Chagas' disease: selective affinity and cytotoxicity of Trypanosoma cruzi-immune lymphocytes to parasympathetic ganglion cells

The megaesophagus and megacolon endemic in South America are related , to Chagas' disease. These mega conditions are found in patients with chronic Chagas's infection, when the parasite is not demonstrable in the lesions. These are characterized by depopulation of parasympathetic ganglion cells, dilation and hypertrophy of the viscera. In the experiments described here we deminstrate a selective affinity and adherence of Trypanosoma cruzi-immune lymphocytes to myenteric, parasympathetic ganglion cells, leading to neuronolysis. None of these features are observed when non-immune lymphocytes from control rabbits are used, or when the immune lymphocytes are allowed to react with CNS neurons. This demonstration is an indication of the high degree of specificity of the destruction of parasympathetic neurons in Chagas' disease. We postulate that the T. cruzi-immune lymphocyte rejection of parasympathetic neurons, but not of CNS neurons, might be related to recognition of a cross-reacting antigenic determinant secreted only by the target neurons. In favor of this interpretation is the observation of lymphocytic infiltrates and parasympathetic ganglion cell destruction in chronic Chagas' infection in the absence of encephalitis.

Long-term citalopram administration reduces responsiveness of HPA axis in patients with major depression: relationship with S-citalopram concentrations in plasma and cerebrospinal fluid (CSF) and clinical response.

RATIONALE: A dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is a well-documented neurobiological finding in major depression. Moreover, clinically effective therapy with antidepressant drugs may normalize the HPA axis activity. OBJECTIVE: The aim of this study was to test whether citalopram (R/S-CIT) affects the function of the HPA axis in patients with major depression (DSM IV). METHODS: Twenty depressed patients (11 women and 9 men) were challenged with a combined dexamethasone (DEX) suppression and corticotropin-releasing hormone (CRH) stimulation test (DEX/CRH test) following a placebo week and after 2, 4, and 16 weeks of 40 mg/day R/S-CIT treatment. RESULTS: The results show a time-dependent reduction of adrenocorticotrophic hormone (ACTH) and cortisol response during the DEX/CRH test both in treatment responders and nonresponders within 16 weeks. There was a significant relationship between post-DEX baseline cortisol levels (measured before administration of CRH) and severity of depression at pretreatment baseline. Multiple linear regression analyses were performed to identify the impact of psychopathology and hormonal stress responsiveness and R/S-CIT concentrations in plasma and cerebrospinal fluid (CSF). The magnitude of decrease in cortisol responsivity from pretreatment baseline to week 4 on drug [delta-area under the curve (AUC) cortisol] was a significant predictor (p<0.0001) of the degree of symptom improvement following 16 weeks on drug (i.e., decrease in HAM-D21 total score). The model demonstrated that the interaction of CSF S-CIT concentrations and clinical improvement was the most powerful predictor of AUC cortisol responsiveness. CONCLUSION: The present study shows that decreased AUC cortisol was highly associated with S-CIT concentrations in plasma and CSF. Therefore, our data suggest that the CSF or plasma S-CIT concentrations rather than the R/S-CIT dose should be considered as an indicator of the selective serotonergic reuptake inhibitors (SSRIs) effect on HPA axis responsiveness as measured by AUC cortisol response.

The Azadirachtins: potent insect growth inhibitors

In the course of their coevolution with insects, plants have learnt to protect themselves by chemical means. Semiochemical act as antifeedants or deterrents, others by disrupting growth and development. By use of the Epilachna varivestis bioassay we isolated from Azadirachta indica seed a group of triterpenoids which interfee with larval growth and development in ppm range. Main components are the azadirachtins A and B with identical biological activity. Various other azadirachtins were obtained, either as minor seed components or by chemical modification of the naturally occuring compounds. Structure vs. activity relation studies enabled us to postulate a basic structural element that should still be biologically active and with much simpler chemical structure than natural compounds. What underlies the biological activity of these insect growth inhibitors? Their interference with the hormonal regulation of development and reproduction has been studied in Locusta migratoria and Rhodnius prolixus. In addition, tritiated dihydroazadirachtin A was used. With this approach, a precise correlation between administered dose, resulting effects, and retention of the compound was established. The azadirachtins either interrupt, delay, or deviate whole developmental programs. Results from these studies provide another chemical probe for studies in insect endocrinology and physiology.

Clinical benefit of fulvestrant in postmenopausal women with advanced breast cancer and primary or acquired resistance to aromatase inhibitors: final results of phase II Swiss Group for Clinical Cancer Research Trial (SAKK 21/00).

