A DNA Vaccine Encoding Multiple HIV CD4 Epitopes Elicits Vigorous Polyfunctional, Long-Lived CD4(+) and CD8(+) T Cell Responses

T-cell based vaccines against HIV have the goal of limiting both transmission and disease progression by inducing broad and functionally relevant T cell responses. Moreover, polyfunctional and long-lived specific memory T cells have been associated to vaccine-induced protection. CD4(+) T cells are important for the generation and maintenance of functional CD8(+) cytotoxic T cells. We have recently developed a DNA vaccine encoding 18 conserved multiple HLA-DR-binding HIV-1 CD4 epitopes (HIVBr18), capable of eliciting broad CD4(+) T cell responses in multiple HLA class II transgenic mice. Here, we evaluated the breadth and functional profile of HIVBr18-induced immune responses in BALB/c mice. Immunized mice displayed high-magnitude, broad CD4(+)/CD8(+) T cell responses, and 8/18 vaccine-encoded peptides were recognized. In addition, HIVBr18 immunization was able to induce polyfunctional CD4(+) and CD8(+) T cells that proliferate and produce any two cytokines (IFN gamma/TNF alpha, IFN gamma/IL-2 or TNF alpha/IL-2) simultaneously in response to HIV-1 peptides. For CD4(+) T cells exclusively, we also detected cells that proliferate and produce all three tested cytokines simultaneously (IFN gamma/TNF alpha/IL-2). The vaccine also generated long-lived central and effector memory CD4(+) T cells, a desirable feature for T-cell based vaccines. By virtue of inducing broad, polyfunctional and long-lived T cell responses against conserved CD4(+) T cell epitopes, combined administration of this vaccine concept may provide sustained help for CD8(+) T cells and antibody responses-elicited by other HIV immunogens.

Expression of eight genes of nuclear factor-kappa B pathway in multiple myeloma using bone marrow aspirates obtained at diagnosis

Purpose: To evaluate the expression of NF-kappa B pathway genes in total bone marrow samples obtained from MM at diagnosis using real-time quantitative PCR and to evaluate its possible correlation with disease clinical features and survival. Material and methods: Expression of eight genes related to NF-kappa B pathway (NFKB1, IKB, RANK, RANKL, OPG, IL6, VCAM1 and ICAM1) were studied in 53 bone marrow samples from newly diagnosed MM patients and in seven normal controls, using the Taqman system. Genes were considered overexpressed when tumor expression level was at least four times higher than that observed in normal samples. Results: The percentages of overexpression of the eight genes were: NFKB1 0%, IKB 22.6%, RANK 15.1%, RANKL 31.3%, OPG 7.5%, IL6 39.6%, VCAM1 10% and ICAM1 26%. We found association between IL6 expression level and International Staging System (ISS) (p = 0.01), meaning that MM patients with high ISS scores have more chance of overexpression of IL6. The mean value of ICAM1 relative expression was also associated with the ISS score (p = 0.02). Regarding OS, cases with IL6 overexpression present worse evolution than cases with IL6 normal expression (p = 0.04). Conclusion: We demonstrated that total bone marrow aspirates can be used as a source of material for gene expression studies in MM. In this context, we confirmed that IL6 overexpression was significantly associated with worse survival and we described that it is associated with high ISS scores. Also, ICAM1 was overexpressed in 26% of cases and its level was associated with ISS scores.

