerebrospinal fluid was performed by Cr release and tetramer staining assays in 15 patients. RESULTS: Of 11 PML patients with analyzable cerebrospinal fluid (CSF), two had no detectable JCV-specific CTL in the blood and CSF and died 3.7 and 7.2 months later. The nine remaining patients had an inactive course of PML and detectable JCV-specific CTL in the blood. In addition, four of them (44%) also had detectable JCV-specific CTL in the CSF. Both HIV-positive patients with OND had detectable JCV-specific CTL in the blood and one in the CSF. Using tetramer technology, we obtained highly enriched JCV-specific CTL lines that were able to kill target cells presenting JCV peptides. The breadth of the TCR repertoire was CTL epitope dependent. CONCLUSIONS: These results indicate that JCV-specific CTL are present in the CNS of PML patients and pave the way for an immune-based therapeutic approach.

Protein kinase C-activating tumor promoters enhance the differentiation of astrocytes in aggregating fetal brain cell cultures.

Serum-free aggregating cell cultures of fetal rat telencephalon treated with the potent tumor promoter phorbol 12-myristate 13-acetate (PMA) showed a marked, rapid, and sustained increase in the activity of the astrocyte-specific enzyme glutamine synthetase (GS). This effect was accompanied by a small increase in RNA synthesis and a progressive reduction in DNA synthesis. Only mitotically active cultures were responsive to PMA treatments. Since in aggregate cultures astrocytes are the preponderant cell type, both in number and mitotic activity, it can be concluded that PMA induces and/or enhances the terminal differentiation of astrocytes. The developmental expression of GS was also greatly stimulated by mezerein, a potent nonphorbol tumor promoter, but not by 4 alpha-phorbol 12,13-didecanoate, a nonpromoting phorbol ester. Since both tumor promoters, PMA and mezerein, are potent and specific activators of C-kinase, it is suggested that C-kinase plays a regulatory role in the growth and differentiation of normal astrocytes.

Insulin Resistance as a Therapeutic Target for Chronic Kidney Disease.

Insulin resistance (IR) is a prevalent metabolic feature in chronic kidney disease (CKD). Postreceptor insulin-signaling defects have been observed in uremia. A decrease in the activity of phosphatidylinositol 3-kinase appears critical in the pathophysiology of CKD-associated IR. Lipotoxicity due to ectopic accumulation of lipid moieties has recently emerged as another mechanism by which CKD and/or associated metabolic disorders may lead to IR through impairment of various insulin-signaling molecules. Metabolic acidosis, anemia, excess of fat mass, inflammation, vitamin D deficiency, adipokine imbalance, physical inactivity, and the accumulation of nitrogenous compounds of uremia all contribute to CKD-associated IR. The clinical impacts of IR in this setting are numerous, including endothelial dysfunction, increased cardiovascular mortality, muscle wasting, and possibly initiation and progression of CKD. This is why IR may be a therapeutic target in the attempt to improve outcomes in CKD. General measures to improve IR are directed to counteract causal factors. The use of pharmaceutical agents such as inhibitors of the renin-angiotensin system may improve IR in hypertensive and CKD patients. Pioglitazone appears a safe and promising therapeutic agent to reduce IR and uremic-associated abnormalities. However, interventional studies are needed to test if the reduction and/or normalization of IR may actually improve outcomes in these patients.

Body temperature regulation and outcome after cardiac arrest and therapeutic hypothermia.

OBJECTIVE: Therapeutic temperature modulation is recommended after cardiac arrest (CA). However, body temperature (BT) regulation has not been extensively studied in this setting. We investigated BT variation in CA patients treated with therapeutic hypothermia (TH) and analyzed its impact on outcome. METHODS: A prospective cohort of comatose CA patients treated with TH (32-34°C, 24h) at the medical/surgical intensive care unit of the Lausanne University Hospital was studied. Spontaneous BT was recorded on hospital admission. The following variables were measured during and after TH: time to target temperature (TTT=time from hospital admission to induced BT target <34°C), cooling rate (spontaneous BT-induced BT target/TTT) and time of passive rewarming to normothermia. Associations of spontaneous and induced BT with in-hospital mortality were examined. RESULTS: A total of 177 patients (median age 61 years; median time to ROSC 25 min) were studied. Non-survivors (N=90, 51%) had lower spontaneous admission BT than survivors (median 34.5 [interquartile range 33.7-35.9]°C vs. 35.1 [34.4-35.8]°C, p=0.04). Accordingly, time to target temperature was shorter among non-survivors (200 [25-363]min vs. 270 [158-375]min, p=0.03); however, when adjusting for admission BT, cooling rates were comparable between the two outcome groups (0.4 [0.2-0.5]°C/h vs. 0.3 [0.2-0.4]°C/h, p=0.65). Longer duration of passive rewarming (600 [464-744]min vs. 479 [360-600]min, p<0.001) was associated with mortality. CONCLUSIONS: Lower spontaneous admission BT and longer time of passive rewarming were associated with in-hospital mortality after CA and TH. Impaired thermoregulation may be an important physiologic determinant of post-resuscitation disease and CA prognosis. When assessing the benefit of early cooling on outcome, future trials should adjust for patient admission temperature and use the cooling rate rather than the time to target temperature.

Therapeutic failure of praziquantel in the treatment of Schistosoma haematobium infection in Brazilians returning from Africa

Several cases of therapeutic failure of praziquantel used for the treatment of urinary schistosomiasis have been reported. Alternative drugs, like niridazol and metrifonate, have shown a lower therapeutic effect and more side effects than praziquantel. Twenty-six Brazilian military men (median age of 29 years) with a positive urine parasitological exam who were part of a United Nation peace mission in Mozambique in 1994 were treated with 40 mg/kg body weight praziquantel, single dose. They swimmed in Licungo river (Mocuba city, Mozambique) during the weekends. After this, they presented haematuria, dysuria, polakiuria, and lumbar pain. Control cystoscopy examinations carried out between 6 and 24 months after each treatment (including two additional treatments at a minimum interval of 6 months) revealed the presence of viable eggs. Granulomas in the vesical submucosa were observed in 46.2% (12/26) of the individuals. A vesical biopsy confirmed the presence of granulomas in all of these patients and the presence of viable eggs in 34.3% (9/26) of individuals who no longer excreted eggs in urine. The eggs filled with miracidia showed characteristics of viability. Histopathological examination using different strains demonstrated therapeutic failure and the need for repeated treatment. In this study, we demonstrated a low efficacy of praziquantel in the treatment of schistosomiasis haematobia, and the necessity of the urinary bladder biopsy as criterion of cure.

CC-chemokine receptors: a potential therapeutic target for Trypanosoma cruzi-elicited myocarditis

The comprehension of the pathogenesis of Trypanosoma cruzi-elicited myocarditis is crucial to delineate new therapeutic strategies aiming to ameliorate the inflammation that leads to heart dysfunction, without hampering parasite control. The augmented expression of CCL5/RANTES and CCL3/MIP-1alpha, and their receptor CCR5, in the heart of T. cruzi-infected mice suggests a role for CC-chemokines and their receptors in the pathogenesis of T. cruzi-elicited myocarditis. Herein, we discuss our recent results using a CC-chemokine receptor inhibitor (Met-RANTES), showing the participation of CC-chemokines in T. cruzi infection and unraveling CC-chemokine receptors as an attractive therapeutic target for further evaluation in Chagas disease.