Tumor necrosis factor region polymorphisms are associated with AIDS and with cytomegalovirus retinitis

Background: The tumor necrosis factor (TNF) gene is located within the highly polymorphic major histocompatibility complex region, exhibiting the -308 CA promoter region polymorphism and six microsatellites (TNFa-f) spanning the region nearby the TNF locus. Objective: In the present Study, we evaluated the frequency of -308 CA and TNFa-e polymorphisms and respective haplotypes (in chromosomal sequence: TNFd-TNFe-308GA-TNFc-TNFa-TNFb), in 222 patients with AIDS, 52 of whom exhibited cytomegalovirus retinitis, and in 202 healthy HIV-negative individuals. Method: TNF microsatellite and single nucleotide polymorphism typings were performed by PCR followed by polyacrylamide gel electrophoresis. Results: The TNF-308A allele and the 4-3-C-2-7-1 haplotype were associated with susceptibility to AIDS, whereas the TNFb4 allele and the 3-3-C-1-11-4 haplotype were associated with protection against AIDS development. The TNFc2 allele and the 4-1-G-2-2-1 haplotype, which contains the TNFc2 allele, were associated with cytomegalovirus retinitis. Conclusion: This study highlights that polymorphic sites spanning the region nearby the TNF locus are associated with AIDS per se and with cytomegalovirus retinitis in AIDS patients. (C) 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins

Precise Temporal Regulation of roughest is Required for Correct Salivary Gland Autophagic Cell Death in Drosophila

The Drosophila roughest (rst) locus encodes an immunoglobulin superfamily transmembrane glycoprotein implicated in a variety of embryonic and postembryonic developmental processes. Here we demonstrate a previously unnoticed role for this gene in the autophagic elimination of larval salivary glands during early pupal stages by showing that overexpression of the Rst protein ectodomain in early pupa leads to persistence of salivary glands up to at least 12 hours after head eversion, although with variable penetrance. The same phenotype is observed in individuals carrying the dominant regulatory allele rst(D), but not in loss of function alleles. Analysis of persistent glands at the ultrastructural level showed that programmed cell death starts at the right time but is arrested at an early stage of the process. Finally we describe the expression pattern and intracellular distribution of Rst in wild type and rstD mutants, showing that its downregulation in salivary glands at the beginning of pupal stage is an important factor in the correct implementation of the autophagic program of this tissue in space and time. genesis 47:492-504, 2009. (C) 2009 Wiley-Liss, Inc.

Vascular endothelial growth factor genotypes and haplotypes are associated with pre-eclampsia but not with gestational hypertension

Vascular endothelial growth factor (VEGF) is relevant for normal pregnancy, and abnormalities in VEGF functions are associated with hypertensive disorders of pregnancy. Because there are few studies on how VEGF genetic polymorphisms affect susceptibility to pre-eclampsia (PE), and no studies on how they affect susceptibility to gestational hypertension (GH), we compared VEGF genotype and haplotype distributions in normotensive and hypertensive pregnancies. Genotypes and haplotypes for VEGF polymorphisms (C-2578A, G-1154A and G-634C) were determined in 303 pregnant women (108 healthy pregnant, HP; 101 with GH and 94 with PE). When white and non-white pregnant women were considered together, no significant differences were found in the distributions of VEGF genotypes or haplotypes (P > 0.05) in the three groups. However, with only white subjects, significant differences were found in genotypes distributions for two (C-2578A and G-634C) VEGF polymorphisms (both P < 0.05) between the HP and the PE groups. Importantly, the haplotype including the variants C-2578, G-1154 and C-634, which is associated with higher VEGF gene expression, was less common in the PE group compared with the HP group (4% versus 16%; P = 0.0047). However, we found no significant differences in VEGF haplotypes distributions when the HP and GH groups were compared (P > 0.05). These findings suggest a protective effect for the `C-2578, G-1154 and C-634` haplotype against the development of PE, but no major effects of VEGF gene variants on susceptibility to GH.

