Synthesis, structure-activity relationship and trypanocidal activity of pyrazole-imidazoline and new pyrazole-tetrahydropyrimidine hybrids as promising chemotherapeutic agents for Chagas disease.

Drug therapy for Chagas disease remains a major challenge as potential candidate drugs have failed clinical trials. Currently available drugs have limited efficacy and induce serious side effects. Thus, the discovery of new drugs is urgently needed in the fight against Chagas' disease. Here, we synthesized and evaluated the biological effect of pyrazole-imidazoline (1a-i) and pyrazole-tetrahydropyrimidine (2a-i) derivatives against relevant clinical forms of Trypanosoma cruzi. The structure-activity relationship (SAR), drug-target search, physicochemical and ADMET properties of the major active compounds in vitro were also assessed in silico. Pyrazole derivatives showed no toxicity in Vero cells and also no cardiotoxicity. Phenotypic screening revealed two dichlorinated pyrazole-imidazoline derivatives (1c and 1d) with trypanocidal activity higher than that of benznidazole (Bz) against trypomastigotes; these were also the most potent compounds against intracellular amastigotes. Replacement of imidazoline with tetrahydropyrimidine in the pyrazole compounds completely abolished the trypanocidal activity of series 2(a-i) derivatives. The physicochemical and ADMET properties of the compounds predicted good permeability, good oral bioavailability, no toxicity and mutagenicity of 1c and 1d. Pyrazole nucleus had high frequency hits for cruzipain in drug-target search and structure activity relationship (SAR) analysis of pyrazole-imidazoline derivatives revealed enhanced activity when chlorine atom was inserted in meta-positions of the benzene ring. Additionally, we found evidence that both compounds (1c and 1d) have the potential to interact non-covalently with the active site of cruzipain and also inhibit the cysteine proteinase activity of T. cruzi. Collectively, the data presented here reveal pyrazole derivatives with promise for further optimization in the therapy of Chagas disease.

Efficacy of 2-hydroxy-3-phenylsulfanylmethyl-[1,4]-naphthoquinone derivatives against different Trypanosoma cruzi discrete type units: Identification of a promising hit compound.

The limited efficacy of benznidazole (Bz) indicated by failures of current Phase II clinical trials emphasizes the urgent need to identify new drugs with improved safety and efficacy for treatment of Chagas disease (CD). Herein, we analyzed the efficacy of a series of 2-hydroxy-3-phenylsulfanylmethyl-[1,4]-naphthoquinones against different Trypanosoma cruzi discrete type units (DTUs) of relevant clinical forms of CD. Cytotoxic and trypanocidal effect of naphthoquinone derivatives were assessed in mammalian cells, trypomastigotes and intracellular amastigotes using, luminescent assays (CellTiter-Glo and T. cruzi Dm28c-luciferase) and/or counting with a light microscope. Reactive oxygen species (ROS) production and intracellular targets of promising compounds were assessed with 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) probe and ultrastructural analysis, respectively. ADMET properties were analyzed by in silico modeling. Most of the compounds showed low cytotoxic effect. Only two compounds (Compounds 2 and 11) had IC50 values lower than Bz, showing higher susceptibility of bloodstream trypomastigotes. Compound 2 exhibited greater efficacy against trypomastigotes from different T. cruzi DTUs, even better than Bz against Brazil and CL strains. Ultrastructural analysis revealed changes in intracellular compartments, suggesting autophagy as one possible mechanism of action. Oxidative stress, induced by Compound 2, resulted in elevated level of ROS, leading to parasite death. Compound 2 was also effective against intracellular amastigotes, showing high selectivity index. ADMET analysis predicted good oral bioavailability, reduced drug metabolism and no carcinogenic potential for Compound 2. The data highlight Compound 2 as a hit compound and stimulate further structural and pharmacological optimization to potentiate its trypanocidal activity and selectivity.

Trypanosoma cruzi heparin-binding proteins mediate the adherence of epimastigotes to the midgut epithelial cells of Rhodnius prolixus.

