Mycobacterium tuberculosis Cell Wall Antigens Induce the Formation of Immune Complexes and the Development of Vasculitis in an Experimental Murine Model.

Tuberculosis (TB) of the central nervous system (CNS) presents high mortality due to brain damage and inflammation events. The formation and deposition of immune complexes (ICs) in the brain microvasculature during Mycobacterium tuberculosis (Mtb) infection are crucial for its pathobiology. The relevance of ICs to Mtb antigens in the pathogenesis of CNS-TB has been poorly explored. Here, we aimed to establish a murine experimental model of ICs-mediated brain vasculitis induced by cell wall antigens of Mtb. We administered a cell wall extract of the prototype pathogenic Mtb strain H37Rv to male BALB/c mice by subcutaneous and intravenous routes. Serum concentration and deposition of ICs onto blood vessels were determined by polyethylene glycol precipitation, ELISA, and immunofluorescence. Histopathological changes in the brain, lung, spleen, liver, and kidney were evaluated by hematoxylin and eosin staining. Our results evidenced that vasculitis developed in the studied tissues. High serum levels of ICs and vascular deposition were evident in the brain, lung, and kidneys early after the last cell wall antigen administration. Cell wall Mtb antigens induce strong type III hypersensitivity reactions and the development of systemic vasculitis with brain vascular changes and meningitis, supporting a role for ICs in the pathogenesis of TB.

Mycobacterium tuberculosis Infection Induces BCSFB Disruption but No BBB Disruption In Vivo: Implications in the Pathophysiology of Tuberculous Meningitis.

Central nervous system (CNS) tuberculosis is the most lethal and devastating form among the diseases caused by Mycobacterium tuberculosis. The mechanisms by which M. tuberculosis bacilli enter the CNS are still unclear. However, the BBB and the BCSFB have been proposed as possible routes of access into the brain. We previously reported that certain strains of M. tuberculosis possess an enhanced ability to cause secondary CNS infection in a mouse model of progressive pulmonary tuberculosis. Here, we evaluated the morphostructural and molecular integrity of CNS barriers. For this purpose, we analyzed through transmission electron microscopy the ultrastructure of brain parenchymal microvessels and choroid plexus epithelium from animals infected with two mycobacterial strains. Additionally, we determined the expression of junctional proteins and cytokines by immunological techniques. The results showed that the presence of M. tuberculosis induced disruption of the BCSFB but no disruption of the BBB, and that the severity of such damage was related to the strain used, suggesting that variations in the ability to cause CNS disease among distinct strains of bacteria may also be linked to their capacity to cause direct or indirect disruption of these barriers. Understanding the pathophysiological mechanisms involved in CNS tuberculosis may facilitate the establishment of new biomarkers and therapeutic targets.

Evaluation of New Benzimidazole Derivatives as Cysticidal Agents: In Vitro, in Vivo and Docking Studies.

Based on our previous research on cysticidal drugs, we report the synthesis and evaluation of three new benzimidazole derivatives. In these compounds, the amido group was used as a bioisosteric replacement of the ester group. The molecular docking on ß-tubulin revealed that the derivatives interacted through hydrogen bonding with N165, E198 and V236. All compounds showed in vitro activity against Taenia crassiceps cysts. Among them, benzimidazole 3 was found to be the most potent of the series (EC50 0.86 µM). This compound also exhibited the highest probability of binding and the lowest binding free energy score and was therefore selected for in vivo evaluation. Results indicated that the efficacy of compound 3 was comparable to that of the reference drug, albendazole (50.39 vs. 47.16% parasite reduction). Animals treated with compound 3 seemed to tolerate this benzimidazole well, with no changes in behavior, or food and water consumption. These findings are consistent with the in silico prediction results, which indicated low toxicity risks. The pharmacokinetic study showed that the half-life and mean residence time (6.06 and 11.9 h, respectively) were long after oral administration. Together, these results indicate that this new benzimidazole derivative represents a promising structure with cysticidal activity.

Quinine and carbenoxolone enhance the anticonvulsant activity of some classical antiepileptic drugs.

Objective Quinine (QUIN) and carbenoxolone (CNX) elicit anticonvulsant effects typically characterized by the reduction of the epileptiform activity as well as changes in behavioral parameters related to seizures. Therefore, the aim of this study was to analyze the effects of these molecules on the anticonvulsant activity of some classical antiepileptic drugs. Methods Male Wistar rats were used. Valproate (VPA), phenytoin (PHT), or carbamazepine (CBZ) was administered at sub-therapeutic doses for intraperitoneal via. Subsequently, animals were administered with a single dose of QUIN or CNX. The anticonvulsant activity was evaluated with the maximal electroshock (MES) test and pentylenetetrazole (PTZ) administration. Additionally, the plasma levels of CBZ were determined using an HPLC method. Results All the control rats presented generalized tonic-clonic seizures after the MES test or the administration of PTZ. For the MES test, all of the antiepileptic drugs increased their anticonvulsant activity when were co-administered with QUIN. For the PTZ test, only the combination CBZ plus QUIN significantly increased the percentage of protection against the generalized tonic-clonic seizures. The co-administration of CBZ plus QUIN resulted in an augmented concentration of CBZ in plasma. Discussion The present study shows that QUIN and CNX enhance the anticonvulsant activity of some classical antiepileptic drugs. However, only the combination CBZ/QUIN had significant effects on both MES and PTZ models. Such anticonvulsant activity could be attributed to increased levels of CBZ in plasma. We propose that these molecules could improve the pharmacological actions of antiepileptic drugs administered at sub-therapeutic doses.