BACKGROUND: The aim of this study was to evaluate the efficacy and tolerability of fulvestrant, an estrogen receptor antagonist, in postmenopausal women with hormone-responsive tumors progressing after aromatase inhibitor (AI) treatment. PATIENTS AND METHODS: This is a phase II, open, multicenter, noncomparative study. Two patient groups were prospectively considered: group A (n=70) with AI-responsive disease and group B (n=20) with AI-resistant disease. Fulvestrant 250 mg was administered as intramuscular injection every 28 (+/-3) days. RESULTS: All patients were pretreated with AI and 84% also with tamoxifen or toremifene; 67% had bone metastases and 45% liver metastases. Fulvestrant administration was well tolerated and yielded a clinical benefit (CB; defined as objective response or stable disease [SD] for >or=24 weeks) in 28% (90% confidence interval [CI] 19% to 39%) of patients in group A and 37% (90% CI 19% to 58%) of patients in group B. Median time to progression (TTP) was 3.6 (95% CI 3.0 to 4.8) months in group A and 3.4 (95% CI 2.5 to 6.7) months in group B. CONCLUSIONS: Overall, 30% of patients who had progressed following prior AI treatment gained CB with fulvestrant, thereby delaying indication to start chemotherapy. Prior response to an AI did not appear to be predictive for benefit with fulvestrant.

T-type Ca2+ channels, SK2 channels and SERCAs gate sleep-related oscillations in thalamic dendrites.

T-type Ca2+ channels (T channels) underlie rhythmic burst discharges during neuronal oscillations that are typical during sleep. However, the Ca2+-dependent effectors that are selectively regulated by T currents remain unknown. We found that, in dendrites of nucleus reticularis thalami (nRt), intracellular Ca2+ concentration increases were dominated by Ca2+ influx through T channels and shaped rhythmic bursting via competition between Ca2+-dependent small-conductance (SK)-type K+ channels and Ca2+ uptake pumps. Oscillatory bursting was initiated via selective activation of dendritically located SK2 channels, whereas Ca2+ sequestration by sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) and cumulative T channel inactivation dampened oscillations. Sk2-/- (also known as Kcnn2) mice lacked cellular oscillations, showed a greater than threefold reduction in low-frequency rhythms in the electroencephalogram of non-rapid-eye-movement sleep and had disrupted sleep. Thus, the interplay of T channels, SK2 channels and SERCAs in nRt dendrites comprises a specialized Ca2+ signaling triad to regulate oscillatory dynamics related to sleep.

Phosphodiesterase-5 inhibition mimics intermittent reoxygenation and improves cardioprotection in the hypoxic myocardium.

Although chronic hypoxia is a claimed myocardial risk factor reducing tolerance to ischemia/reperfusion (I/R), intermittent reoxygenation has beneficial effects and enhances heart tolerance to I/R. AIM OF THE STUDY: To test the hypothesis that, by mimicking intermittent reoxygenation, selective inhibition of phosphodiesterase-5 activity improves ischemia tolerance during hypoxia. Adult male Sprague-Dawley rats were exposed to hypoxia for 15 days (10% O₂) and treated with placebo, sildenafil (1.4 mg/kg/day, i. p.), intermittent reoxygenation (1 h/day exposure to room air) or both. Controls were normoxic hearts. To assess tolerance to I/R all hearts were subjected to 30-min regional ischemia by left anterior descending coronary artery ligation followed by 3 h-reperfusion. Whereas hypoxia depressed tolerance to I/R, both sildenafil and intermittent reoxygenation reduced the infarct size without exhibiting cumulative effects. The changes in myocardial cGMP, apoptosis (DNA fragmentation), caspase-3 activity (alternative marker for cardiomyocyte apoptosis), eNOS phosphorylation and Akt activity paralleled the changes in cardioprotection. However, the level of plasma nitrates and nitrites was higher in the sildenafil+intermittent reoxygenation than sildenafil and intermittent reoxygenation groups, whereas total eNOS and Akt proteins were unchanged throughout. CONCLUSIONS: Sildenafil administration has the potential to mimic the cardioprotective effects led by intermittent reoxygenation, thereby opening the possibility to treat patients unable to be reoxygenated through a pharmacological modulation of NO-dependent mechanisms.

The clinical application of converting enzyme inhibitors.

Chronic blockade of the renin angiotensin system became possible when orally active inhibitors of angiotensin converting enzyme, the enzyme which catalyzes the transformation of angiotensin I into angiotensin II, were synthetized. Two compounds, captopril and enalapril, have been investigated in clinical studies. The decrease of the pressor response to exogenous angiotensin I and of the circulating levels of angiotensin II following administration of these inhibitors has been demonstrated to be directly related to the degree of suppression of plasma angiotensin converting enzyme activity. These inhibitors have been shown to normalize blood pressure alone in some hypertensive patients whereas in many others, satisfactory blood pressure control can be achieved only after the addition of a diuretic. Captopril and enalapril also markedly improve cardiac function of patients with chronic congestive heart failure. Chronic blockade of the renin angiotensin system has therefore provided an interesting new approach to the treatment of clinical hypertension and heart failure.