Heparin-binding proteins (HBPs) have been demonstrated in both infective forms of Trypanosoma cruzi and are involved in the recognition and invasion of mammalian cells. In this study, we evaluated the potential biological function of these proteins during the parasite-vector interaction. HBPs, with molecular masses of 65·8 kDa and 59 kDa, were isolated from epimastigotes by heparin affinity chromatography and identified by biotin-conjugated sulfated glycosaminoglycans (GAGs). Surface plasmon resonance biosensor analysis demonstrated stable receptor-ligand binding based on the association and dissociation values. Pre-incubation of epimastigotes with GAGs led to an inhibition of parasite binding to immobilized heparin. Competition assays were performed to evaluate the role of the HBP-GAG interaction in the recognition and adhesion of epimastigotes to midgut epithelial cells of Rhodnius prolixus. Epithelial cells pre-incubated with HBPs yielded a 3·8-fold inhibition in the adhesion of epimastigotes. The pre-treatment of epimastigotes with heparin, heparan sulfate and chondroitin sulfate significantly inhibited parasite adhesion to midgut epithelial cells, which was confirmed by scanning electron microscopy. We provide evidence that heparin-binding proteins are found on the surface of T. cruzi epimastigotes and demonstrate their key role in the recognition of sulfated GAGs on the surface of midgut epithelial cells of the insect vector.

Involvement of host cell heparan sulfate proteoglycan in Trypanosoma cruzi amastigote attachment and invasion.

Cell surface glycosaminoglycans (GAGs) play an important role in the attachment and invasion process of a variety of intracellular pathogens. We have previously demonstrated that heparan sulfate proteoglycans (HSPG) mediate the invasion of trypomastigote forms of Trypanosoma cruzi in cardiomyocytes. Herein, we analysed whether GAGs are also implicated in amastigote invasion. Competition assays with soluble GAGs revealed that treatment of T. cruzi amastigotes with heparin and heparan sulfate leads to a reduction in the infection ratio, achieving 82% and 65% inhibition of invasion, respectively. Other sulfated GAGs, such as chondroitin sulfate, dermatan sulfate and keratan sulfate, had no effect on the invasion process. In addition, a significant decrease in infection occurred after interaction of amastigotes with GAG-deficient Chinese Hamster Ovary (CHO) cells, decreasing from 20% and 28% in wild-type CHO cells to 5% and 9% in the mutant cells after 2 h and 4 h of infection, respectively. These findings suggest that amastigote invasion also involves host cell surface heparan sulfate proteoglycans. The knowledge of the mechanism triggered by heparan sulfate-binding T. cruzi proteins may provide new potential candidates for Chagas disease therapy.

A modified staining technique for arbuscular mycorrhiza compatible with molecular probes.

The effects of the different steps of the root staining on the arbuscular mycorrhizal (AM) fungal rDNA extraction and amplification have been assessed. The results obtained using molecular techniques are compared with those obtained from fresh, non-stained leek roots. A modified staining procedure that eliminates heating, the use of hydrochloric acid and trypan blue, has been proved to be the most adequate to observe the AM colonisation in different plant species with/without lignified roots allowing at the same time the subsequent rDNA extraction and amplification from the stained roots. The staining technique decreased the sensitivity of the process and a higher number of roots had to be used to obtain enough material for a positive amplification. The extraction and amplification process was reliable up to 3 days after staining. A week after staining, the amplification was not dependable and after 2 weeks there was no amplification from stained material.

[Value of MRCP for diagnosis of biliary complications after liver transplantation]. / Evaluation de la cholangiographie par résonance magnétique dans le diagnostic des complications biliaires après transplantation hépatique.

PURPOSE: To assess the value of MRCP in the detection of biliary complications after orthotopic liver transplantation. MATERIALS AND METHODS: 27 transplanted patients with suspected biliary complication underwent a total of 34 MR and direct cholangiography procedures. MRCP were reviewed by 2 independent reviewers blinded to clinical and laboratory findings. The biliary tract was divided into 7 segments, and all lesions were evaluated using this segmental anatomy. Each segment was evaluated for the presence of dilatation, stenosis and intra-ductal debris. MRCP results were compared to results frpm direct cholangiography. RESULTS: 216 (98%) of 221 biliary segments could be evaluated on MRCP, with good to excellent visualization in 179 (80%) cases. Segmental analysis showed sensitivity, specificity and accuracy values of 85%, 81% and 83% for the detection of biliary stenosis, 82%, 81% and 81% for the detection of biliary dilatation, and 60%, 88% and 80% for the detection of inyraductal debris. CONCLUSION: MRCP is accurate for the detection of biliary stenosis and dilatation in patients after liver transplantation and provides an alternative to direct cholangiography.