Concomitant treatment with pertussis toxin plus temozolomide increases the survival of rats bearing intracerebral RG2 glioma.

PURPOSE: Glioblastoma multiforme is the most frequent primary brain tumor, it has poor prognosis, and it remains refractory to current treatment. The success of temozolomide (TMZ) appears to be limited by the occurrence of chemoresistance. Recently, we report the use of pertussis toxin as adjuvant immunotherapy in a C6 glioma model; showing a decrease in tumoral size, it induced selective cell death in Treg cells, and it elicited less infiltration of tumoral macrophages. Here, we evaluated the cytotoxic effect of pertussis toxin in combination with TMZ for glioma treatment, both in vitro and in vivo RG2 glioma model. METHODS: We determined cell viability, cell cycle, apoptosis, and autophagy on treated RG2 cells through flow cytometry, immunofluorescence, and Western blot assays. Twenty-eight rats were divided in four groups (n = 7) for each treatment. After intracranial implantation of RG2 cells, animals were treated with TMZ (10 mg/Kg/200 µl of apple juice), PTx (2 µg/200 µl of saline solution), and TMZ + PTx. Animals without treatment were considered as control. RESULTS: We found an induction of apoptosis in around 20 % of RG2 cells, in both single treatments and in their combination. Also, we determined the presence of autophagy vesicles, without any modifications in the cell cycle in the TMZ - PTx-treated groups. The survival analyses showed an increase due to individual treatments; while in the group treated with the combination TMZ - PTx, this effect was enhanced. CONCLUSION: We show that the concomitant use of pertussis toxin plus TMZ could represent an advantage to improve the glioma treatment.

Investigación preclínica por microPET en la UNAM / Pre-clinical research at UNAM using microPET

La tomografía por emisión de positrones (PET) es una técnica de imágenes de medicina nuclear ya establecida en México, fundamental en el diagnóstico y seguimiento clínico de enfermedades oncológicas, neurológicas y cardiológicas. Esta modalidad de imagenología molecular está basada en la administración de cantidades muy pequeñas de fármacos marcados con emisores de positrones y en la subsecuente detección de radiación con el fin de obtener imágenes tomográficas que reflejan la distribución del radiofármaco en el paciente. El desarrollo de nuevos radiofármacos para PET requiere de un método para verificar que éstos siguen las rutas metabólicas de interés, que su vida media biológica es suficiente para la realización de un estudio, que no tienen efectos adversos y que es viable para estudios en pacientes. El desarrollo de equipos de microtomografía por emisión de positrones (microPET), dedicados a estudiar animales de laboratorio, ha permitido realizar estas pruebas antes de su aplicación clínica. Además, el microPET es una herramienta de gran utilidad en la investigación preclínica de diversas enfermedades, en el desarrollo de tratamientos innovadores que permite el seguimiento no invasivo en modelos animales. En la Unidad PET/CT-Ciclotrón de la Facultad de Medicina de la UNAM, se cuenta desde hace unos años con un equipo microPET para investigación. En este trabajo se muestran algunos resultados de los estudios que se realizan con mayor frecuencia con el microPET utilizando los radiofármacos de mayor uso en el medio clínico y se muestra la utilidad que puede tener en diversos proyectos de investigación.

Positron emission tomography (PET) is a nuclear medicine imaging technique well established in Mexico, essential for the clinical diagnosis and follow-up of oncological, neurological and cardiac pathologies. This molecular imaging modality is based on the administration of small amounts of drugs labeled with a positron emitting radionuclides and the subsequent radiation detection to obtain tomographic images which reflect the distribution of the radiopharmaceutical in the patient. The development of new radiopharmaceuticals for PET requires a method to verify that they follow the expected metabolic pathways, that they have a long-enough biological half-life for imaging studies, that they have no side effects and that it is viable for use in patients. The development of positron emission microtomography (microPET) systems to be used in small laboratory animale has allowed researchers to perform these tests on radiopharmaceuticals before being used in the clinic. In addition, microPET is a useful tool in preclinical research of different diseases in the development of innovating non-invasive treatments allowing to follow up animal models. At the PET/CT-Ciclotron Unit, Facultad de Medicina, UNAM, a microPET system has been available in the last few years for research purposes. In this work, examples of frequent imaging studies performed with the microPET and in-the-clinic commonly-used radiopharmaceuticals, as well the use it may have in different research projects are shown here.