Inflammation and Overweight in Peritoneal Dialysis: Is There an Association?

More than 30% of the patients on peritoneal dialysis show chronic systemic inflammatory activity with high levels of C-reactive protein. The purpose of this cross-sectional study was to investigate the influence of the inflammatory state on clinical and nutritional markers in patients on peritoneal dialysis. Twenty-seven patients were included: mean age was 57.6 +/- 19 years, 48% were male, and median time on peritoneal dialysis was 16.0 (8.3; 35.8) months. Clinical, dialytic, laboratory, anthropometric and electric bioimpedance data were collected with the sample stratified for C-reactive protein. In patients, the levels of Interleukin-6 and tumor necrosis factor-a were higher, while adiponectin levels were lower than in healthy individuals (p <= 0.001), indicating the presence of inflammatory activity in the sample. When compared to patients with C-reactive protein < 1 mg/dL, those with = 1mg/dL showed higher body mass index (29.4 +/- 6.1 vs. 24.4 +/- 4.5 kg/m(2); p = 0.009), percent of standard body weight (124.5 +/- 25.4 vs. 106.8 +/- 17.9 %; p = 0.012), and percent of body fat as assessed by both anthropometry (31.3 +/- 9.9 vs. 23.9 +/- 9.1%; p = 0.056) and bioimpedance (38.9 +/- 6.3 vs. 26.2 +/- 12.6 %; p < 0.001). Patients with C-reactive protein = 1mg/dL also exhibited higher levels of ferritin (701 +/- 568 vs. 532 +/- 356 ng/mL; p = 0.054) and lower total lymphocyte count (median 1838 vs. 1638 mm(3); p = 0.001). In conclusion, higher body mass index and body fat markers were associated with C-reactive protein = 1mg/dL, and higher C-reactive protein was associated with immunocompetence impairment evidenced by the lower total lymphocyte count. Our findings confirm the relationship between inflammation, body fat, and immunocompetence, which may be superimposed potentializing the inflammatory status.

Loss of samples in the tissue microarray technique: comparison between slides using adhesive tape and silanized slides

The tissue microarray (TMA) technique allows multiple tissue samples in a single block. Commercial adhesive tape is used to avoid the loss of tissue samples during the immunostaining process. Few reports exist in the literature comparing the use of these adhesive tapes to other adhesive techniques. The objective of this study was to compare loss of sections adhered to slides using commercial adhesive tapes versus using silanized only slides. TMA was constructed with varying tissues using a fixed-base device (Beecher Instruments), placing 108 cylinders of 1 mm diameter in duplicate, spaced 1.2 mm apart. Section of 4 mu m were cut from the TMA block and adhered to 30 silanized slides and 30 commercial glass slides using adhesive tape, according to manufacturer`s recommendations. Vimentin immunoexpression was evaluated by immunohistochemistry. Antigenic recovery was realized in citrate buffer using a microwave oven. Cylinder loss in the immunohistochemical process was quantified and expressed as: total (>80%), almost complete (75-79%), or partial (50-74%). The commercial adhesive tape group presented lesser total loss (1.1 versus 6.4%), almost complete loss (2.2 versus 3.5%), and partial loss (2.1 versus 3.8%) than the silanized slide group (ANOVA, P < 0.05). The sum of total and almost complete losses in the silanized slide group was 9.9%, greater than the losses in slides using commercial adhesive tapes (3.3%) and less than reported and considered acceptable in the literature (10-30%). In conclusion, the use of silanized only slides presents very satisfactory results, requires less training, and reduces costs significantly, thus justifying their use in research.