Les complications de la nephrectomie laparoscopique

Objectif: Depuis l'avenement de la chirurgie minimale invasive; la voie laparoscopique devient la reference pour le traitement chirurgical des pathologies renales. L'objectif de ce travail est d'analyser les complications de la nephrectomie laparoscopique. Patients et methodes: Les nephrectomies laparoscopiques realisees en 10 ans (1996 - 2005) ont ete revues. Les caracteres demographiques; les incidents per-operatoires et les complications post-operatoires ont ete etudies. Resultats: Il y avait 181 patients operes dont 83 nephrectomies radicales; 80 nephrectomies simples et 18 nephro-ureterectomies. L'age moyen est de 59 ans. Des antecedents de chirurgie et/ou de radiotherapie etaient observes chez 103 patients (56;91). Chez 25 patients (13;81); on notait au moins une complication. Une conversion etait necessaire chez 6 patients (3;31) avec 4 plaies vasculaires et 2 difficultes de dissection. Dans 10 cas (5;52); les complications etaient majeures (8 cas de saignements; 1 cas d'odeme aigu des poumons; 1 cas de fistule digestive) avec 2 deces. Chez 15 patients (8;29); elles etaient mineures avec 9 cas d'hematome; 3 cas d'infection parietale; 2 cas d'emphyseme sous cutanee et 1 cas d'eventration lombaire. Conclusion: Le taux global de complications est de 13;81. Il semblerait que l'existence de facteurs de risque associes (age avance; tares; antecedents chirurgicaux ou irradiation) augmente le taux de complication. Le profil de ces patients est un des elements a prendre en compte pour prevoir et prevenir ces complications

Trypanosoma cruzi-cardiomyocyte interaction: role of fibronectin in the recognition process and extracellular matrix expression in vitro and in vivo.

We investigated the involvement of fibronectin (FN) in Trypanosoma cruzi-cardiomyocyte invasion and the extracellular matrix (ECM) components expression during T. cruzi infection in vivo and in vitro. Treatment of trypomastigotes with FN or a synthetic peptide (MRGDS) prior to cardiomyocyte interaction reduced T. cruzi infection, indicating that FN mediates the parasite invasion through its RGD sequence. In murine experimental Chagas' disease, an enhancement of the ECM components was detected in the myocardium during the late acute infection, coinciding with inflammatory infiltrates accumulation. In contrast, highly infected cardiomyocytes displayed a reduction in FN expression in vitro, while laminin spatial distribution was altered. Although it has been demonstrated that cardiomyocytes are able to synthesize cytokines upon T. cruzi infection, our data suggest that matrix remodeling is dependent on cytokines secreted by inflammatory cells recruited in immune response.

[Tumor-associated significant pericardiac effusions: analysis of 18 cases]. / Derrames pericárdicos significativos asociados a tumores: análisis de 18 casos.

Metastatic infiltration is most frequent than primary pericardiac tumors. Most frequent tumors are adenocarcinoma and lymphomas. A retrospective analysis of 18 oncological patients with significant pericardiac effusion (SPE) is carried out. The conclusions of the study are: SPE can be the first manifestation of a neoplasm; frequently, pericardiac tamponade (PT) has a neoplastic origin; thorax is the most frequent localization of the primary tumor; pericardiac fluid (PF) cytology analysis has low diagnostic yield; most useful diagnostic tests are thoracocentesis, thorax computerized tomography (CT) and bronchoscopy; SPE in a neoplasm suggest poor short-term prognosis; poor prognosis variables in this series were primary tumor unfavorable histology, advanced tumor disease and (probably) presentation as PT.

Surgical treatment of bladder diverticula: laparoscopic approach.

