Rapid cohort generation and analysis of disease spectrum of large animal model of cone dystrophy.

Large animal models are an important resource for the understanding of human disease and for evaluating the applicability of new therapies to human patients. For many diseases, such as cone dystrophy, research effort is hampered by the lack of such models. Lentiviral transgenesis is a methodology broadly applicable to animals from many different species. When conjugated to the expression of a dominant mutant protein, this technology offers an attractive approach to generate new large animal models in a heterogeneous background. We adopted this strategy to mimic the phenotype diversity encounter in humans and generate a cohort of pigs for cone dystrophy by expressing a dominant mutant allele of the guanylate cyclase 2D (GUCY2D) gene. Sixty percent of the piglets were transgenic, with mutant GUCY2D mRNA detected in the retina of all animals tested. Functional impairment of vision was observed among the transgenic pigs at 3 months of age, with a follow-up at 1 year indicating a subsequent slower progression of phenotype. Abnormal retina morphology, notably among the cone photoreceptor cell population, was observed exclusively amongst the transgenic animals. Of particular note, these transgenic animals were characterized by a range in the severity of the phenotype, reflecting the human clinical situation. We demonstrate that a transgenic approach using lentiviral vectors offers a powerful tool for large animal model development. Not only is the efficiency of transgenesis higher than conventional transgenic methodology but this technique also produces a heterogeneous cohort of transgenic animals that mimics the genetic variation encountered in human patients.

A novel mucosal orthotopic murine model of human papillomavirus-associated genital cancers.

Cervical cancer results from infection with high-risk type human papillomaviruses (HPV). Therapeutic vaccines aiming at controlling existing genital HPV infections and associated lesions are usually tested in mice with HPV-expressing tumor cells subcutaneously implanted into their flank. However, effective vaccine-induced regression of these ectopic tumors strongly contrasts with the poor clinical results of these vaccines produced in patients with HPV-associated genital neoplasia. To assess HPV therapeutic vaccines in a more relevant setting, we have, here, established an orthotopic mouse model where tumors in the genital mucosa (GM) develop after an intravaginal instillation of HPV16 E6/E7-expressing tumor cells transduced with a luciferase-encoding lentiviral vector for in vivo imaging of tumor growth. Tumor take was 80-90% after nonoxynol-9 induced damage of the epithelium. Tumors remained localized in the genital tract, and histological analysis showed that most tumors grew within the squamous epithelium of the vaginal wall. Those tumors induced (i) E7-specific CD8 T cells restricted to the GM and draining lymph nodes, in agreement with their mucosal location and (ii) high Foxp3+ CD4+ infiltrates, similarly to those found in natural non-regressing HPV lesions. This novel genital HPV-tumor model by requiring GM homing of vaccine-induced immune responses able to overcome local immuno-suppression may be more representative of the situation occurring in patients upon therapeutic vaccination.

Upregulation of the GABA transporter GAT-1 in the gracile nucleus in the spared nerve injury model of neuropathic pain.

Neuropathic pain is a major health issue and is frequently accompanied by allodynia (painful sensations in response to normally non-painful stimulations), and unpleasant paresthesia/dysesthesia, pointing to alterations in sensory pathways normally dedicated to the processing of non-nociceptive information. Interestingly, mounting evidence indicate that central glial cells are key players in allodynia, partly due to changes in the astrocytic capacity to scavenge extracellular glutamate and gamma-aminobutyric acid (GABA), through changes in their respective transporters (EAAT and GAT). In the present study, we investigated the glial changes occurring in the dorsal column nuclei, the major target of normally innocuous sensory information, in the rat spared nerve injury (SNI) model of neuropathic pain. We report that together with a robust microglial and astrocytic reaction in the ipsilateral gracile nucleus, the GABA transporter GAT-1 is upregulated with no change in GAT-3 or glutamate transporters. Furthermore, [(3)H] GABA reuptake on crude synaptosome preparation shows that transporter activity is functionally increased ipsilaterally in SNI rats. This GAT-1 upregulation appears evenly distributed in the gracile nucleus and colocalizes with astrocytic activation. Neither glial activation nor GAT-1 modulation was detected in the cuneate nucleus. Together, the present results point to GABA transport in the gracile nucleus as a putative therapeutic target against abnormal sensory perceptions related to neuropathic pain.

