Preeclampsia is the most common serious disorder during pregnancy and studies show several immune-related processes in its pathophysiology. The role of cytokines and their expression remains controversial in this field. One of the cytokines of interest in recent studies has been TNF-α, which has been shown to have a higher level in maternal plasma of preeclamptic women.This study was designed to evaluate the role of TNF-α polymorphism at position -238 in the risk of developing preeclampsia during pregnancy.One hundred fifty three preeclamptic cases and 140 healthy pregnant women were retrieved from two major hospitals of Mashhad, Iran. Methods a case-control study were designed. Anyone with a history of inflammatory disease, hypertension, or chronic kidney disease was excluded. DNA was extracted from peripheral blood leukocytes. Both groups were genotyped for the polymorphism of the TNF-α gene at position -238 by the RFLP method with Ava II enzyme. Allele and genotype frequencies were compared using one-way ANOVA and the Fisher’s exact test.There were significant differences between the two groups in TNF-α genotype at position -238 (P < 0.001). In the preeclamptic group, the frequency of the AA genotype was higher (P < 0.001) and the frequency of the GG genotype was lower (P < 0.001). The overall prevalence of the A allele at position -238 was higher in preeclamptic cases (P < 0.001).In this study group, TNF-α -238 polymorphism was shown to be different in preeclamptic and non-preeclamptic pregnant women. The AA genotype and the A allele may carry an increased risk for developing of preeclampsia. Preeclampsia is the most common serious disorder during pregnancy and having a prevalence of 2-5 percent, it leads to significant maternal and neonatal morbidity and mortality . AlthougTNF- α is a T helper-1 cytokine produced by lymphocytes, macrophages and trophoblasts during pregnancy. It functions in several processes that have been found to take part in pathogenesis of preeclampsia, including abnormal placental invasion, endothelial cell damage, and oxidative stress . In the Considering the difference in the prevalence of the TNF-α genotype in different ethnicities , we studIn a case-control study, 153 preeclamptic cases and 140 healthy pregnant women were collected from two major hospitals (Qaem and Omolbanin) of Mashhad, Iran. Preeclampsia was defined as newly diagnosed blood pressure after the 20th week of gestation accompanied by proteinuria (more than 300 mg protein concentration in the 24hr urine) . HyperteA 71 base pair sequence of TNF-α promoter was amplified by PCR using the device Biometra- T3-Thermoblock model. A primer sequence of 20 nucleotides was designed by the Gene Runner software and checked with the Gene Bank and EMBL (European Molecular and Biology Library). The Primer sequence was as follows: TNF-α (-238) Forward: 5´ GGT CCT ACA CAC AAA TCA GT 3´TNF-α (-238) Reverse: 5´ CAC TCC CCA TCC TCC CTG GTC 3´The PCR cycles included a primary 3-min cycle at 94˚C, 43 cycles of three parts , and a final 5-min cycle at 72˚C. The PCR product underwent RFLP and electrophoresis and was dyed by silver nitrate where the sliced parts were clarified by applying the Ava II enzyme. Afterwards, the -238 TNF-α genotype was analyzed. The entire studied variables for both groups were collected and recorded in special forms.Sample size was calculated using frequency data of previous studies with a power of 80% and a minimum detectable odds ratio of 2.5. A p-value of less than 0.01 was considered significant. The demographic and laboratory obtained data were analyzed by the SPSS software (version 11.5). One-way ANOVA was used for comparing frequencies between the three genotype groups. Allele and genotype frequencies were compared using a 2x2 contingency table with Fisher’s exact test. T-test was applied on quantitative variables with normal distribution (age and blood pressure) and Chi-square test was used for proportion analysis of qualitative variables (parity and family history of hypertension). The deviation from Hardy-Weinberg equilibrium was also assessed using chi-square test.The clinical characteristics of the study population are presented in . There wLike other inflammatory diseases with familial and genetic components , there hThe idea of TNF-α having a part in the process has been initiated and supported by several findings. A meta-analysis of 24 studies has shown that the level of TNF-α in the maternal plasma is higher in preeclamptic women . HoweverGenetic studies have been conducted on polymorphisms at different locations in the promoter region of TNF-α, that could possibly have an effect on its level of expression. Single nucleotide polymorphisms at positions -850 , 23, 24,Our study faced some limitations. The significant cost of laboratory tests limited the number of participants and lowered the power, making the negative results unreliable. We also couldn’t make a random sampling as a result of limited available cases. Our study, according to its type (case-control), was prone to some bias concerning the history of patients. Our data lacked a baseline protein excretion and blood pressure level to assist in making the diagnosis of preeclampsia. We recommend conducting a larger study with sufficient participants and more power on different polymorphisms of TNF-α and other theoretically supported molecules.