BACKGROUND AND PURPOSE: Bladder diverticulectomy is classically performed by open surgery (extravesical, intravesical, or combined) or, less frequently, by an endoscopic approach for small diverticula. We used a celioscopic approach to diverticulectomy in order to assess its feasibility and the operative and postoperative complications. PATIENTS AND METHODS: Five patients aged 55 to 76 years (mean 64.2 years) were treated by celioscopy between October 1999 and October 2001. All the diverticula had occurred as a result of infravesical obstruction by benign prostatic hyperplasia, which was treated at the same time by endoscopic resection of the prostate. An ipsilateral ureteral catheter was inserted during endoscopy. After creation of an umbilical minilaparotomy with the patient in the dorsal decubitus position, a 10-mm optical trocar was inserted, then two 5-mm trocars into the right and left iliac fossae, and a 10-mm subpubic trocar. Diverticular dissection was performed with a peritoneal approach in order to free the diverticular neck. After resection, the neck was closed in two planes by interrupted absorbable sutures, and a tightness test was performed. RESULTS: The average operating time was 160 minutes (range 120-230 minutes), and the average blood loss was 150 mL (range 80-200 mL). There was no conversion to open surgery. The probe was removed on day 5 (range 3-7 days). No complication occurred, and the mean hospital stay was 5 days (range 4-6 days) with resumption of satisfactory micturation. CONCLUSION: This technique is a promising alternative to classical surgery, as it is less aggressive and uses a smaller incision. Operative bleeding is minimal, and the technique is reproducible in experienced hands. However, the indications are limited with regard to the associated pathologies (size of the prostate in the present cases), the morphology, the site of the diverticulum, and the surgical history of the patient.

[Analysis of 73 cases of significant pericardiac effusion]. / Análisis de 73 casos de derrame pericárdico significativo.

Seventy-three patients with significant pericardiac effusion (SPE) are analyzed retrospectively. The results concerning etiology, clinical findings, evolution, echocardiography findings and pericardiac effusion (PE) findings are summarized. Conclusions drawn are: 1) the pericardiac effusion (PE) is a difficult diagnosis without the assistance of the echocardiogram; 2) the echocardiogram signs of hemodynamic alterations have prognostic value; 3) the most frequent causes of SPE are: tumors, idiopathic acute pericarditis, and iatrogenesis; 4) in an important percentage of DPS patients the cause is not identified; 5) the clinical presentation as pericardiac tamponade (PT) is most frequent in the tumors; 6) the analysis of the PE has a low yield, which means that diagnostic pericardicentesis is not justified in all patients with SPE; 7) the pericardiac biopsy hasa low diagnostic yield; 8) the predictive mortality factors are: presentation as PT and tumor etiology, and 9) because of the dynamic character of the SPE, it is important to carry out a progress follow-up of it.

Linfadenitis histiocítica necrotizante de Kikuchi / Kikuchi's histiocytic necrotizing lymphadenitis

La linfadenitis histiocítica necrotizante (LHN) o enfermedad de Kikuchi-Fujimoto es una entidad anatomoclínica poco frecuente; afecta preferentemente a mujeres jóvenes e individuos de razas orientales y se caracteriza por fiebre, adenopatías preferentemente cervicales, afectación sistémica y, ocasionalmente, extraganglionar.Se presentan dos casos de LHN en mujeres jóvenes con afectación cutánea y en uno de los casos con meningitis linfocitaria.Se discuten los aspectos etiológicos, la relación de la LHN con las enfermedades colágeno-vasculares (fundamentalmente el lupus eritematoso sistémico [LES]), los problemas del diagnóstico anatomopatológico y las posibilidades terapéuticas (AU)

[Kikuchi's histiocytic necrotizing lymphadenitis]. / Linfadenitis histiocítica necrotizante de Kikuchi.

Necrotizing histiocytic lymphadenitis (NHL) or Kikuchi-Fujimoto disease is an infrequent seen clinicopathologic entity that affects most frequently young women and individuals of eastern races, and that is characterized by fever and adenopathies (basically cervical) and systemic disease even though occasional patients present extranodal disease. We present two young women with NHL and cutaneous affectation, and with lymphocytic meningitis in one of the patients. We discuss the etiology of NHL, the relation of NHL with collagen-vascular diseases (basically systemic erythematous lupus) the problem of the pathologic diagnosis, and the therapeutic possibilities in these patients.