Inhibition of COX 1 and 2 prior to Renal Ischemia/Reperfusion Injury Decreases the Development of Fibrosis

Ischemia and reperfusion injury (IRI) contributes to the development of chronic interstitial fibrosis/tubular atrophy in renal allograft patients, Cyclooxygenase (COX) 1 and 2 actively participate in acute ischemic injury by activating endothelial cells and inducing oxidative stress. Furthermore, blockade of COX I and 2 has been associated with organ improvement after ischemic damage. The aim of this study was to evaluate the role of COX I and 2 in the development of fibrosis by performing a COX I and 2 blockade immediately before IRI We subjected C57BI/6 male mice to 60 min of unilateral renal pedicle occlusion, Prior to surgery mice were either treated with indomethacin (IMT) at days -1 and 0 or were untreated. Blood and kidney samples were collected 6 wks after IRI. Kidney samples were analyzed by real-time reverse transcription-poly me rase chain reaction for expression of transforming growth factor beta (TGF-beta), monocyte chemoattractant protein 1 (MCP-1), osteopontin (OPN), tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1 beta, IL-10, heme oxygenose 1 (HO-1), vimentin, connective-tissue growth factor (CTGF), collagen 1, and bone morphogenic protein 7 (BMP-7), To assess tissue fibrosis we performed morphometric analyses and Sirius red staining. We also performed immunohistochemical analysis of anti-actin smooth muscle, Renal function did not significantly differ between groups. Animals pretreated with IMT showed significantly less interstitial fibrosis than nontreated animals. Gene transcript analyses showed decreased expression of TGF-beta, MCP-1,TNF-alpha, IL-1-beta, vimentin, collagen 1, CTGF and IL-10 mRNA (all P < 0.05), Moreover, HO-I mRNA was increased in animals pretreated with IMT (P < 0.05) Conversely, IMT treatment decreased osteopontin expression and enhanced BMP-7 expression, although these levels did rot reach statistical significance when compared with control expression levels, I he blockade of COX 1 and 2 resulted in less tissue fibrosis, which was associated with a decrease in proinflammatory cytokines and enhancement of the protective cellular response.

MTA-induced neutrophil recruitment: a mechanism dependent on IL-1 beta, MIP-2, and LTB(4)

Objective. The objective of this study was to investigate the mediators and the resident peritoneal cells involved in the neutrophil migration (NM) induced by mineral trioxide aggregate (MTA) in mice. Study design. MTA (25 mg/cavity) was injected into normal and pretreated peritoneal cavities (PC) with indomethacin (IND), dexamethasone (DEX), BWA4C, U75302, antimacrophage inflammatory protein-2 (MIP-2), and anti-interleukin-1 beta (IL-1 beta) antibodies and the NM was determined. The role of macrophage (MO) and mast cells (MAST) was determined by administration of thioglycollate 3% or 48/80 compound, respectively. The concentration of IL-1 beta and MIP-2 exudates was measured by ELISA. Results. MTA induced dose-and time-dependent NM into mice PC, with the participation of MO and MAST. NM was inhibited by DEX, BWA4C, and U75302, as well as anti-MIP-2 and anti-IL-1 beta antibodies. In the exudates, IL-1 beta and MIP-2 were detected. Conclusions. This study suggests that MTA induces NM via a mechanism dependent on MAST and MO mediated by IL-1 beta, MIP-2, and LTB(4).

Sensitivity and uncertainty analyses for burden of disease and risk factor estimates

Epidemiological studies report confidence or uncertainty intervals around their estimates. Estimates of the burden of diseases and risk factors are subject to a broader range of uncertainty because of the combination of multiple data sources and value choices. Sensitivity analysis can be used to examine the effects of social values that have been incorporated into the design of the disability–adjusted life year (DALY). Age weight, where a year of healthy life lived at one age is valued differently from at another age, is the most controversial value built into the DALY. The discount rate, which addresses the difference in value of current versus future health benefits, also has been criticized. The distribution of the global disease burden and rankings of various conditions are largely insensitive to alternate assumptions about the discount rate and age weighting. The major effects of discounting and age weighting are to enhance the importance of neuropsychiatric conditions and sexually transmitted infections. The Global Burden of Disease study also has been criticized for estimating mortality and disease burden for regions using incomplete and uncertain data. Including uncertain results, with uncertainty quantified to the extent possible, is preferable, however, to leaving blank cells in tables intended to provide policy makers with an overall assessment of burden of disease. No estimate is generally interpreted as no problem. Greater investment in getting the descriptive epidemiology of diseases and injuries correct in poor countries will do vastly more to reduce uncertainty in disease burden assessments than a philosophical debate about the appropriateness of social value