Antibody PG9 is a prototypical member of a class of V1/V2-directed antibodies that effectively neutralizes diverse strains of HIV-1. The crystal structure of PG9 bound to scaffolded V1/V2 has provided insight into its mode of recognition. We sought to gain a more complete understanding of the interaction of PG9 with the functional viral spike, and to extend our understanding to other antibodies of this class.We analyzed amino acid frequencies in the V2 region of PG9 sensitive and resistant strains to identify potentially important residues. We also used the crystal structure of PG9 with scaffolded V1/V2 to identify potential contact sites. Based on these analyses we designed mutations in PG9 resistant strains with the goal of “knocking in” sensitivity. Parent and mutant Envs were used to make pseudoviruses. The potency of PG9 as well as V1/V2 antibodies PG16, CH01, CH04, PGT141, and PGT145 against the pseudoviruses was assessed by TZM-bl neutralization assay.For 20/20 of resistant strains, mutations in a short segment of V1/V2 resulted in gain of sensitivity to at least one antibody of the PG9 class, and 13/20 showed gain of sensitivity to 3 or more. Mutations in V2 strand C, particularly the addition or substitution of lysine at positions 168, 169, or 171, had the greatest effects.These results highlight the importance of strand C contacts for neutralization by V1/V2 antibodies, provide functional confirmation of the crystal structure, and suggest a general mechanism of resistance to V1/V2-directed broadly neutralizing antibodies.


Scientific articles are retracted at increasing rates, with the highest rates among top journals. Here we show that a single retraction triggers citation losses through an author's prior body of work. Compared to closely-matched control papers, citations fall by an average of 6.9% per year for each prior publication. These chain reactions are sustained on authors' papers (a) published up to a decade earlier and (b) connected within the authors' own citation network by up to 4 degrees of separation from the retracted publication. Importantly, however, citation losses among prior work disappear when authors self-report the error. Our analyses and results span the range of scientific disciplines. The science community regularly experiences instances of major scientific mistakes or misconduct. Prominent examples include retracted claims about cloning human embryos and harvesting their stem cells, a claimed link between the MMR vaccine and autism, and claims about super-conducting plastics that misled scientists for years across many top physics laboratories1Prior literature on retractions primarily examines biomedical journals, using PubMed data, and finds that retractions result in a 35–65% decrease in the retracted paper's citations compared to control papers22678910prior work, thus quantifying a potentially critical consequence, and disincentive, for being associated with false scientific results. Our analysis further shows how chain reactions to retraction hinge on whether authors self-report errors.In this paper, we draw on all retraction notices in the Web of Science (WOS) database. We focus on the post-2000 period when WOS indexing of retractions appears relatively complete  with the counterfactual citation path of “control” papers. Control papers are those that have similar citations paths to a treated paper prior to the retraction event. The effect of retraction is thus determined by examining the divergence, after retraction, between a treated paper and its ex-ante controls. As described below (see Methods), we use the entire WOS to find the most closely matched control papers within each field, allowing for substantially closer matches to the treated papers than can be determined using more limited databases.prior work. To isolate the effect of single retractions, we exclude cases where authors have multiple retractions, leaving 667 retracted papers and 1,737 authors with prior work. We build the sample of prior work using the WOS database. Specifically, we trace citations from each retracted article to prior articles by the same author (a 1st degree self-citation), citations from these prior articles to other prior articles by the same author (a 2nd degree self-citation), and so on up to the 11th degree, at which point additional prior work is no longer revealed. We identify additional prior publications by tracing forward this citation network – locating papers by the same author that cite these past publications. The average number of prior articles per author generated is 25.9, creating a sample of 45,039 prior papers. Note that none of the prior work was itself retracted.not self-reported. When retractions are not self-reported, the annual flow of citations to a prior publication falls 4.7% (p < .0001) in the first two years after the retraction and 12.5% (p < .0001) five or more years after the retraction, compared to