A rapid method for testing in vivo the susceptibility of different strains of Trypanosoma cruzi to active chemotherapeutic agents

A method is described which permits to determine in vivo an in a short period of time (4-6 hours) the sensitivity of T. cruzo strains to known active chemotherapeutic agents. By using resistant- and sensitive T. cruzi stains a fairly good correlation was observed between the results obtained with this rapid method (which detects activity against the circulating blood forms) and those obtained with long-term schedules which involve drug adminstration for at least 20 consecutive days and a prolonged period of assessment. This method may be used to characterize susceptibility to active drugs used clinically, provide infomation on the specific action against circulating trypomastigotes and screen active compounds. Differences in the natural susceptibility of Trypanosoma cruzi strains to active drugs have been already reported using different criteria, mostly demanding long-term study of the animal (Hauschka, 1949; Bock, Gonnert & Haberkorn, 1969; Brener, Costa & Chiari, 1976; Andrade & Figueira, 1977; Schlemper, 1982). In this paper we report a method which detects in 4-6 hours the effect of drugs on bloodstream forms in mice with established T. cruzi infections. The results obtained with this method show a fairly good correlation with those obtained by prolonged treatment schedules used to assess the action of drugs in experimental Chagas' disease and may be used to study the sensitivity of T. cruzi strains to active drugs.

Chronic murine myocarditis due to Trypanosoma cruzi: an ultrastructural study and immunochemical characterization of cardiac interstitial matrix

In an attempt to define the mouse-model for chronic Chagas' disease, a serological, histopathological and ultrastructural study as well as immunotyping of myocardium collagenic matrix were performed on Swiss mice, chronically infected with Trypanosoma cruzi strains: 21 SF and mambaí (Type II); PMN and Bolivia (Type III), spontaneously surviving after 154 to 468 days of infection. Haemagglutination and indirect immunofluorescence tests showed high titres of specific antibodies. The ultrastructural study disclosed the cellular constitution of the inflammatory infiltrate showing the predominance of monocytes, macrophages with intense phagocytic activity, fibroblasts, myofibroblasts and abundant collagen matrix suggesting the association of the inflammatory process with fibrogenesis in chronic chagasic cardiomyopathy. Artertolar and blood capillary alterations together with dissociation of cardiac cells from the capillary wall by edema and inflammation were related to ultrastructural lesions of myocardial cells. Rupture of parasitized cardiac myocells contribute to intensify the inflammatory process in focal areas. Collagen immunotyping showed the predominance of Types III and IV collagen. Collagen degradation and phagocytosis were present suggesting a reversibility of the fibrous process. The mouse model seems to be valuable in the study of the pathogenetic mechanisms in Chagas cardiomyopathy, providing that T. cruzi strains of low virulence and high pathogenecity are used.

CLOCK-controlled polyphonic regulation of circadian rhythms through canonical and noncanonical E-boxes.

In mammalian circadian clockwork, the CLOCK-BMAL1 complex binds to DNA enhancers of target genes and drives circadian oscillation of transcription. Here we identified 7,978 CLOCK-binding sites in mouse liver by chromatin immunoprecipitation-sequencing (ChIP-Seq), and a newly developed bioinformatics method, motif centrality analysis of ChIP-Seq (MOCCS), revealed a genome-wide distribution of previously unappreciated noncanonical E-boxes targeted by CLOCK. In vitro promoter assays showed that CACGNG, CACGTT, and CATG(T/C)G are functional CLOCK-binding motifs. Furthermore, we extensively revealed rhythmically expressed genes by poly(A)-tailed RNA-Seq and identified 1,629 CLOCK target genes within 11,926 genes expressed in the liver. Our analysis also revealed rhythmically expressed genes that have no apparent CLOCK-binding site, indicating the importance of indirect transcriptional and posttranscriptional regulations. Indirect transcriptional regulation is represented by rhythmic expression of CLOCK-regulated transcription factors, such as Krüppel-like factors (KLFs). Indirect posttranscriptional regulation involves rhythmic microRNAs that were identified by small-RNA-Seq. Collectively, CLOCK-dependent direct transactivation through multiple E-boxes and indirect regulations polyphonically orchestrate dynamic circadian outputs.