Drosophila melanogaster lipins are tissue-regulated and developmentally regulated and present specific subcellular distributions

Lipins constitute a novel family of Mg2+-dependent phosphatidate phosphatases that catalyze the dephosphorylation of phosphatidic acid to yield diacylglycerol, an important intermediate in lipid metabolism and cell signaling. Whereas a single lipin is detected in less complex organisms, in mammals there are distinct lipin isoforms and paralogs that are differentially expressed among tissues. Compatible with organism tissue complexity, we show that the single Drosophila Lpin1 ortholog (CG8709, here named DmLpin) expresses at least three isoforms (DmLpinA, DmLpinK and DmLpinJ) in a temporal and spatially regulated manner. The highest levels of lipin in the fat body, where DmLpinA and DmLpinK are expressed, correlate with the highest levels of triacylglycerol (TAG) measured in this tissue. DmLpinK is the most abundant isoform in the central nervous system, where TAG levels are significantly lower than in the fat body. In the testis, where TAG levels are even lower, DmLpinJ is the predominant isoform. Together, these data suggest that DmLpinA might be the isoform that is mainly involved in TAG production, and that DmLpinK and DmLpinJ could perform other cellular functions. In addition, we demonstrate by immunofluorescence that lipins are most strongly labeled in the perinuclear region of the fat body and ventral ganglion cells. In visceral muscles of the larval midgut and adult testis, lipins present a sarcomeric distribution. In the ovary chamber, the lipin signal is concentrated in the internal rim of the ring canal. These specific subcellular localizations of the Drosophila lipins provide the basis for future investigations on putative novel cellular functions of this protein family.

Age-related deregulation of Aire and peripheral tissue antigen genes in the thymic stroma of non-obese diabetic (NOD) mice is associated with autoimmune type 1 diabetes mellitus (DM-1)

Gene expression of peripheral tissue antigens (PTAs) in stromal medullary thymic epithelial cells (mTECs) is a key process to the negative selection of autoreactive thymocytes. This phenomenon was termed ""promiscuous gene expression"" (PGE), which is partially controlled by the Aire gene. Nevertheless, reasons for the correlation of Aire and PTAs with the emergence of autoimmune diseases are largely unknown, though it may be a result of a chronological effect. Although the effect of Aire mutations in pathogenic autoimmunity is well know, it could not be a unique cause for autoimmunity. Independently of mutations, temporal deregulation of Aire expression may imbalance Aire-dependent PTAs and/or wide PGE. This deregulation may be an early warning sign for autoimmune diseases as it guarantees autoantigen representation in the thymus. To assess this hypothesis, we studied the expression levels of Aire, Aire-dependent (Ins2) and Aire-independent (Gad67 and Col2a1) PTAs using real-time-PCR of the thymic stromal cells of NOD mice during the development of autoimmune type 1 diabetes mellitus (DM-1). Wide PGE was studied by microarrays in which the PTA genes were identified through parallel CD80(+) mTEC 3.10 cell line expression profiling. The results show that Aire gene was down-regulated in young pre-autoimmune (pre-diabetic) NOD mice. PGE and specific PTA genes were down-regulated in adult autoimmune diabetic animals. These findings represent evidence indicating that chronological deregulation of genes important to negative selection may be associated with the development of an autoimmune disease (DM-1) in mice.