the control papers  database. We focus on the post-2000 period when WOS indexing of retractions appears relatively complete and use the WOS to expand our analysis across the known universe of fields. Our analysis can thus provide a more comprehensive cross-field view of retractions than the existing literature.To analyze retraction effects, we use a “treatment” and “control” methodology2f with publication year p as fpN. For a treated paper itc denotes the citations paper i receives in year t and r is the year of retraction. Computationally, we define fields based on the 252 field categories in the WOS and locate, for each treated paper, ten control papers (with the same field and publication year as the treated paper) with the lowest ijD. In our main analysis, we take the two nearest neighbors, one from above and one from below the treated paper in terms of average citations prior to the retraction event. Because we access over 26 million articles in the WOS, this control strategy succeeds for the majority of treated papers (66.4%) in finding control paper pairs that on average have exactly the same citation pattern prior to the retraction event. We use this set for our primary analysis. The Control papers are those that minimize the distance from the treated papers' citation pattern prior to the retraction year. Specifically, define the set of papers in field ity, is the number of citations received by paper i in year t. Fixed effects for each paper (iα) and each year since publication (tμ) capture the mean citation pattern of articles. iTreat is a dummy variable that equals 1 if article i is a treatment paper, and ktPost is a dummy variable that equals 1 if year t is after the retraction event for a given treatment and control group k. The coefficient of interest (difβ) captures any difference in citations for the treated paper, after the retraction event, compared to its control papers. In practice, we can use variations ofThe regression calculates the decline in citations to treated papers, after the retraction event, compared to the counterfactual citation path of the control papers. Following standard methodology, we estimate regressions of the form B.J. wrote the main manuscript text, S.L. prepared all figures and tables, G.J. wrote the supporting materials and B.U. prepared the data for analysis. All authors helped define the research questions and methodologies. All authors reviewed and edited the manuscript and Supplementary Information


Tumor necrosis factor-alpha (TNF-α) is a potent proinflammatory cytokine that inhibits osteoblast differentiation while stimulating osteoclast differentiation and bone resorption. TNF-α activates MAP kinase pathway leading to inhibition of osterix (Osx) expression. TNF-α also induces the expression of E3 ubiquitin ligase protein Smurf1 and Smurf2 and promotes degradation of Runx2, another key transcription factor regulating osteoblast differentiation and bone formation. We showed previously that overexpression of glucocorticoid (GC)-induced leucine zipper (GILZ) enhances osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs). We and others also showed that GILZ is a GC effecter and mediates GC anti-inflammatory activity. In this study, we asked the question whether GILZ retains its osteogenic activity while functioning as an anti-inflammatory mediator. To address this question, we infected mouse bone marrow MSCs with retroviruses expressing GILZ and induced them for osteogenic differentiation in the presence or absence of TNF-α. Our results show that overexpression of GILZ antagonized the inhibitory effects of TNF-α on MSC osteogenic differentiation and the mRNA and protein expression of Osx and Runx2, two pivotal osteogenic regulators. Further studies show that these antagonistic actions occur via mechanisms involving GILZ inhibition of TNF-α-induced ERK MAP kinase activation and protein degradation. These results suggest that GILZ may have therapeutic potential as a novel anti-inflammation therapy. Chronic inflammation, such as rheumatoid arthritis (RA), causes bone loss. Long-term use of steroid hormone glucocorticoids (GCs), which are potent anti-inflammatory agents and are frequently used to treat these conditions, also causes bone loss and results in osteoporosis (GC-induced osteoporosis), and is thus a major limiting factor for long term GC therapy Bone marrow mesenchymal stem cells (MSCs) are multipotent cells and can differentiate into several distinct cell lineages, including bone-forming osteoblasts and cartilage-forming chondrocytes The osteogenic differentiation of MSC is controlled by two key transcription factors, osterix (Osx) and Runx2 Glucocorticoid-induced leucine zipper (GILZ) is a member of the leucine zipper protein family and belongs to the transforming growth factor β-stimulated clone-22 (TSC-22d3) family of transcription factors We reported previously that overexpression of GILZ in bone marrow