Cortical origin of functional recovery in the somatosensory cortex of the adult mouse after thalamic lesion

Résumé L'accident vasculaire cérébral sensoriel pur est un des syndromes lacunaires, dû à l'occlusion de petits vaisseaux cérébraux, souvent dans le cadre d'une lésion intéressant le noyau ventro-caudal du thalamus. Il produit un hémisyndrome sensitif pur, et parfois un syndrome douloureux se développe à distance de l'événement aigu. Afin d'étudier la récupération fonctionnelle dans le cortex somatosensoriel (SI) après une telle lésion dans le thalamus, un modèle de lésion excitotoxique a été développé dans le système somatosensoriel de la souris adulte, caractérisé par la présence de formations cytoarchitectoniques dans SI appelées "tonneaux". Chacun de ces tonneaux correspond à la représentation corticale d'une vibrisse du museau. L'activité métabolique a été mesurée dans SI à différents intervalles après la lésion, à l'aide de déoxyglucose marqué radioactivement. Dans les deux premiers jours suivant celle-ci, l'activité métabolique diminue de manière importante dans toutes les couches corticales, avec une atteinte plus marquée dans la couche IV, principale projection des axones thalamo-corticaux. Une récupération de l'activité métabolique se produit ensuite, d'autant plus marquée que le délai après la lésion est grand. Cette récupération s'observe dans toutes les couches coticales, les couches I et Vb récupérant plus rapidement que les couches II, III, IV, Va et VI. Cinq semaines après la lésion, l'absence des vibrisses correspondant à la partie déafférentée de SI diminue l'activité métabolique corticale de 32% et démontre l'activation par la périphérie de cette partie de l'écorce, malgré la perte des axones thalamo-corticaux provenant du noyau ventro-caudal. Des expériences de traçage rétrograde ont montré une augmentation des projections intracorticales sur la partie déafférentée de l'écorce, en particulier de longue distance, ainsi que des projections interhémisphériques, mais n'ont pas permis de mettre en évidence de nouvelle projection thalamique, indiquant une origine corticale à la récupération fonctionnelle observée. Abstract To study the degree and time course of the functional recovery in the somatosensory cortex (SI) after an excitotoxic lesion in the adult mouse thalamus, metabolic activity was determined in SI at various times points post lesion. Immediately after the lesion, metabolic activity in the thalamically deafferented part of SI was at its lowest value but increased progressively at subsequent time points. This was seen in all cortical layers, however, layers I and Vb recover more rapidly than layers II, III, IV, Va and VI. Removal of the mystacial whiskers corresponding to the deafferented area, 5 weeks after cortical recovery, produced a subsequent 32% drop in metabolic activity, demonstrating peripheral sensory activation of this part of the cortex. Tracing experiments revealed that the deafferented cortex did not receive a novel thalamic input, but cortico-cortical and contralateral barrel cortex projections to this area were reinforced. We conclude that the cortical functional recovery after a thalamic lesion is, at least partially, due to modified cortico-cortical and callosal projections to the deafferented cortical area.

T-cell receptor V beta use predicts reactivity and tolerance to Mlsa-encoded antigens.

T lymphocytes reactive with the product of the Mlsa-allele of the minor lymphocyte stimulating (Mls) locus use a predominant T-cell receptor beta-chain variable gene segment (V beta 6). Such V beta 6-bearing T cells are selectively eliminated in the thymus of Mlsa-bearing mice, consistent with a model in which tolerance to self antigens is achieved by clonal deletion.

When orthologs diverge between human and mouse.

Despite the common assumption that orthologs usually share the same function, there have been various reports of divergence between orthologs, even among species as close as mammals. The comparison of mouse and human is of special interest, because mouse is often used as a model organism to understand human biology. We review the literature on evidence for divergence between human and mouse orthologous genes, and discuss it in the context of biomedical research.

Proteomic and Metabolomic Analyses of Mitochondrial Complex I-deficient Mouse Model Generated by Spontaneous B2 Short Interspersed Nuclear Element (SINE) Insertion into NADH Dehydrogenase (Ubiquinone) Fe-S Protein 4 (Ndufs4) Gene.