Syndromic and non-syndromic aneurysms of the human ascending aorta share activation of the Smad2 pathway

Common features such as elastic fibre destruction, mucoid accumulation, and smooth muscle cell apoptosis are co-localized in aneurysms of the ascending aorta of various aetiologies. Recent experimental studies reported an activation of TGF-beta in aneurysms related to Marfan (and Loeys-Dietz) syndrome. Here we investigate TGF-beta signalling in normal and pathological human ascending aortic wall in syndromic and non-syndromic aneurysmal disease. Aneurysmal ascending aortic specimens, classified according to aetiology: syndromic MFS (n = 15, including two mutations in TGFBR2), associated with BAV (n = 15) or degenerative forms (n = 19), were examined. We show that the amounts of TGF-beta 1 protein retained within and released by aneurysmal tissue were greater than for control aortic tissue, whatever the aetiology, contrasting with an unchanged TGF-beta 1 mRNA level. The increase in stored TGF-beta 1 was associated with enhanced LTBP-I protein and mRNA levels. These dysiregulations of the extracellular ligand are associated with higher phosphorylated Smad2 and Smad2 mRNA levels in the ascending aortic wall from all types of aneurysm. This activation correlated with the degree of elastic fibre fragmentation. Surprisingly, there was no consistent association between the nuclear location of pSmad2 and extracellular TGF-beta 1 and LTBP-I staining and between their respective mRNA expressions. In parallel, decorin. was focally increased in aneurysmal media, whereas biglycan was globally decreased in aneurysmal aortas. In conclusion, this study highlights independent dysregulations of TGF-beta retention and Smad2 signalling in syndromic and non-syndromic aneurysms of the ascending aorta. Copyright (C) 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Tissue diffusion and retention of metalloproteinases in ascending aortic aneurysms and dissections

Histopathological alterations in human aneurysms and dissections of the thoracic ascending aorta include areas of mucoid degeneration within the medial layer, colocalized with areas of cell disappearance and disruption of extracellular matrix elastic and collagen fibers. We studied the presence of matrix metalloproteinases in relation to their capacity to diffuse through the tissue or to be retained in areas of mucoid degeneration in aneurysms and dissections of the ascending aorta. Ascending aortas from 9 controls, 33 patients with aneurysms, and 14 with acute dissections, all collected at surgery, were analyzed. The morphological aspect was similar whatever the etiology or phenotypic expression of the pathological aortas, involving areas of extracellular matrix breakdown and cell rarefaction associated with mucoid degeneration. Release of proMMP-2, constitutively expressed by smooth muscle cells, was not different between controls and aneurysmal aortas, whereas the aneurysmal aortas released more of the active form. Release of pro and active MMP-9 was also similar between controls and aneurysmal aortas. Immunohistochemical staining of MMP-2 and MMP-9 was weak in both control and pathological aortas. In contrast, released MMP-7 (matrilysin) and MMP-3 (stromelysin-1) could not be detected in conditioned media but were present in tissue extracts with no detectable quantitative difference between controls and pathological aortas. Immunohistochemical staining of MMP-7 and MMP-3 revealed their retention in areas of mucoid degeneration, and semiquantitative evaluation of immunostaining showed more MMP-7 in pathological aortas than in controls. In conclusion, areas of mucoid degeneration, the hallmark of aneurysms, and dissections of thoracic ascending aortas, whatever their etiology, are not inert and can retain specific proteases. (c) 2009 Elsevier Inc. All rights reserved.

Focal and linear endocardial and epicardial catheter-based cryoablation of normal and infarcted ventricular tissue