Primary Hyperparathyroidism as the First Clinical Manifestation of Multiple Endocrine Neoplasia Type 2A in a 5-Year-Old Child

Background: Primary hyperparathyroidism occurs in only 10%-30% of patients with multiple endocrine neoplasia type 2A (MEN2A), rarely as the sole clinical manifestation, and is usually diagnosed after the third decade of life. Summary: A5-year-old girl was referred for prophylactic thyroidectomy as she carried the p.C634R RET mutation. She was clinically asymptomatic, with a normally palpable thyroid and with the cervical region free of lymphadenopathy or other nodules. Preoperative tests revealed hypercalcemia associated with elevation of parathyroid hormone (PTH) (calcium = 11.2mg/dL, calcium ion = 1.48mmol/L, phosphorus = 4.0 mg/dL, alkaline phosphatase = 625U/L, parathyroid hormone (PTH) PTH = 998 pg/mL). A thyroid ultrasound was normal and parathyroid scintigraphy with (99m)Tc-Sestamibi revealed an area of radioconcentration in the upper half of the left thyroid lobe suggesting hyperfunctioning parathyroid tissue. She underwent total thyroidectomy and parathyroidectomy and developed hypocalcemia. The anatomopathological examination showed no histopathological changes in the thyroid tissue and an adenoma of the parathyroid gland, confirming the diagnosis of hyperparathyroidism. Conclusions: Primary hyperparathyroidism can be a precocious manifestation of MEN2A. This case report highlights that asymptomatic hypercalcemia should be scrutinized in children related to patients with MEN2A who carry a mutation in the RET proto-oncogene, especially mutations in the codon 634, before the currently recommended age of 8 years.

RNA interference-mediated knockdown of CD49e (alpha 5 integrin chain) in human thymic epithelial cells modulates the expression of multiple genes and decreases thymocyte adhesion

Background: The thymus is a central lymphoid organ, in which bone marrow-derived T cell precursors undergo a complex process of maturation. Developing thymocytes interact with thymic microenvironment in a defined spatial order. A component of thymic microenvironment, the thymic epithelial cells, is crucial for the maturation of T-lymphocytes through cell-cell contact, cell matrix interactions and secretory of cytokines/chemokines. There is evidence that extracellular matrix molecules play a fundamental role in guiding differentiating thymocytes in both cortical and medullary regions of the thymic lobules. The interaction between the integrin alpha 5 beta 1 (CD49e/CD29; VLA-5) and fibronectin is relevant for thymocyte adhesion and migration within the thymic tissue. Our previous results have shown that adhesion of thymocytes to cultured TEC line is enhanced in the presence of fibronectin, and can be blocked with anti-VLA-5 antibody. Results: Herein, we studied the role of CD49e expressed by the human thymic epithelium. For this purpose we knocked down the CD49e by means of RNA interference. This procedure resulted in the modulation of more than 100 genes, some of them coding for other proteins also involved in adhesion of thymocytes; others related to signaling pathways triggered after integrin activation, or even involved in the control of F-actin stress fiber formation. Functionally, we demonstrated that disruption of VLA-5 in human TEC by CD49e-siRNA-induced gene knockdown decreased the ability of TEC to promote thymocyte adhesion. Such a decrease comprised all CD4/CD8-defined thymocyte subsets. Conclusion: Conceptually, our findings unravel the complexity of gene regulation, as regards key genes involved in the heterocellular cell adhesion between developing thymocytes and the major component of the thymic microenvironment, an interaction that is a mandatory event for proper intrathymic T cell differentiation.

Serotonin receptors are involved in the spinal mediation of descending facilitation of surgical incision-induced increase of Fos-like immunoreactivity in rats