The ability model of emotional intelligence: Searching for valid measures

Current measures of ability emotional intelligence (EI)--including the well-known Mayer-Salovey-Caruso Emotional Intelligence Test (MSCEIT)--suffer from several limitations, including low discriminant validity and questionable construct and incremental validity. We show that the MSCEIT is largely predicted by personality dimensions, general intelligence, and demographics having multiple R's with the MSCEIT branches up to .66; for the general EI factor this relation was even stronger (Multiple R = .76). As concerns the factor structure of the MSCEIT, we found support for four first-order factors, which had differential relations with personality, but no support for a higher-order global EI factor. We discuss implications for employing the MSCEIT, including (a) using the single branches scores rather than the total score, (b) always controlling for personality and general intelligence to ensure unbiased parameter estimates in the EI factors, and (c) correcting for measurement error. Failure to account for these methodological aspects may severely compromise predictive validity testing. We also discuss avenues for the improvement of ability-based tests.

A model of cellular dosimetry for macroscopic tumors in radiopharmaceutical therapy.

PURPOSE: In the radiopharmaceutical therapy approach to the fight against cancer, in particular when it comes to translating laboratory results to the clinical setting, modeling has served as an invaluable tool for guidance and for understanding the processes operating at the cellular level and how these relate to macroscopic observables. Tumor control probability (TCP) is the dosimetric end point quantity of choice which relates to experimental and clinical data: it requires knowledge of individual cellular absorbed doses since it depends on the assessment of the treatment's ability to kill each and every cell. Macroscopic tumors, seen in both clinical and experimental studies, contain too many cells to be modeled individually in Monte Carlo simulation; yet, in particular for low ratios of decays to cells, a cell-based model that does not smooth away statistical considerations associated with low activity is a necessity. The authors present here an adaptation of the simple sphere-based model from which cellular level dosimetry for macroscopic tumors and their end point quantities, such as TCP, may be extrapolated more reliably. METHODS: Ten homogenous spheres representing tumors of different sizes were constructed in GEANT4. The radionuclide 131I was randomly allowed to decay for each model size and for seven different ratios of number of decays to number of cells, N(r): 1000, 500, 200, 100, 50, 20, and 10 decays per cell. The deposited energy was collected in radial bins and divided by the bin mass to obtain the average bin absorbed dose. To simulate a cellular model, the number of cells present in each bin was calculated and an absorbed dose attributed to each cell equal to the bin average absorbed dose with a randomly determined adjustment based on a Gaussian probability distribution with a width equal to the statistical uncertainty consistent with the ratio of decays to cells, i.e., equal to Nr-1/2. From dose volume histograms the surviving fraction of cells, equivalent uniform dose (EUD), and TCP for the different scenarios were calculated. Comparably sized spherical models containing individual spherical cells (15 microm diameter) in hexagonal lattices were constructed, and Monte Carlo simulations were executed for all the same previous scenarios. The dosimetric quantities were calculated and compared to the adjusted simple sphere model results. The model was then applied to the Bortezomib-induced enzyme-targeted radiotherapy (BETR) strategy of targeting Epstein-Barr virus (EBV)-expressing cancers. RESULTS: The TCP values were comparable to within 2% between the adjusted simple sphere and full cellular models. Additionally, models were generated for a nonuniform distribution of activity, and results were compared between the adjusted spherical and cellular models with similar comparability. The TCP values from the experimental macroscopic tumor results were consistent with the experimental observations for BETR-treated 1 g EBV-expressing lymphoma tumors in mice. CONCLUSIONS: The adjusted spherical model presented here provides more accurate TCP values than simple spheres, on par with full cellular Monte Carlo simulations while maintaining the simplicity of the simple sphere model. This model provides a basis for complementing and understanding laboratory and clinical results pertaining to radiopharmaceutical therapy.

Deciphering genetic disease in the genomic era: the model of GnRH deficiency.

Isolated gonadotropin-releasing hormone (GnRH) deficiency is a treatable albeit rare form of reproductive failure that has revealed physiological mechanisms controlling human reproduction, but despite substantial progress in discovering pathogenic single-gene defects, most of the genetic basis of GnRH deficiency remains uncharted. Although unbiased genetic investigations of affected families have identified mutations in previously unsuspected genes as causes of this disease in some cases, their application has been severely limited because of the negative effect of GnRH deficiency on fertility; moreover, relatively few of the many candidate genes nominated because of biological plausibility from in vitro or animal model experiments were subsequently validated in patients. With the advent of exciting technological platforms for sequencing, homozygosity mapping, and detection of structural variation at the whole-genome level, human investigations are again assuming the leading role for gene discovery. Using human GnRH deficiency as a paradigm and presenting original data from the screening of numerous candidate genes, we discuss the emerging model of patient-focused clinical genetic research and its complementarities with basic approaches in the near future.