Background: This study of a chronic porcine postinfarction model examined whether linear epicardial cryoablation was capable of creating large, homogenous lesions in regions of the myocardium including scarred ventricle. Endocardial and epicardial focal cryolesions were also compared to determine if there were significant differences in lesion characteristics. Methods: Eighty focal endocardial and 28 focal epicardial cryoapplications were delivered to eight normal caprine and four normal porcine ventricular myocardium, and 21 linear cryolesions were applied along the border of infarcted epicardial tissue in a chronic porcine infarct model in six swines. Results: Focal endocardial cryolesions in normal animals measured 9.7 +/- 0.4 mm (length) by 7.3 +/- 1.4 mm (width) by 4.8 +/- 0.2 mm (depth), while epicardial lesions measured 10.2 +/- 1.4 mm (length) by 7.7 +/- 2 mm (width) by 4.6 +/- 0.9 mm (depth); P > 0.05. Linear epicardial cryolesions in the chronic porcine infarct model measured 36.5 +/- 7.8 mm (length) by 8.2 +/- 1.3 mm (width) by 6.0 +/- 1.2 mm (depth). The mean depth of linear cryolesions applied to the border of the infarct scar was 7 +/- 0.7 mm, as measured by magnetic resonance imaging. Conclusions:Cryoablation can create deep lesions when delivered to the ventricular epicardium. Endocardial and epicardial cryolesions created by a focal cryoablation catheter are similar in size and depth. The ability to rapidly create deep linear cryolesions may prove to be beneficial in substrate-based catheter ablation of ventricular arrhythmias.

Characterization of Equine Adipose Tissue-Derived Progenitor Cells Before and After Cryopreservation

In horses, stem cell therapies are a promising tool to the treatment of many injuries, which are common consequences of athletic endeavor, resulting in high morbidity and often compromising the performance. In spite of many advantages, the isolation of stem cells similar to human, from equine adipose tissue, occurred only recently. The aim of this study was to isolate equine adipose tissue-derived progenitor cells (eAT-PC), to characterize their proliferative potential, and to study their differentiation capacity before and after cryopreservation. The cells, isolated from horse adipose tissue, presented similar fibroblast-like cell morphology in vitro. Their proliferation rate was evaluated during 63 days (23 passages) before and after cryopreservation. After the induction of osteogenic differentiation, von Kossa staining and positive immunostaining studies revealed the formation of calcified extracellular matrix confirming the osteogenic potential of these cells. Adipogenic differentiation was induced using two protocols: routine and other one developed by us, while our protocol requires a shorter time (Oil Red O staining revealed significant accumulation of lipid droplets after 7 days). Chondrogenic differentiation was observed after 21 days of induced pellet culture, as evidenced by histological (toluidine blue) and immunohistochemistry studies. Our data demonstrate that eAT-PC can be easily isolated and successfully expanded in vitro while presenting significant proliferating rate. These cells can be maintained undifferentiated in vitro and can efficiently undergo differentiation at least into mesodermal derivates. These eAT-PC properties were preserved even after cryopreservation. Our findings classify eAT-PC as a promising type of progenitor cells that can be applied in different cell therapies in equines.

Characteristics of Filiform, Fungiform and Vallate Papillae and Surface of Interface Epithelium-Connective Tissue of the Maned Sloth Tongue Mucosa (Bradypus torquatus, Iliger, 1811): Light and Scanning Electron Microscopy Study

The study of lingual surfaces and the surface of interface epithelium-connective tissue of the tongue of Bradypus torquatus was performed by employing the light and scanning electron microscopy (SEM) techniques. The results revealed that the rostral part of the tongue presents a round apex and covered by filiform and fungiform lingual papillae and a ventral smooth surface. It was observed that the epithelial layer of the dorsal surface possesses the basal, spinosum, granular and cornified epithelial cells. The lamina propria is characterized by a dense connective tissue forming the long, short and round papillae. Numerous typical filiform papillae are located especially in the rostral part intermingled for few fungiform papillae, which were revealed in three-dimensional SEM images. Usually, the fungiform papillae are located in the border of rostral apex of the tongue exhibiting the rounded form. They are covered by keratinized epithelial cells. In the fungiform papillae, several taste pores were observed on the surface. The vallate papillae presented numerous taste buds in the wall of epithelial cells, being that the major number of taste buds is located on the superior half of vallate papilla. The taste pores are surrounded by several laminae of keratinized epithelial cells. The samples treated with NaOH solution and examined by SEM revealed, after removal of the epithelial layer, the dense connective core in original disposition, presenting different sizes and shapes. The specimens stained with Picrosirius and examined by polarized light microscopy revealed the connective tissue, indicating the collagen fibres type I and type III.