Estudi d’alteracions genètiques predictores de resposta al tractament amb cetuximab en càncer colorectal

Projecte de recerca elaborat a partir d’una estada al Cancer Center, Massachusetts General Hospital- Harvard School of Medicine, Boston, Estats Units entre gener i juny 2007. El desenvolupament de nous fàrmacs dirigits a dianes moleculars específiques ha suposat un gran avanç en el tractament del càncer. El millor coneixement dels mecanismes que determinen la sensibilitat a aquests tractaments biològics és crucial per poder oferir tractaments selectius, optimitzar l'índex terapèutic i controlar l'elevat cost d'aquests fàrmacs. Tal com ha demostrat el grup liderat pel Dr. Settleman i altres, la sensibilitat del tumor a nous fàrmacs dirigits a diana molecular ve determinada en part per alteracions genètiques de les cèl.lules tumorals. El paradigma és la resposta clínica a fàrmacs inhibidors tirosin-cinasa d’ EGFR dels pacients amb càncer de pulmó amb mutacions d'EGFR. Donada la importància de trobar marcadors predictors de sensibilitat a les noves teràpies biològiques, la detecció a gran escala d' alteraciones genètiques i las seva correlació amb la sensibilitat al tractament amb aquests fàrmacs en models preclínics és un primer pas essencial per a un posterior desenvolupament a nivell clínic. En aquest estudi vam establir una plataforma de cribatge d’alta densitat (high throughput screening) de línies cel.lulars que ens permet detectar alteracions genètiques predictores de resposta a fàrmacs dirigits a diana molecular específica. Presentem el desenvolupament d'aquesta plataforma i el resultat de dues aplicacions específiques(... )

A new chemical tool (C0036E08) supports the role of adenosine A(2B) receptors in mediating human mast cell activation.

Asthma is a chronic inflammatory disease of the airways that involves many cell types, amongst which mast cells are known to be important. Adenosine, a potent bronchoconstricting agent, exerts its ability to modulate adenosine receptors of mast cells thereby potentiating derived mediator release, histamine being one of the first mediators to be released. The heterogeneity of sources of mast cells and the lack of highly potent ligands selective for the different adenosine receptor subtypes have been important hurdles in this area of research. In the present study we describe compound C0036E08, a novel ligand that has high affinity (pK(i) 8.46) for adenosine A(2B) receptors, being 9 times, 1412 times and 3090 times more selective for A(2B) receptors than for A(1), A(2A) and A(3) receptors, respectively. Compound C0036E08 showed antagonist activity at recombinant and native adenosine receptors, and it was able to fully block NECA-induced histamine release in freshly isolated mast cells from human bronchoalveolar fluid. C0036E08 has been shown to be a valuable tool for the identification of adenosine A(2B) receptors as the adenosine receptors responsible for the NECA-induced response in human mast cells. Considering the increasing interest of A(2B) receptors as a therapeutic target in asthma, this chemical tool might provide a base for the development of new anti-asthmatic drugs.

Combinatorial Synthesis and Biological Evaluation of Inhibitors of D-Ala-D-Ala-Ligase

Report for the scientific sojourn carried out at the Max Planck Institut of Molecular Phisiology, Germany, from 2006 to 2008.The work carried out during this postdoctoral stage was focused on two different projects. Firstly, identification of D-Ala D-Ala Inhibitors and the development of new synthethic approaches to obtain lipidated peptides and proteins and the use of these lipidated proteins in biological and biophysical studies. In the first project, new D-Ala D-Ala inhibitors were identified by using structural alignments of the ATP binding sites of the bacterial ligase DDl and protein and lipid kinases in complex with ATP analogs. We tested a series of commercially available kinase inhibitors and found LFM-A13 and Tyrphostine derivatives to inhibit DDl enzyme activity. Based on the initial screening results we synthesized a series of malononitrilamide and salicylamide derivatives and were able to confirm the validity of these scaffolds as inhibitors of DDl. From this investigation we gained a better understanding of the structural requirements and limitations necessary for the preparation of ATP competitive DDl inhibitors. The compounds in this study may serve as starting points for the development of bi-substrate inhibitors that incorporate both, an ATP competitive and a substrate competitive moiety. Bisubstrate inhibitors that block the ATP and D-Ala binding sites should exhibit enhanced selectivity and potency profiles by preferentially inhibiting DDl over kinases. In the second project, an optimized synthesis for tha alkylation of cysteins using the thiol ene reaction was establisehd. This new protocol allowed us to obtain large amounts of hexadecylated cysteine that was required for the synthesis of differently lipidated peptides. Afterwards the synthesis of various N-ras peptides bearing different lipid anchors was performed and the peptides were ligated to a truncated N-ras protein. The influence of this differently lipidated N-ras proteins on the partioning and association of N-Ras in model membrane subdomains was studied using Atomic Force Microscopy.