Background: Descending pronociceptive pathways may be implicated in states of persistent pain. Paw skin incision is a well-established postoperative pain model that causes behavioral nociceptive responses and enhanced excitability of spinal dorsal horn neurons. The number of spinal c-Fos positive neurons of rats treated intrathecally with serotonin, noradrenaline or acetylcholine antagonists where evaluated to study the descending pathways activated by a surgical paw incision. Results: The number of c-Fos positive neurons in laminae I/II ipsilateral, lamina V bilateral to the incised paw, and in lamina X significantly increased after the incision. These changes: remained unchanged in phenoxybenzamine-treated rats; were increased in the contralateral lamina V of atropine-treated rats; were inhibited in the ipsilateral lamina I/II by 5-HT(1/2B/2C) (methysergide), 5-HT(2A) (ketanserin) or 5-HT(1/2A/2C/5/6/7) (methiothepin) receptors antagonists, in the ipsilateral lamina V by methysergide or methiothepin, in the contralateral lamina V by all the serotonergic antagonists and in the lamina X by LY 278,584, ketanserin or methiothepin. Conclusions: We conclude: (1) muscarinic cholinergic mechanisms reduce incision-induced response of spinal neurons inputs from the contralateral paw; (2) 5-HT(1/2A/2C/3) receptors-mediate mechanisms increase the activity of descending pathways that facilitates the response of spinal neurons to noxious inputs from the contralateral paw; (3) 5-HT(1/2A/2C) and 5-HT(1/2C) receptors increases the descending facilitation mechanisms induced by incision in the ipsilateral paw; (4) 5-HT(2A/3) receptors contribute to descending pronociceptive pathways conveyed by lamina X spinal neurons; (5) alpha-adrenergic receptors are unlikely to participate in the incision-induced facilitation of the spinal neurons.

Trends in dermatomyositis- and polymyositis-related mortality in the state of Sao Paulo, Brazil, 1985-2007: multiple cause-of-death analysis

Background: Dermatomyositis (DM) and polymyositis (PM) are rare systemic autoimmune rheumatic diseases with high fatality rates. There have been few population-based mortality studies of dermatomyositis and polymyositis in the world, and none have been conducted in Brazil. The objective of the present study was to employ multiple-cause of-death methodology in the analysis of trends in mortality related to dermatomyositis and polymyositis in the state of Sao Paulo, Brazil, between 1985 and 2007. Methods: We analyzed mortality data from the Sao Paulo State Data Analysis System, selecting all death certificates on which DM or PM was listed as a cause of death. The variables sex, age and underlying, associated or total mentions of causes of death were studied using mortality rates, proportions and historical trends. Statistical analysis were performed by chi-square and H Kruskal-Wallis tests, variance analysis and linear regression. A p value less than 0.05 was regarded as significant. Results: Over a 23-year period, there were 318 DM-related deaths and 316 PM-related deaths. Overall, DM/PM was designated as an underlying cause in 55.2% and as an associated cause in 44.8%; among 634 total deaths females accounted for 71.5%. During the study period, age-and gender-adjusted DM mortality rates did not change significantly, although PM as an underlying cause and total mentions of PM trended lower (p < 0.05). The mean ages at death were 47.76 +/- 20.81 years for DM and 54.24 +/- 17.94 years for PM (p = 0.0003). For DM/PM, respectively, as underlying causes, the principal associated causes of death were as follows: pneumonia (in 43.8%/33.5%); respiratory failure (in 34.4%/32.3%); interstitial pulmonary diseases and other pulmonary conditions (in 28.9%/17.6%); and septicemia (in 22.8%/15.9%). For DM/PM, respectively, as associated causes, the following were the principal underlying causes of death: respiratory disorders (in 28.3%/26.0%); circulatory disorders (in 17.4%/20.5%); neoplasms (in 16.7%/13.7%); infectious and parasitic diseases (in 11.6%/9.6%); and gastrointestinal disorders (in 8.0%/4.8%). Of the 318 DM-related deaths, 36 involved neoplasms, compared with 20 of the 316 PM-related deaths (p = 0.03). Conclusions: Our study using multiple cause of deaths found that DM/PM were identified as the underlying cause of death in only 55.2% of the deaths, indicating that both diseases were underestimated in the primary mortality statistics. We observed a predominance of deaths in women and in older individuals, as well as a trend toward stability in the mortality rates. We have confirmed that the risk of death is greater when either disease is accompanied by neoplasm, albeit to lesser degree in individuals with PM. The investigation of the underlying and associated causes of death related to DM/PM broaden the knowledge of the natural history of both diseases and could help integrate mortality data for use in the evaluation of control measures for DM/PM.