Eukaryotic cells generate energy in the form of ATP, through a network of mitochondrial complexes and electron carriers known as the oxidative phosphorylation system. In mammals, mitochondrial complex I (CI) is the largest component of this system, comprising 45 different subunits encoded by mitochondrial and nuclear DNA. Humans diagnosed with mutations in the gene NDUFS4, encoding a nuclear DNA-encoded subunit of CI (NADH dehydrogenase ubiquinone Fe-S protein 4), typically suffer from Leigh syndrome, a neurodegenerative disease with onset in infancy or early childhood. Mitochondria from NDUFS4 patients usually lack detectable NDUFS4 protein and show a CI stability/assembly defect. Here, we describe a recessive mouse phenotype caused by the insertion of a transposable element into Ndufs4, identified by a novel combined linkage and expression analysis. Designated Ndufs4(fky), the mutation leads to aberrant transcript splicing and absence of NDUFS4 protein in all tissues tested of homozygous mice. Physical and behavioral symptoms displayed by Ndufs4(fky/fky) mice include temporary fur loss, growth retardation, unsteady gait, and abnormal body posture when suspended by the tail. Analysis of CI in Ndufs4(fky/fky) mice using blue native PAGE revealed the presence of a faster migrating crippled complex. This crippled CI was shown to lack subunits of the "N assembly module", which contains the NADH binding site, but contained two assembly factors not present in intact CI. Metabolomic analysis of the blood by tandem mass spectrometry showed increased hydroxyacylcarnitine species, implying that the CI defect leads to an imbalanced NADH/NAD(+) ratio that inhibits mitochondrial fatty acid β-oxidation.

High-resolution magnetic resonance imaging quantitatively detects individual pancreatic islets.

OBJECTIVE-We studied whether manganese-enhanced high-field magnetic resonance (MR) imaging (MEHFMRI) could quantitatively detect individual islets in situ and in vivo and evaluate changes in a model of experimental diabetes.RESEARCH DESIGN AND METHODS-Whole pancreata from untreated (n = 3), MnCl(2) and glucose-injected mice (n = 6), and mice injected with either streptozotocin (STZ; n = 4) or citrate buffer (n = 4) were imaged ex vivo for unambiguous evaluation of islets. Exteriorized pancreata of MnCl(2) and glucose-injected mice (n = 6) were imaged in vivo to directly visualize the gland and minimize movements. In all cases, MR images were acquired in a 14.1 Testa scanner and correlated with the corresponding (immuno)histological sections.RESULTS-In ex vivo experiments, MEHFMRI distinguished different pancreatic tissues and evaluated the relative abundance of islets in the pancreata of normoglycemic mice. MEHFMRI also detected a significant decrease in the numerical and volume density of islets in STZ-injected mice. However, in the latter measurements the loss of beta-cells was undervalued under the conditions tested. The experiments on the externalized pancreata confirmed that MEHFMRI could visualize native individual islets in living, anesthetized mice.CONCLUSIONS-Data show that MEHFMRI quantitatively visualizes individual islets in the intact mouse pancreas, both ex vivo and in vivo. Diabetes 60:2853-2860, 2011

Cells with neuronal characteristics differentiate and persist for one year in rat optic nerve explants cultures.

Evidence concerning the presence or absence of common neuronglia lineages in the postnatal mammalian central nervous system is still a matter of speculation. We address this problem using optic nerve explants, which show an extremely long survival in culture. Morphological, immunocytochemical and immunochemical methods were applied. The results obtained from in vitro tissue were compared with optic nerves (ONs) and whole-brain samples from animals of different ages. Newborn rat ONs represented the starting material of our tissue culture; they are composed of unmyelinated axons, astrocytes and progenitor cells but devoid of neuronal cell bodies. At this age, Western blots of ONs were positively stained by neurofilament and synapsin I specific antibodies. These bands increased in intensity during postnatal in situ development. In explant cultures, the glia cells reach a stage of functional differentiation and they maintain, together with undifferentiated cells, a complex histotypic organization. After 6 days in vitro, neurofilaments and synapsin I could not be detected on immunoblots, indicating that 1) axonal degeneration was completed, and 2) neuronal somata were absent at the time. Surprisingly, after about 4-5 weeks in culture, a new cell type appeared, which showed characteristics typical of neurons. After 406 days in vitro, neurofilaments and synapsin I were unequivocally detectable on Western blots. Furthermore, both immunocytochemical staining and light and electron microscopic examinations corroborated the presence of this earlier-observed cell type. These in vitro results clearly show the high developmental plasticity of ON progenitor cells, even late in development. The existence of a common neuron-glia precursor, which never gives rise to neurons in situ, is suggested.

