MHC-restricted cytotoxic T-lymphocyte assay: an improved method based on normal and SV40-immortalized rabbit epidermal target cells

Although several techniques are available to evaluate cell-mediated immunity, numerous difficulties have prevented their use in rabbits. Cytotoxic T-lymphocyte (CTL) assays have been used to determine the ex vivo cytolytic activity of CD8+ T-lymphocytes in immunization protocols. However, this assay cannot be performed with rabbit peripheral blood mononuclear cell (PBMC) targets because of their high spontaneous (51)Cr release. To overcome this intrinsic difficulty shown by rabbit cells, syngeneic normal and SV40-immortalized cells were prepared from skin biopsies. The results show that: (i) skin-derived rabbit fibroblasts can be used as target cells after infection with a fowlpox virus recombinant; (ii) SV40-immortalized skin fibroblasts appear to be more appropriate for repeated assays; (iii) antigen-expanded T-cells and fresh PBMCs can be used as effectors with a similar efficiency; and (iv) dissociation of adherent skin fibroblast target cells with EDTA is to be preferred over TrypLE enzymatic treatment.     

Collagen concentration on the facial skin of postmenopausal women after topical treatment with estradiol and genistein: a randomized double-blind controlled trial

Objective: The objective of this study is to compare the effects of topical estrogen and genistein (a soy isoflavone) on the facial skin collagen of postmenopausal women not undergoing systemic hormonal therapy.

Methods: This is a prospective, double blind, randomized, controlled clinical trial. Volunteer women (N?=?30) 45–55?year old from the Endocrine Gynecology sector of the Gynecology Department of the Federal University of São Paulo (UNIFESP). The Ethical Committee of the Federal University of São Paulo approved the study (report no. 386/2004; registration on ClinicalTrials.gov NCT01553773), were assigned to topical treatment with either estrogen or genistein for 24?weeks. We quantified and compared facial collagen concentration before and after each treatment by performing pre-auricular skin biopsies.

Results: Our data showed an increase in the amount of both type I and type III facial collagen by the end of both treatments. However, the outcomes of the estrogen GI (ER) group were superior to the genistein GII (GEN) group, with statistical significance p?Conclusion: Treatment with topical estrogen is superior to genistein, but both have positive impacts on facial skin collagen. Nevertheless, it is still unclear whether prolonged use of genistein and other topical phytoestrogens could produce systemic effects and further research is needed to clarify this question.     

Visually cued motor synchronization: modulation of fMRI activation patterns by baseline condition

A well-known issue in functional neuroimaging studies, regarding motor synchronization, is to design suitable control tasks able to discriminate between the brain structures involved in primary time-keeper functions and those related to other processes such as attentional effort. The aim of this work was to investigate how the predictability of stimulus onsets in the baseline condition modulates the activity in brain structures related to processes involved in time-keeper functions during the performance of a visually cued motor synchronization task (VM). The rational behind this choice derives from the notion that using different stimulus predictability can vary the subject's attention and the consequently neural activity. For this purpose, baseline levels of BOLD activity were obtained from 12 subjects during a conventional-baseline condition: maintained fixation of the visual rhythmic stimuli presented in the VM task, and a random-baseline condition: maintained fixation of visual stimuli occurring randomly. fMRI analysis demonstrated that while brain areas with a documented role in basic time processing are detected independent of the baseline condition (right cerebellum, bilateral putamen, left thalamus, left superior temporal gyrus, left sensorimotor cortex, left dorsal premotor cortex and supplementary motor area), the ventral premotor cortex, caudate nucleus, insula and inferior frontal gyrus exhibited a baseline-dependent activation. We conclude that maintained fixation of unpredictable visual stimuli can be employed in order to reduce or eliminate neural activity related to attentional components present in the synchronization task.     

Detection and quantification of<Emphasis Type="Italic"> Mycobacterium leprae</Emphasis> in tissue samples by real-time PCR

Real-time PCR technology has improved molecular diagnostics of many pathogens, but no such test is available for Mycobacterium leprae. In this report we describe the establishment and the pre-clinical evaluation of such an assay. The test achieved a theoretical analytical sensitivity limit of 194 M. leprae cells per skin biopsy specimen and facilitated quantification of mycobacteria in tissue over a range of 54–54,000,000 cells per sample. In punch skin biopsies from 39 untreated Ugandan patients with newly diagnosed leprosy, the clinical diagnosis could be confirmed in 88.9% of multibacillary and 33.3% of paucibacillary (microscopically negative) patients. Real-time detection thus did not increase the clinical sensitivity of PCR as compared to conventional protocols, in spite of its evidently high analytical sensitivity. On the other hand, as still no culture system exists for M. leprae, the assay appears to be a robust tool for detection of the bacterium in selected clinical situations, as well as for quantitation in experimental settings.     