ATP-competitive inhibitors of mTOR: new perspectives in the treatment of renal cell carcinoma.

Targeting mTOR (mammalian target of rapamycin) is an effective approach in the treatment of advanced RCC (renal cell carcinoma). Rapamycin-like drugs (rapalogues) have shown clinical activities and have been approved for the treatment of RCC. Recently, with the development of ATP-competitive inhibitors of mTOR, therapies targeting mTOR have entered a new era. Here, we discuss the biological relevance of blocking mTOR in RCC and review the mechanisms of action of rapalogues in RCC. We also advance some perspectives on the use of ATP-competitive inhibitors of mTOR in RCC.

Predators promote defence of rhizosphere bacterial populations by selective feeding on non-toxic cheaters.

Soil pseudomonads increase their competitiveness by producing toxic secondary metabolites, which inhibit competitors and repel predators. Toxin production is regulated by cell-cell signalling and efficiently protects the bacterial population. However, cell communication is unstable, and natural populations often contain signal blind mutants displaying an altered phenotype defective in exoproduct synthesis. Such mutants are weak competitors, and we hypothesized that their fitness depends on natural communities on the exoproducts of wild-type bacteria, especially defence toxins. We established mixed populations of wild-type and signal blind, non-toxic gacS-deficient mutants of Pseudomonas fluorescens CHA0 in batch and rhizosphere systems. Bacteria were grazed by representatives of the most important bacterial predators in soil, nematodes (Caenorhabditis elegans) and protozoa (Acanthamoeba castellanii). The gacS mutants showed a negative frequency-dependent fitness and could reach up to one-third of the population, suggesting that they rely on the exoproducts of the wild-type bacteria. Both predators preferentially consumed the mutant strain, but populations with a low mutant load were resistant to predation, allowing the mutant to remain competitive at low relative density. The results suggest that signal blind Pseudomonas increase their fitness by exploiting the toxins produced by wild-type bacteria, and that predation promotes the production of bacterial defence compounds by selectively eliminating non-toxic mutants. Therefore, predators not only regulate population dynamics of soil bacteria but also structure the genetic and phenotypic constitution of bacterial communities.

Development of natural products as drugs acting on central nervous system

We have recenty studied several natural product constituents which have effects on the CNS. (1) Tetrahydropalmatine (THP) and its analogues were isolated from Corydalis ambigua and various species of Stephania. (+)-THP and (-)-THP posses not only analgesic activity, but also exert sedative-tranquillizing and hypnotic actions. Results of receptor binding assay and their pre-and post-synaptic effects on dopaminergic system indicate that (-)-THP and (-)-stepholidine are dopamine receptor antagonists while (+)-THP is a selective dopamine depletor. (2) 3-Acetylaconitine (AAC) is an alkaloid isolated from Aconitum flavum. The relative potency of analgesic action of AAC was 5.1-35.6 and 1250-3912 times that of morphine and aspirin, respectively. The analgesic effect of AAC was antagonized by naloxone, but was eliminated by reserpine. In monkeys, after AAC was injected for 92 days, no abstinence syndrome was seen after sudden AAC withdrawal or when challenged with nalorphine. (3) Huperzine A (Hup-A) is an alkaloid isolated from Huperzia serrata which was found to be a selective ChE inhibitor and could improve learning and retrieval process. Preliminary clinical studies showed that Hup-A improve short-and long-term memory in patients of cerebral arteriosclerosis with memory impairment. (4) Ranamargarin is a new tetradecapeptide isolated from the skin of the Chines frog Rana margaratae. This peptide may mainly act on NK-1 receptor.

Proteinase inhibitors in Brazilian leguminosae

Serine proteinase inhitors, in the seeds of several Leguminosae from the Pantanal region (West Brazil), were studied using bovine trypsin, a digestive enzyme, Factor XIIa and human plasma Kallikrein, two blood clotting factors. The inhibitors were purified from Enterolobium contortisiliquum (Mr=23,000), Torresea cearensis (Mr = 13,000), Bauhinia pentandra (Mr = 20,000) and Bauhinia bauhinioides (Mr = 20,000). E. contortisiliquum inhibitor inactivates all three enzymes, whereas the T. cearensis inhibitor inactivates trypsin and Factor XSSa, but does nor affect plasma kallikrein; both Bauhinia inhibitors, on the other hand, inactivate trypsin and plasma kallikrein but only the Bpentandra inhibitor affects Factor XIIa. Ki values were calculated between 10 [raised to the power of] -7 and 10 [raised to the power of] -8 M.