Multiple classical limits in relativistic and nonrelativistic quantum mechanics

The existence of a classical limit describing the interacting particles in a second-quantized theory of identical particles with bosonic symmetry is proved. This limit exists in addition to the previously established classical limit with a classical field behavior, showing that the limit h -> 0 of the theory is not unique. An analogous result is valid for a free massive scalar field: two distinct classical limits are proved to exist, describing a system of particles or a classical field. The introduction of local operators in order to represent kinematical properties of interest is shown to break the permutation symmetry under some localizability conditions, allowing the study of individual particle properties.

Human Dental Pulp Cells: A New Source of Cell Therapy in a Mouse Model of Compressive Spinal Cord Injury

Strategies aimed at improving spinal cord regeneration after trauma are still challenging neurologists and neuroscientists throughout the world. Many cell-based therapies have been tested, with limited success in terms of functional outcome. In this study, we investigated the effects of human dental pulp cells (HDPCs) in a mouse model of compressive spinal cord injury (SCI). These cells present some advantages, such as the ease of the extraction process, and expression of trophic factors and embryonic markers from both ecto-mesenchymal and mesenchymal components. Young adult female C57/BL6 mice were subjected to laminectomy at T9 and compression of the spinal cord with a vascular clip for 1 min. The cells were transplanted 7 days or 28 days after the lesion, in order to compare the recovery when treatment is applied in a subacute or chronic phase. We performed quantitative analyses of white-matter preservation, trophic-factor expression and quantification, and ultrastructural and functional analysis. Our results for the HDPC-transplanted animals showed better white-matter preservation than the DMEM groups, higher levels of trophic-factor expression in the tissue, better tissue organization, and the presence of many axons being myelinated by either Schwann cells or oligodendrocytes, in addition to the presence of some healthy-appearing intact neurons with synapse contacts on their cell bodies. We also demonstrated that HDPCs were able to express some glial markers such as GFAP and S-100. The functional analysis also showed locomotor improvement in these animals. Based on these findings, we propose that HDPCs may be feasible candidates for therapeutic intervention after SCI and central nervous system disorders in humans.

Supersymmetric and Kaluza-Klein particles multiple scattering in the Earth

Neutrino telescopes with cubic kilometer volumes have the potential to discover new particles. Among them are next to lightest supersymmetric (NLSPs) and next to lightest Kaluza-Klein (NLKPs) particles. Two NLSPs or NLKPs will transverse the detector simultaneously producing parallel charged tracks. The track separation inside the detector can be a few hundred meters. As these particles might propagate a few thousand kilometers before reaching the detector, multiple scattering could enhance the pair separation at the detector. We find that the multiple scattering will alter the separation distribution enough to increase the number of NLKP pairs separated by more than 100 meters (a reasonable experimental cut) by up to 46% depending on the NLKP mass. Vertical upcoming NLSPs will have their separation increased by 24% due to multiple scattering.

Lossless filter for multiple repeats with bounded edit distance

Background: Identifying local similarity between two or more sequences, or identifying repeats occurring at least twice in a sequence, is an essential part in the analysis of biological sequences and of their phylogenetic relationship. Finding such fragments while allowing for a certain number of insertions, deletions, and substitutions, is however known to be a computationally expensive task, and consequently exact methods can usually not be applied in practice. Results: The filter TUIUIU that we introduce in this paper provides a possible solution to this problem. It can be used as a preprocessing step to any multiple alignment or repeats inference method, eliminating a possibly large fraction of the input that is guaranteed not to contain any approximate repeat. It consists in the verification of several strong necessary conditions that can be checked in a fast way. We implemented three versions of the filter. The first is simply a straightforward extension to the case of multiple sequences of an application of conditions already existing in the literature. The second uses a stronger condition which, as our results show, enable to filter sensibly more with negligible (if any) additional time. The third version uses an additional condition and pushes the sensibility of the filter even further with a non negligible additional time in many circumstances; our experiments show that it is particularly useful with large error rates. The latter version was applied as a preprocessing of a multiple alignment tool, obtaining an overall time (filter plus alignment) on average 63 and at best 530 times smaller than before (direct alignment), with in most cases a better quality alignment. Conclusion: To the best of our knowledge, TUIUIU is the first filter designed for multiple repeats and for dealing with error rates greater than 10% of the repeats length.