The personality profile of borderline personality disordered patients using the five-factor model of personality

The purpose of this ex post facto study is to analyze the personality profile of outpatients who met criteria for borderline personality disorder according to the Five-Factor Model of personality. All patients (N = 52) completed the International Personality Disorder Examination (IPDE) Screening Questionnaire, the Big Five Questionnaire (BFQ), the Beck Depression Inventory (BDI), and the Beck Hopelessness Scale (BHS). The results show a high comorbidity with other DSM-IV-TR Axis II disorders, in particular with those from Cluster C. The BFQ average score indicates that the outpatients who met borderline criteria score lower than controls on all five dimensions, and especially on emotional stability. Correlations were computed between the BFQ and the IPDE scales in our sample. These results suggest that specific personality profile are linked to different comorbidity patterns. More than a half of our sample has clinically significant scores on Beck's scales. Surprisingly, depression and hopelessness are neither correlated with the borderline scale, nor have an effect in the relationship between personality and personality disorders.

Hégémonie du modèle de l'autisme: carrefour epistémologique entre médecine, familles et politique [Hegemonic model of autism: epistemological crossroads between medicine, family and politics].

The aim of this article is to make a contribution to the regional reflection with regard to autism spectrum disorders (ASDs) at a key moment in which the authorities are requested by the users, professionals in the fields of health, pedagogy and education to put forward a structured answer to a multitude of expressed needs. The question for the creation of a competence pole of an academic tertiary level is posed in order to advise in the best possible way the families who do not know how to orient themselves in the maze and contradictions of the proposed solutions and to help the professionals who are submerged by an ever increasing demand of services exceeding the means of the existing institutions and who cannot justify their choices among the various existing theoretical and scientific models.

The neuroanatomical model of post-stroke depression: towards a change of focus?

One third of all stroke survivors develop post-stroke depression (PSD). Depressive symptoms adversely affect rehabilitation and significantly increase risk of death in the post-stroke period. One of the theoretical views on the determinants of PSD focuses on psychosocial factors like disability and social support. Others emphasize biologic mechanisms such as disruption of biogenic amine neurotransmission and release of proinflammatory cytokines. The "lesion location" perspective attempts to establish a relationship between localization of stroke and occurrence of depression, but empirical results remain contradictory. These divergences are partly related to the fact that neuroimaging methods, unlike neuropathology, are not able to assess precisely the full extent of stroke-affected areas and do not specify the different types of vascular lesions. We provide here an overview of the known phenomenological profile and current pathogenic hypotheses of PSD and present neuropathological data challenging the classic "single-stroke"-based neuroanatomical model of PSD. We suggest that vascular burden due to the chronic accumulation of small macrovascular and microvascular lesions may be a crucial determinant of the development and evolution of PSD.

Spinal cord T-cell infiltration in the spared nerve injury model of neuropathic pain: a time course study

Background : Numerous studies have shown that immune cells infiltrate the spinal cord after peripheral nerve injury and that they play a major contribution to sensory hypersensitivity in rodents. In particular, the role of monocyte-derived cells and T lymphocytes seems to be prominent in this process. This exciting new perspective in research on neuropathic pain opens many different areas of work, including the understanding of the function of these cells and how they impact on neural function. However, no systematic description of the time course or cell types that characterize this infiltration has been published yet, although this seems to be the rational first step of an overall understanding of the phenomenon. Objective : Describe the time course and cell characteristics of T lymphocyte infiltration in the spinal cord in the Spared Nerve Injury (SNI) model of neuropathic pain in rats. Methods : Collect of lumbar spinal cords of rats at days 2, 7, 21 and 40 after SNI or sham operation (n=4). Immunofluorescence detecting different proteins of T cell subgroups (CD2+CD4+, CD2+CD8+, Th1 markers, Th2 markers, Th17 markers). Quantification of the infiltration rate of the different subgroups. Expected results : First, we expect to see an infiltration of T cells in the spinal cord ipsilateral to nerve injury, higher in SNI rats than in sham animals. Second, we anticipate that different subtypes of T cells penetrate at different time points. Finally, the number of T lymphocytes are expected to decrease at the latest time point, showing a resolution of the process underlying their infiltrating the spinal cord in the first place. Impact : A systematic description of the infiltration of T cells in the spinal cord after peripheral nerve injury is needed to have a better understanding of the role of immune cells in neuropathic pain. The time course that we want to establish will provide the scientific community with new perspectives. First, it will confirm that T cells do indeed infiltrate the spinal cord after SNI in rats. Second, the type of T cells infiltrating at different time points will give clues about their function, in particular their inflammatory or anti-inflammatory profile. From there on, other studies could be lead, investigating the functional side of the specific subtypes put to light by us. Ultimately, this could lead to the discovery of new drugs targeting T cells or their infiltration, in the hope of improving neuropathic pain.

Regulation of peptidase activity in a three-dimensional aggregate model of brain tumor vasculature.