MSCs enhances osteogenic differentiation by shifting MSC lineage commitment towards the osteoblast pathway To investigate whether GILZ is capable of enhancing MSC osteogenic differentiation while serving as an anti-inflammatory mediator, we infected MSCs with retroviruses expressing GILZ or GFP  and induced them with osteogenic induction media (OS) in the absence or presence of different concentrations of TNF-α. To elucidate the molecular mechanisms underlying GILZ antagonizing TNF-α inhibition of osteoblast differentiation, we examined the expression levels of Osx and Runx2 mRNA and protein. MSC-GFP and MSC-GILZ cells were cultured in OS in the absence or presence of TNF-α (1 ng/ml). Eight days after the treatment, one set of cells was harvested for RNA analysis and the other set for protein analysis. To determine whether the protection of Osx by GILZ  was due Next, we examined the levels of E3 ubiquitin ligases Smurf1 and Smurf2 since studies have shown that TNF-α up-regulates Smurf1 and Smurf2, which, in turn, promotes Runx2 protein degradation Since GILZ antagonizes TNF-α-induced ERK phosphorylation , an evenin vivo study showed that mice with bone-specific glucocorticoid receptor knockout resist to glucocorticoid-induced bone loss In this study we have demonstrated that GILZ antagonizes the inhibitory effect of TNF-α on MSC osteogenic differentiation. Evidence presented in this study showed that GILZ antagonizes TNF-α effect in osteoblast differentiation by inhibiting TNF-α-induced ERK/MAP kinase activation and by inhibiting the expression of E3 ubiquitin ligase Smurf proteins. This antagonistic effect resulted in a partial rescue of TNF-α inhibition on Osx transcription, and a deceleration of TNF-α-induced Runx2 degradation. Attempts to confirm these results by knocking down GILZ were proven to be unsuccessful due to the low basal levels of endogenous GILZ expression (barely detectable by Western blot), and approaches that can increase GILZ levels, such as treatment of cells with glucocorticoid, activates glucocorticoid receptor and induces the expression of MAP kinase phosphatase-1 (MKP-1) in vitro luciferase reporter assays) Osx and Runx2 are pivotal transcription factors regulating osteogenic differentiation and bone formation. These two genes are primary targets for TNF-α in osteoblasts. In addition to activating the ERK pathway, TNF-α also activates NF-κB and induces p65/NF-κB nuclear translocation The inhibition of Runx2 by TNF-α, on the other hand, is primarily post-translational, involving TNF-α up-regulation of Smurf proteins and resulting in accelerated Runx2 protein degradation in vivo study by Beaulieu et al showed that the levels of GILZ is increased by therapeutic doses of glucocorticoids in the synovium of collagen-induced arthritis (CIA) mice, and that knockdown of GILZ using siRNA increases the clinical and histologic severity of CIA and synovial expression of TNF-α and IL-1 in vivo study directly supports our findings reported here regarding the role and therapeutic potential of GILZ in RA. TNF-α is a potent proinflammatory cytokine and triggers inflammatory reactions and further amplifies inflammatory signals by inducing other inflammatory cytokines , chemokines , and inducible enzymes . Importantly, TNF-α inhibits genes that directly regulate osteoblast differentiation and bone formation Although the causes of rheumatoid arthritis (RA), which is characterized by inflammation, pain, and swelling TNF-α (#410-MT) was purchased from R&D Systems Inc.; PD98059 (#PHZ1164) from BioSource International, Inc.; One-Step NBT/BCIP solution (#34042) from Pierce; SYBR Green PCR Master Mix (#4367659) from Applied Biosystems; Osx antibody (#ab22552) from Abcam Inc.; Runx2 (#130-3) monoclonal antibody from MBL International; Smurf1 (#AP2104a) and Smurf2 (#AP2105a) antibodies from Abgent Inc.; ERK1/2 (#9102) and phospho-ERK1/2 (#9106) antibodies from Cell Signaling Technology, Inc. All chemicals, except where specified, were purchased from Sigma-Aldrich.Isolation, characterization and infection of MSCs were described previously 4 cells/cm2 the previous day and then treated with osteogenic supplements (OS) consisting of DMEM supplemented with 2% FBS, 5 mM β-glycerophosphate, and 50 µM L-ascorbic acid-2-phosphate for the indicated time period.Cells were plated in 96-well plates in triplicate at a density of 1×10For alkaline phosphatase (ALP) staining, cells were rinsed 3 times with calcium and phosphate-free saline solution, fixed in 4% paraformaldehyde for 30 min at RT, washed 3 times with double-distilled water, and stained with One-Step™ NBT/BCIP solution  for 15 min at RT. Cells were washed again with double-distilled water and the plates were scanned using a CanoScan LiDE 80 flat-bed scanner.For alizarin red S (ARS) staining, cells were rinsed with calcium and phosphate-free saline solution, and fixed with ice-cold 70% ethanol for 1 hr. After a brief wash