Sequential injection analysis implementing multiple standard additions for As speciation by liquid chromatography and atomic fluorescence spectrometry (SIA-HPLC-AFS)

An analytical procedure for multiple standard additions of arsenic species using sequential injection analysis (SIA) is proposed for their quantification in seafood extracts. SIA presented flexibility for generating multiple specie standards at the ng mL(-1) concentration level by adding different volumes of As(III), As(V), monomethylarsonic (MMA) and dimethylarsinic (DMA) to the sample. The mixed sample plus standard solutions were delivered from SIA to fill the HPLC injection loop. Subsequently, As species were separated by HPLC and analyzed by atomic fluorescence spectrometry (AFS). The proposed system comprised two independently controlled modules, with the HPLC loop acting as the intermediary device. The analytical frequency was enhanced by combining the actions of both modules. While the added sample was flowing through the chromatographic column towards the detection system, the SIA program started performing the standard additions to another sample. The proposed method was applied to spoiled seafood extracts. Detection limits based on 3 sigma for As(III), As(V), MMA and DMA were 0.023, 0.39, 0.45 and 1.0 ng mL(-1), respectively. (C) 2011 Elsevier B.V. All rights reserved.

Evaluation of the use of multiple lines for determination of metals in water by inductively coupled plasma optical emission spectrometry with axial viewing

Inductively coupled plasma optical emission spectrometers (ICP DES) allow fast simultaneous measurements of several spectral lines for multiple elements. The combination of signal intensities of two or more emission lines for each element may bring such advantages as improvement of the precision, the minimization of systematic errors caused by spectral interferences and matrix effects. In this work, signal intensities for several spectral lines were combined for the determination of Al, Cd, Co, Cr, Mn, Pb, and Zn in water. Afterwards, parameters for evaluation of the calibration model were calculated to select the combination of emission lines leading to the best accuracy (lowest values of PRESS-Predicted error sum of squares and RMSEP-Root means square error of prediction). Limits of detection (LOD) obtained using multiple lines were 7.1, 0.5, 4.4, 0.042, 3.3, 28 and 6.7 mu g L(-1) (n = 10) for Al, Cd. Co, Cr, Mn, Pb and Zn, respectively, in the presence of concomitants. On the other hand, the LOD established for the most intense emission line were 16. 0.7, 8.4, 0.074. 23, 26 and 9.6 mu g L(-1) (n = 10) for these same elements in the presence of concomitants. The accuracy of the developed procedure was demonstrated using water certified reference material. The use of multiple lines improved the sensitivity making feasible the determination of these analytes according to the target values required for the current environmental legislation for water samples and it was also demonstrated that measurements in multiple lines can also be employed as a tool to verify the accuracy of an analytical procedure in ICP DES. (C) 2009 Elsevier B.V. All rights reserved.

Contuse lesion of the rat spinal cord of moderate intensity leads to a higher time-dependent secondary neurodegeneration than severe one - An open-window for experimental neuroprotective interventions

Secondary neurodegeneration takes place in the surrounding tissue of spinal cord trauma and modifies substantially the prognosis, considering the small diameter of its transversal axis. We analyzed neuronal and glial responses in rat spinal cord after different degree of contusion promoted by the NYU Impactor. Rats were submitted to vertebrae laminectomy and received moderate or severe contusions. Control animals were sham operated. After 7 and 30 days post surgery, stereological analysis of Nissl staining cellular profiles showed a time progression of the lesion volume after moderate injury, but not after severe injury. The number of neurons was not altered cranial to injury. However, same degree of diminution was seen in the caudal cord 30 days after both severe and moderate injuries. Microdensitometric image analysis demonstrated a microglial reaction in the white matter 30 days after a moderate contusion and showed a widespread astroglial reaction in the white and gray matters 7 days after both severities. Astroglial activation lasted close to lesion and in areas related to Wallerian degeneration. Data showed a more protracted secondary degeneration in rat spinal cord after mild contusion, which offered an opportunity for neuroprotective approaches. Temporal and regional glial responses corroborated to diverse glial cell function in lesioned spinal cord. (C) 2007 Elsevier Ltd. All rights reserved.