Enantioseparation of basic pharmaceutical compounds by capillary electrophoresis using sulfated cyclodextrins. Application to E-6006, a novel antidepressant.

In this study, a chiral capillary electrophoresis method was optimized and validated for E-6006, a thienylpyrazolylethanamine derivative (pKa 8.9). Enantioselectivity of neutral and anionic cyclodextrins (CDs) was evaluated at acid pH (3), obtaining cathodic and anodic migration, respectively. Hydroxypropyl-beta-CD, carboxymethyl-beta-CD and sulfobutyl ether-beta-CD led to similar and partial selectivity, whereas sulfate (S)-beta-CD produced baseline separation of the enantiomers. Four types of sulfated CDs were compared considering: cavity size (alpha, beta, gamma) and random substitution versus unique derivative (S-beta-CD, 6-heptakis-S-beta-CD). Complete peak separation was obtained in all cases, but with different affinity and binding strength. Some factors that play a role in the complex formation include: position/region/degree of substitution, size of CD cavity and proportion of derivatives in mixtures. Enantioaffinity and enantioselectivity increased with the average of sulfate groups/mol. Beta cavity size complexed better, although alpha and gamma cavities did not compromise separation. 6-Heptakis-S-beta-CD had less affinity and separation efficiency, attributed to its lower degree and unique position of substitution. The method was optimized with S-beta-CD (Aldrich, randomly substituted, 7-11 groups/mol). With this selector, the effect of pH value (3-9) was evaluated. Around pH 7 the cross-over point with change in the direction and order of migration was observed, associated with great enantioselectivity and long migration times. Fine tuning was done by adjusting the CD concentration and the buffer counterion. Definitive conditions were: uncoated silica capillary, 10 mM S-beta-CD-25 mM sodium phosphate, pH 3. Validation parameters are included.

Crystallization and preliminary structural analysis of an antibody complex formed with PfMSP1-19, a malaria vaccine candidate.

The 11 kDa C-terminal fragment of the proteolyticly matured surface antigen, PfMSP1, from Plasmodium falciparum is a promising malaria vaccine candidate. The soluble recombinant form of this naturally occurring fragment has been crystallized as a complex with the Fab of a specific murine monoclonal antibody. The crystals belong to the space group P2(1), with unit-cell parameters a = 51.8, b = 213.5,c = 60.0 A, beta =101.0 degrees, and with Z = 4. Diffraction data have been measured to 2.9 A resolution and a preliminary model of the complex has been determined by molecular replacement. The epitope recognised by G17.12 is located on the N-terminal EGF-like domain of the antigen.

Simultaneous separation of the enantiomers of cizolirtine and its degradation products by capillary electrophoresis.

The simultaneous enantioselective separation of (+/-)-cizolirtine and its impurities: (+/-)-N-desmethylcizolirtine, (+/-)-cizolirtine-N-oxide and (+/- )-5-(alpha-hydroxybenzyl)-1-methylpyrazole was investigated by capillary electrophoresis. Electrokinetic chromatography with carboxymethyl-beta-CD (CM-beta-CD) and sulfobutyl-ether-beta-CD was tried, showing good enantioseparation but poor chemical selectivity. The four racemic pairs were baseline separated, in a single run, by cyclodextrin-modified micellar electrokinetic chromatography. The migration buffer composition was: (60 mM hydroxypropyl-beta-cyclodextrin-150 mM sodium dodecyl sulfate-50 mM disodium tetraborate, pH 9.2, in water)-butanol 95:5, v/v). Work was done to determine the effect of buffer components and their optimal concentration on selectivity. The method was validated with respect to enantioselectivity of cizolirtine as well as its degradation products and separation selectivity between the different components. Linearity, limit of detection, limit of quantitation and precision were also determined. This method is suitable for the enantiomeric purity determination and stability control of cizolirtine (racemic mixture or enantiomers) and its degradation products. Examples of electropherograms of (R)-cizolirtine degraded under stressed conditions are shown.

Aracnoiditis y lesión intramedular. Complicaciones de la meningitis tuberculosa en dos pacientes con infección por el virus de la inmunodeficiencia humana / Arachnoiditis and intraspinal lesion

Escanear (AU)

Enfermedad de Still del adulto e hipofisitis linfocitaria

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