Genetic isolates in Corsica (France): linkage disequilibrium extension analysis on the Xq13 region

Genetic isolates with a history of a small founder population, long-lasting isolation and population bottlenecks represent exceptional resources in the identification of genes involved in the pathogenesis of multifactorial diseases. In these populations, the disease allele reveals linkage disequilibrium (LD) with markers over significant genetic intervals, therefore facilitating disease locus identification. This study has been designed to examine the background LD extension in some subpopulations of Corsica. Our interest in the island of Corsica is due to its geographical and genetic proximity to the other Mediterranean island of Sardinia. Sardinian isolates in which the extension of the background LD is particularly high have been recently identified and are now the object of studies aimed at the mapping of genes involved in complex diseases. Recent evidence has highlighted that the genetic proximity between the populations of Corsica and Sardinia is particularly true for the internal conservative populations. Given these considerations, Sardinia and Corsica may represent a unique system to carry out parallel association studies whose results could be validated by comparison. In the present study, we have analyzed the LD extension on the Xq13 genomic region in three subpopulations of Corsica: Corte, Niolo and Bozio, all located in the mountainous north-center of the island. Our results show a strong degree of LD over long distance for the population of Bozio and to a less extent for the population of Niolo. Their LD extent is comparable to or higher than that reported for other isolates.     

Murine polyomavirus-like particles induce maturation of bone marrow-derived dendritic cells and proliferation of T cells

We analysed the effects of murine polyomavirus-like particles (PLPs) on bone marrow-derived dendritic cells (BMDCs) and T cells in vitro. BMDCs activated with PLPs up-regulated CD40, CD80, CD86 and major histocompatibility complex (MHC) class II surface markers and produced proinflammatory cytokines. Chimeric PLPs [expressing the ovalbumin (OVA)-peptides OVA(257-264) or OVA(323-339)], but not wildtype PLPs, activated OVA-specific CD8 T cells and OVA-specific CD4 T cells, respectively, indicating both MHC class I and II presentation of the peptides by antigen-presenting cells. Our results suggest that PLPs may be used as vaccine adjuvants priming dendritic cells to induce potent T cell responses.     

Effect of footwear on the external knee adduction moment - A systematic review

ContextFootwear modifications have been investigated as conservative interventions to decrease peak external knee adduction moment (EKAM) and pain associated with knee osteoarthritis (OA).ObjectiveTo evaluate the literature on the effect of different footwear and orthotics on the peak EKAM during walking and/or running.MethodsA systematic search of databases resulted in 348 articles of which 33 studies were included.ResultsSeventeen studies included healthy individuals and 19 studies included subjects with medial knee OA. Quality assessment (modified Downs and Black quality index) showed an (average ± SD) of 73.1 ± 10.1%. The most commonly used orthotic was the lateral wedge, with three studies on the medial wedge. Lateral wedging was associated with decreased peak EKAM in healthy participants and participants with medial knee OA while there is evidence for increased peak EKAM with the use of medial wedges. Modern footwear (subjects' own shoe, “stability” and “mobility” shoes, clogs) were likely to increase the EKAM compared to barefoot walking in individuals with medial knee OA. Walking in innovative shoes (“variable stiffness”) decreased the EKAM compared to control shoes. Similarly, shoes with higher heels, sneakers and dress shoes increased EKAM in healthy individuals compared to barefoot walking.ConclusionsFurther development may be needed toward optimal footwear for patients with medial knee OA with the aim of obtaining similar knee moments to barefoot walking.     

Expression of mRNA of neurotrophic factors and their receptors are significantly altered after subchronic ketamine treatment

The neurotrophic factors play an important role in the maintenance of neurone viability and neuronal communication which are considered to be altered in schizophrenia. Subchronic application of ketamine (Ket) was found to be a useful model in schizophrenia research. To further validate this model the mRNA levels of neurotrophic factors NGF, NT-3, and BDNF and their receptors TrkA, TrkB, and TrkC, respectively, were measured in different brain areas in Ket-pretreated rats subchronically dosed with the atypical antipsychotic drug risperidone (Ris). With the exception of NGF in the frontal cortex, Ket pretreatment did change NGF, NT-3, and BDNF mRNA levels in the frontal cortex, the hippocampus, the striatum, the thalamus/hypothalamus region, and in the cerebellum. These changes correspond with changes at their tyrosine kinase receptors. Ris treatment normalised altered NT-3 levels in the hippocampus and balanced BDNF levels in the same structure. It was concluded that the Ket model might reflect distinct alterations in neurotrophic factor activity as found in schizophrenic patients and, moreover, that Ris treatment rebalances disturbed neurotrophic factor activity.