Aerobic exercise is safe and effective in systemic sclerosis

Several studies have established that systemic sclerosis patients have a reduced exercise capacity when compared to healthy individuals. It is relevant to evaluate whether aerobic exercise in systemic sclerosis patients is a safe and effective intervention to improve aerobic capacity. Seven patients without pulmonary impairment and seven healthy controls were enrolled in an 8-week program consisting of moderate intensity aerobic exercise. Patients and controls had a significant improvement in peak oxygen consumption (19.72 +/- 3.51 vs. 22.27 +/- 2.53 and 22.94 +/- 4.70 vs. 24.55 +/- 3.00, respectively, p = 0.006), but difference between groups was not statistically significant (p = 0.149). This finding was reinforced by the fact that at the end of the study both groups were able to perform a significantly higher exercise intensity when compared to baseline, as measured by peak blood lactate (1.43 +/- 0.51 vs. 1.84 +/- 0.33 and 1.11 +/- 0.45 vs. 1.59 +/- 0.25, respectively, p = 0.01). Patients improved the peak exercise oxygen saturation comparing to the baseline (84.14 +/- 9.86 vs. 90.29 +/- 5.09, p = 0.048). Rodnan score was similar before and after the intervention(15.84 +/- 7.84 vs. 12.71 +/- 4.31, p = 0.0855). Digital ulcers and Raynaud`s phenomenon remained stable. Our data support the notion that improving aerobic capacity is a feasible goal in systemic sclerosis management. The long term benefit of this intervention needs to be determined in large prospective studies.

Efficacy and safety of concurrent training in systemic sclerosis

Pinto, ALS, Oliveira, NC, Gualano, B, Christmann, RB, Painelli, VS, Artioli, GG, Prado, DML, and Lima, FR. Efficacy and safety of concurrent training in systemic sclerosis. J Strength Cond Res 25(5): 1423-1428, 2011-The optimal training model for patients with systemic sclerosis (SSc) is unknown. In this study, we aimed to investigate the effects of a 12-week combined resistance and aerobic training program (concurrent training) in SSc patients. Eleven patients with no evidence of pulmonary involvement were recruited for the exercise program. Lower and upper limb dynamic strengths (assessed by 1 repetition maximum [1RM] of a leg press and bench press, respectively), isometric strength (assessed by back pull and handgrip tests), balance and mobility (assessed by the timed up-and-go test), muscle function (assessed by the timed-stands test), Rodnan score, digital ulcers, Rayland`s phenomenon, and blood markers of muscle inflammation (creatine kinase and aldolase) were assessed at baseline and after the 12-week program. Exercise training significantly enhanced the 1RM leg press (41%) and 1RM bench press (13%) values and back pull (24%) and handgrip strength (11%). Muscle function was also improved (15%), but balance and mobility were not significantly changed. The time-to-exhaustion was increased (46.5%, p = 0.0004), the heart rate at rest condition was significantly reduced, and the workload and time of exercise at ventilatory thresholds and peak of exercise were increased. However, maximal and submaximal (V)over dotO(2) were unaltered (p > 0.05). The Rodnan score was unchanged, and muscle enzymes remained within normal levels. No change was observed in digital ulcers and Raynaud`s phenomenon. This is the first study to demonstrate that a 12-week concurrent training program is safe and substantially improves muscle strength, function, and aerobic capacity in SSc patients.