with water, cells were stained for 10 min with 40 mM ARS solution (pH 4.2) at room temperature. Cells were rinsed five times with water followed by a 15-min wash with PBS (with rotation) to reduce nonspecific ARS staining. After scanning of the plate for digital images, ARS dye was dissolved in 10% cetylpyridinium chloride (CPC) in 10 mM sodium phosphate (pH 7.0) for 15 min at RT and the concentrations of ARS determined by absorbance measurement as previously described Cells were cultured in osteogenic induction media in the presence of TNF-α (1 ng/ml) for 8 days before they were harvested. Equal amounts of total protein were separated on SDS-PAGE and analyzed by Western blot as previously described Total cellular RNA was isolated using TRIzol reagent according to the manufacturer's instructions (Invitrogen Corp.). Equal amounts of total RNA (2 µg) were reverse transcribed using TaqMan Reverse Transcription Reagents (Applied Biosystems), and the mRNA levels of the indicated genes were analyzed in triplicate using SYBR Green Master Mix and a Chromo-4 real-time RT-PCR instrument (MJ Research). The mRNA levels were normalized to β-actin  and gene expression was presented as fold changes. The primer sequences used in the PCR reactions are listed in Osx gene was PCR amplified from mouse genomic DNA. The primer sequences used in PCR amplification are: GGTACCTTGTTCCTTTTCCCTCCTGTTC-3′5′-ATAGC (forward), and AAGCTTCTGGGGACCGGGTCCCAAG -3′5′- GTATC (reverse). A Kpn I and a Hind III restriction site (underlined) were included in the forward and reverse primers, respectively. The resulting PCR product was digested with Kpn I and Hind III and inserted into the corresponding cloning sites in the pGL3 Basic luciferase reporter vector .A 791-nucleotide promoter sequence corresponding to −700 to +91 region of the Renilla luciferase to correct variations in transfection efficiency.Osx-Luc was cotransfected with the indicated amounts of expression vectors and an internal control plasmid (pRL-null) into C3H10T1/2 cells using Lipofectamine Plus reagents (Invitrogen Corp.). Total amount of DNA in each transfection was kept constant by adding empty vector DNA (pcDNA3). Twenty-four hours after transfection, the cells were lysed and luciferase activity measured with separate substrates to detect the luciferase (firefly) in the pGL3-basic plasmid and to the second luciferase (Renilla) encoded by the pRL-null vector  as previously described p value less than 0.05 (p<0.05) was considered to be statistically significant. Fold differences are indicated and are presented as mean ± SD.Data were analyzed with InStat Software  using one-way analysis of variance (ANOVA) followed by Bonferroni multiple comparison test. A Figure S1Continuous glucocorticoid treatment inhibits MSC osteogenic differentiation. MSC-GFP and MSC-GILZ cells were cultured in OS without or with 100 nM dexamethasone (Dex) for the first 2, 4, 8, or 12 days and then switched to OS without Dex for the remaining days indicated. Cells were stained with ARS on day 22.(TIF)Click here for additional data file.


Since TNF-α may influence GCs production, not only through the hypothalamus-pituitary axis, but also at the adrenal level, we now investigated the role of this cytokine on the adrenal GCs production. Wild type (WT) and TNF-R1−/− mice undergoing acute infection (Tc-WT and Tc-TNF-R1−/− groups), displayed adrenal hyperplasia together with increased GCs levels. Notably, systemic ACTH remained unchanged in Tc-WT and Tc-TNF-R1−/− compared with uninfected mice, suggesting some degree of ACTH-independence of GCs synthesis. TNF-α expression was increased within the adrenal gland from both infected mouse groups, with Tc-WT mice showing an augmented TNF-R1 expression. Tc-WT mice showed increased levels of P-p38 and P-ERK compared to uninfected WT animals, whereas Tc-TNF-R1−/− mice had increased p38 and JNK phosphorylation respect to Tc-WT mice. Strikingly, adrenal NF-κB and AP-1 activation during infection was blunted in Tc-TNF-R1−/− mice. The accumulation of mRNAs for steroidogenic acute regulatory protein and cytochrome P450 were significantly increased in both Tc-WT and Tc-TNF-R1−/− mice; being much more augmented in the latter group, which also had remarkably increased GCs levels. TNF-α emerges as a potent modulator of steroidogenesis in adrenocortical cells during T. cruzi infection in which MAPK pathways, NF-κB and AP-1 seem to play a role in the adrenal synthesis of pro-inflammatory cytokines and enzymes regulating GCs synthesis. These results suggest the existence of an intrinsic immune-adrenal interaction involved in the dysregulated synthesis of GCs during murine Chagas disease.Adrenal steroidogenesis is under a complex regulation involving extrinsic and intrinsic adrenal factors. TNF-α is an inflammatory cytokine produced in response to tissue injury and several other stimuli. We have previously demonstrated that TNF-R1 knockout (TNF-R1 The immune system plays a key role in the recognition and elimination of pathogens as well as in the regulation of the inflammatory response and tissue repair. In conjunction with the neuro-endocrine system this serves to maintain internal homeostasis in situ, in an ACTH-independently fashion, by growth factors or cytokines such as TNF-α, IL-6 and IL-1β in situ that in turn modulate the expression of enzymes involved in GCs synthesis. Steroidogenesis is the main function of adrenocortical cells in the zone fasciculata. In this regard, studies indicate that TNF-α is able to influence the expression of steroidogenic enzymes Evidence indicates that adrenal GCs secretion can be modulated Trypanosoma cruzi (T. cruzi) suggests that the immune-endocrine response accompanying the development of the infection strongly affects the progression of the disease Tulahuen strain of T. cruzi resulted in an acute fatal disease accompanied by a severe thymic atrophy and higher levels of TNF-α, compatible with an excessive inflammatory reaction arising from an unfavorable host-parasite relationship −/− and TNF-R2−/−) for this cytokine. Infected TNF-R1−/− mice showed a more pronounced stimulation of the HPA axis than wild type mice Our previous work in mice experimentally infected with T. cruzi infection. Since TNF-R1 is the form expressed in the adrenal cortex Trypanosoma cruzi acutely-infected mice.The existence of an intra-adrenal paracrine regulation of GCs release, in which components of the immune response, like TNF-α, may play effects beyond the classical neuro-endocrine-immune interactions, provides a stimulating background for exploring its features in the context of Tnfrsf1a tm1Imx or TNF-R1−/−) originally obtained from The Jackson Laboratory and gently provided by Dr. Silvia Di Genaro, were used throughout the studies. Protocols for animal studies were approved by the Faculty of Medical Sciences Institutional Ethical Committee (Resolution N° 2003–2012). Mice were bred at the animal facilities from the School of Medicine of Rosario, had free access to food and water, and were handled according to institutional guidelines. Sixty to 90-day-old mice (3–6/group) were used in each experiment. The Tulahuén strain of T. cruzi employed in these studies was maintained by serial passages in C57BL/6 suckling mice. Infection was carried out by injecting 200 viable trypomastigotes of T. cruzi by the subcutaneous route.Wild type (WT) male C57BL/6 mice and those lacking TNF-R1 . Data are expressed as number of parasites/mL.Bloodstream forms of Mice were bled by cardiac puncture, and blood was collected in sterile, endotoxin-free tubes and kept refrigerated until centrifugation. Plasma was separated into small plastic vials and stored frozen at −20°C until used. Plasma cytokines were measured by specific two-site enzyme-linked immunosorbent assay (ELISA) according to the manufactureŕs specifications. ELISA kits for TNF-α, IL-1β and IL-6 , were purchased from Pharmingen . All samples were assayed in duplicate.Mice were housed individually for one week before experiments started and kept single-caged throughout the experiments in temperature, and light-controlled rooms (light cycle from 7∶00 a.m. to 7∶00 p.m.). Plasma samples for hormone measurements were obtained from the tip of the tail between 8∶00 and 10∶00 a.m. as previously published Adrenal glands were removed at day 17 post-infection (pi) and fixed in buffered formalin. In parallel, glands from uninfected animals were also processed. Five paraffin-embedded 5 µm sections from each animal were stained with haematoxylin and eosin for evaluation of morphological changes. Serial sections from the adrenals were examined in blind by an experienced pathologist. For morphometrical alterations, three areas of the largest sections in each gland were evaluated.T. cruzi serum . The adrenal glands were removed 17 days after infection, embedded in Tissue-Tek  and frozen in liquid nitrogen. Five 5 mm-thick cryostat sections were settled on poly- L-lysine (Sigma)-covered glass slides, acetone fixed and blocked with PBS-BSA 1%. The sections were incubated for 1 h at room temperature with one of the following primary antibodies: rat anti-mouse PE-labeled CD120b/TNF-R2  or anti-T.cruzi serum obtained from a human collection of positive sera. After 4−5 PBS rinses, polyclonal rabbit anti-human immunoglobulin G (IgG) conjugated with FITC  was added for 1 h in the dark at room temperature. Following PBS washes, the sections were mounted in 25% glycerol/75% PBS and viewed with a laser scanning confocal microscope . Optimal confocal settings  were determined at the beginning of each imaging session and then held constant during the analysis of all samples. The images obtained were subsequently analyzed using the Image J software . The fluorescence intensity was calculated as an average of the area  and the values were recorded as arbitrary