The abnormal vascular system of brain cancers inappropriately expresses membrane proteins, including proteolytic enzymes, ultimately resulting in blood extravasation. The production of inflammatory mediators, such as cytokines and nitric oxide, and tumor hypoxia have been implicated in these effects. We have previously shown that the activity of aminopeptidase A is increased in the abnormal vascular system of human and rat brain tumors. To study the mechanisms regulating the activities of peptidases in cerebral vasculature in brain tumors, we have developed a three-dimensional model of differentiated rat brain cells in aggregate cultures in which rat brain microvessels were incorporated. The secretion of interleukin-6 (IL-6) in the culture medium of aggregates was used as an indicator of inflammatory activation. Addition to these aggregates of C6 glioma cell medium (C6-CM) conditioned under hypoxic or normoxic conditions or serum mimicked tumor-dependent hypoxia or conditions of dysfunction of brain tumor vasculature. Hypoxic and normoxic C6-CM, but not serum, regulated peptidase activity in aggregates, and in particular it increased the activity of aminopeptidase A determined using histoenzymography. Serum, but not C6-CM, increased IL-6 production, but did not increase aminopeptidase A activity in aggregates. Thus soluble glioma-derived factors, but not serum-derived factors, induce dysfunctions of cerebral vasculature by directly regulating the activity of peptidases, not involving inflammatory activation. Tumor hypoxia is not necessary to modulate peptidase activity.

Identification and characterization of novel classes of macrophage migration inhibitory factor (MIF) inhibitors with distinct mechanisms of action.

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is considered an attractive therapeutic target in multiple inflammatory and autoimmune disorders. In addition to its known biologic activities, MIF can also function as a tautomerase. Several small molecules have been reported to be effective inhibitors of MIF tautomerase activity in vitro. Herein we employed a robust activity-based assay to identify different classes of novel inhibitors of the catalytic and biological activities of MIF. Several novel chemical classes of inhibitors of the catalytic activity of MIF with IC(50) values in the range of 0.2-15.5 microm were identified and validated. The interaction site and mechanism of action of these inhibitors were defined using structure-activity studies and a battery of biochemical and biophysical methods. MIF inhibitors emerging from these studies could be divided into three categories based on their mechanism of action: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro(1); 2) a novel class of catalytic site inhibitors; and finally 3) molecules that disrupt the trimeric structure of MIF. Importantly, all inhibitors demonstrated total inhibition of MIF-mediated glucocorticoid overriding and AKT phosphorylation, whereas ebselen, a trimer-disrupting inhibitor, additionally acted as a potent hyperagonist in MIF-mediated chemotactic migration. The identification of biologically active compounds with known toxicity, pharmacokinetic properties, and biological activities in vivo should accelerate the development of clinically relevant MIF inhibitors. Furthermore, the diversity of chemical structures and mechanisms of action of our inhibitors makes them ideal mechanistic probes for elucidating the structure-function relationships of MIF and to further determine the role of the oligomerization state and catalytic activity of MIF in regulating the function(s) of MIF in health and disease.

Selective in vivo visualization of immune-cell infiltration in a mouse model of autoimmune myocarditis by fluorine-19 cardiac magnetic resonance.

BACKGROUND: The goal of this study was to characterize the performance of fluorine-19 ((19)F) cardiac magnetic resonance (CMR) for the specific detection of inflammatory cells in a mouse model of myocarditis. Intravenously administered perfluorocarbons are taken up by infiltrating inflammatory cells and can be detected by (19)F-CMR. (19)F-labeled cells should, therefore, generate an exclusive signal at the inflamed regions within the myocardium. METHODS AND RESULTS: Experimental autoimmune myocarditis was induced in BALB/c mice. After intravenous injection of 2×200 µL of a perfluorocarbon on day 19 and 20 (n=9) after immunization, in vivo (19)F-CMR was performed at the peak of myocardial inflammation (day 21). In 5 additional animals, perfluorocarbon combined with FITC (fluorescein isothiocyanate) was administered for postmortem immunofluorescence and flow-cytometry analyses. Control experiments were performed in 9 animals. In vivo (19)F-CMR detected myocardial inflammation in all experimental autoimmune myocarditis-positive animals. Its resolution was sufficient to identify even small inflammatory foci, that is, at the surface of the right ventricle. Postmortem immunohistochemistry and flow cytometry confirmed the presence of perfluorocarbon in macrophages, dendritic cells, and granulocytes, but not in lymphocytes. The myocardial volume of elevated (19)F signal (rs=0.96; P<0.001), the (19)F signal-to-noise ratio (rs=0.92; P<0.001), and the (19)F signal integral (rs=0.96; P<0.001) at day 21 correlated with the histological myocarditis severity score. CONCLUSIONS: In vivo (19)F-CMR was successfully used to visualize the inflammation specifically and robustly in experimental autoimmune myocarditis, and thus allowed for an unprecedented insight into the involvement of inflammatory cells in the disease process.