The conduction system of the heart in mice chronically infected with Trypanosoma cruzi: histopathological lesions and electrocardiographic correlations

Chronic focal and diffuse myiocarditis with interstitial fibrosis developed in Swiss outbred mice and in the inbred AKR and A/J strains of mice which were chronically infected with several Trypanosoma cruzi strains belonging to three biological types (Type I, II and III). High incidence of electrocardiographic changes with predominance of intraventricular conduction disturbances, 1st. and 2nd. degree AV block, arrhythmias, comparable with those found in human Chagas' disease, were also present. Morphological study of the conduction tissue of the heart revealed inflammatory and fibrotic changes. The presence of inflammation in the inter-atrial septum almost always coincided with the inflammatory involvement of the ventricular conduction system. Focal inflammation was associated with vacuolization and focal necrosis of the specific fibers. Most of the lesions were seen affecting the His bundel (76.3% of the cases), the right bundle branch (73.3%), AV node (43.9%) and left bundle branch (37.5%). Correlation between morphological changes in the conduction tissue and electrocardiographic alteration occured in 53.0 to 62.5% of the cases, according to the experimental groups.

Functional and histopathological identification of the respiratory failure in a DMSXL transgenic mouse model of myotonic dystrophy.

Acute and chronic respiratory failure is one of the major and potentially life-threatening features in individuals with myotonic dystrophy type 1 (DM1). Despite several clinical demonstrations showing respiratory problems in DM1 patients, the mechanisms are still not completely understood. This study was designed to investigate whether the DMSXL transgenic mouse model for DM1 exhibits respiratory disorders and, if so, to identify the pathological changes underlying these respiratory problems. Using pressure plethysmography, we assessed the breathing function in control mice and DMSXL mice generated after large expansions of the CTG repeat in successive generations of DM1 transgenic mice. Statistical analysis of breathing function measurements revealed a significant decrease in the most relevant respiratory parameters in DMSXL mice, indicating impaired respiratory function. Histological and morphometric analysis showed pathological changes in diaphragmatic muscle of DMSXL mice, characterized by an increase in the percentage of type I muscle fibers, the presence of central nuclei, partial denervation of end-plates (EPs) and a significant reduction in their size, shape complexity and density of acetylcholine receptors, all of which reflect a possible breakdown in communication between the diaphragmatic muscles fibers and the nerve terminals. Diaphragm muscle abnormalities were accompanied by an accumulation of mutant DMPK RNA foci in muscle fiber nuclei. Moreover, in DMSXL mice, the unmyelinated phrenic afferents are significantly lower. Also in these mice, significant neuronopathy was not detected in either cervical phrenic motor neurons or brainstem respiratory neurons. Because EPs are involved in the transmission of action potentials and the unmyelinated phrenic afferents exert a modulating influence on the respiratory drive, the pathological alterations affecting these structures might underlie the respiratory impairment detected in DMSXL mice. Understanding mechanisms of respiratory deficiency should guide pharmaceutical and clinical research towards better therapy for the respiratory deficits associated with DM1.

The change of behavior of two strains of Leishmania after cultivation in a defined medium

Attempts have been made to characterize two strains of Leishmania that became infective to golden hamsters only after they had been maintained for several years in a chemically defined culture medium. Observations were made on the growth rates of promastigotes in vitro, course of infection in hamsters, morphology of amastigotes, and electrophoretic mobility patterns of eight isoenzymes. Information was obtained about the buoyant densities of n-DNA and k-DNA, and one strain was tested against monoclonal antibodies. The identity of both strains remains obscure.