units.Immunofluorescence labeling and quantitative confocal microscopy were used to investigate the distribution and quantity of TNF-R2. The presence of antigens was investigated by employing an anti Adrenal gland nuclear fractions were obtained by homogenization of frozen tissues in lysis buffer . After centrifugation at 5,000 rpm for 5 min, the pellet was resuspended in the same lysis buffer and centrifuged to 5,000 rpm for 10 min again. Briefly, pellets were resuspended in buffer Hepes . Proteins were quantified by Lowry’s method For TNF-R1, phosphoriled(P)-p38, P-ERK and P-JNK detection, tissue lysates from adrenal glands were prepared by homogenization in 3 volumes of lysating RIPA buffer containing PBS, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride and 10 µg/ml leupeptin. After a 30-min incubation at 0°C and three freeze–thaw cycles, lysates were cleared by centrifugation at 15,000 rpm for 30 min, and supernatants were kept at −70°C. Proteins were quantified according to Lowry et al. T.cruzi was assessed by comparing the expression of each element with the normalizer RPL13 using the Ct method  as previously described For the evaluation of gene expression, RNA was isolated from adrenals using TRIzol , according to the manufacturer’s instructions. RNA quality and quantity were assessed using an Agilent bioanalyzer . First strand cDNA synthesis was prepared with 1.0 µg total RNA, oligo primer, using the SuperScript TM III Reverse Transcriptase . Real-time PCR was performed with approximately 50 ng of cDNA for each sample and Fast SYBR Green Master Mix . cDNA was amplified using specific murine primer sequences reported in p value was <0.05.Data are presented as mean ± standard error (SEM), unless otherwise stated. Statistical analysis was performed by the non parametric analysis of variance followed by Dunn post-hoc comparison when applicable. Correlation analysis was carried out using Spearman correlation test. The GraphPad Instat 4.0 software  was applied for statistical analyses, and differences were considered significant was when −/− infected mice start to die around during the 3rd week of infection and between-group differences during the first two weeks pi are not well flourished but become quite noticeable at day 17 pi.Based on data recorded in an earlier report −/− and wild type mice, but their survival time was different, being significantly shortened in infected TNF-R1−/− mice, when compared to Tc-WT mice . Moreover, parasitological studies at day 17 pi revealed that Tc-TNF-R1−/− mice exhibited higher parasitemias [median (rank): 24150 (36800-20470)] than Tc-WT . Studies at the same time point indicated that the relative adrenal weight in Tc-TNF-R1−/− and Tc-WT mice was significantly higher than their uninfected counterparts , mortality was 100% in TNF-R1−/− mice. Plasma levels of TNF-α, IL-1β and IL-6 in Tc-TNF-R1−/− were 5.1-, 4.5 and 1.16-fold increased respectively, when compared to the Tc-WT group  depicted in The morphometrical analysis was performed to assess the size of different zones in adrenal cortex. Values (pixel−/− mice) showed no significant differences in the basal levels of CT. In contrast, CT concentrations in Tc-TNF-R1−/− mice [12.47±1.95 µg/dl] were 2-fold higher (p<0.01) than those observed in Tc-WT mice , suggesting a certain degree of ACTH-independency of GCs production. Increase in CT levels during infection in both groups of mice was accompanied by marked morphological alterations in the adrenal cortex. In line with morphometrical studies, both infected group had an increased cortical thickness, largely because of an enlarged zone fasciculata. In Tc-WT, the zone glomerulosa was also increased with polyhedral non-vacuolated eosinophilic cells, whereas the zone reticularis showed a diffuse hyperplasia. In the central cortical area some nodular hyperplasia was also seen. Conversely, no hyperplasia of the zone glomerulosa was seen in TNF-R1−/− mice, showing a remarkable hypertrophy of the zone reticularis. No parasite nests were observed. Adrenal glands from uninfected mice had a normal morphology .To assess whether the increase of systemic pro-inflammatory cytokine levels from infected mice was accompanied by changes in the HPA axis, levels of CT and ACTH were measured. Uninfected mice . These results indicate that TNF-R1 deficiency during T. cruzi infection enhances MAPK activation in adrenal cells.TNF-α may lead to the activation of various MAPK signaling networks, contributing to the induction of NF-κB and AP-1 nuclear factors implicated in the synthesis of pro-inflammatory cytokines and expression of steroidogenic enzymes. Results presented in de novo GCs synthesis. Interestingly, as seen in −/− mice; much more augmented in the latter group. By opposite, mRNA expression levels of CYP11A1 remained unchanged in Tc-WT, but were significantly increased in Tc-TNF-R1−/− group.Genes encoding for key steroidogenic enzymes like acute regulatory protein (StAR), cytochrome P450 enzymes like family 11  are involved in −/−  suggests that changes in cytokine and GCs production in the latter group are mediated by a different signaling pathway. The lower TNF-R2 expression is suggestive of a non significant role of this signaling pathway. In present studies, infection with T. cruzi does lead to the activation of NF-κB and different MAPK cascades within the adrenal gland. Besides NF-κB, MAPK have been implicated in the synthesis of pro-inflammatory cytokines, acting as positive regulator for the expression of a variety of genes such as TNF-α, IL-6 and other inducible enzymes i.e., the ones involved in GCs production −/− mice showed a remarkably increased p-JNK and p-38 MAPK expression together with an augmented p-ERK expression as seen in the Tc-WT group. Evidence indicates that p-JNK is associated to other transcription factors, like AP-1, positively influencing the synthesis of pro-inflammatory cytokines and GCs −/− mice had increased levels of IL-6 respect WT. Therefore, the higher serum CT levels observed in Tc-TNF-R1−/− mice compared with Tc-WT mice may result both from a direct negative effect of TNF-α on adrenal CT synthesis, together with the non mutually excluding influence of increased IL-6 levels, known to up-regulate adrenal GCs synthesis.This cytokine mediates the inflammatory response mainly via the TNF-R1 expressed by various cell types, including lymphocytes and macrophages. In WT mice, TNF-R1 was expressed in the adrenal glands under basal conditions, markedly increasing during de novo synthesizing enzymes were found in both groups of T. cruzi-infected mice. Also, an even higher expression of enzymes driving GCs secretion was seen in Tc-TNF-R1−/− mice consistent with their more augmented levels of GCs in circulation. Both groups had a decreased expression 11β-HSD2 which converts active GCs to inactive form, more pronounced in Tc-TNF-R1−/− mice.In line with the increased plasma levels of CT, high expression level of GCs-related −/− mice revealed increased parasitemia, accelerated death and more augmented TNF-α, IL-1 and IL-6 concentrations in circulation. Extending these findings, present study suggest that reasons for the accompanying highly increased corticosterone levels may also include processes other than the classical anti-inflammatory reflex resulting from HPA activation. Our data indicate that TNF-α is able to differentially modulate CT secretion at the adrenal level. In situations of a cytokine-mediated intense HPA stimulation at the central level, TNF-α may exert a counterbalancing effect in situ, resulting in a highly augmented GCs response and the corresponding adverse effects. Whatever the case, our study adds novel information on the effect of TNF-α on the adrenal gland function during inflammation, particularly within the context of T. cruzi infection.Corroborating the critical role of TNF-α for host defense, Tc-TNF-R1


We found that RA patients tend to have higher levels of TNF-α than both healthy individuals and AS patients prior to treatment (P < 0.05). We measured greatly increased levels of TNF-α in both the AS and RA etanercept patient groups during the course of treatment, while in the infliximab treated patients, the amount of TNF-α measured remained unchanged. Elevated TNF-α in the etanercept treated patients does not appear to be a significant risk factor for the spontaneous development of further autoimmune diseases in our study group. Increased levels of TNF-R1 were determined in both AS (P < 0.05) and RA (P < 0.001) patients when compared to healthy controls. In AS patients, the levels of TNF-R1 dropped significantly when treated with either infliximab (P < 0.01) or etanercept (P < 0.001). In contrast, the levels of this receptor remained unchanged in RA patients treated with either compound.The effects of the TNF- TNF-α exerts its activity through binding to the membrane bound TNF-α receptors TNF-R1 and TNF-R2. In addition to their cell-bound forms, these receptors also exist as soluble molecules (sTNF-Rs) that are a result of enzymatic cleavage of the extracellular portions of the receptors [α antagonists. It is thought that sTNF-R1 and sTNF-R2 modulate and balance the activity of TNF-α in the course of inflammatory events [Proinflammatory signaling that is activated by TNF-y events .α biologicals that complex TNF-α and neutralize its disease driving activities are beneficial drugs in the treatment of chronic inflammatory disorders. Infliximab, a humanized mouse monoclonal antibody against TNF-α, and etanercept, a soluble fusion protein comprising an epitope derived from the human TNF-receptor 2 fused to the Fc portion of human IgG, represent two different types of biotechnical engineered molecules developed for therapeutic targeting of TNF-α in inflammatory diseases. Both drugs are approved for the treatment of AS and RA as well as for other autoimmune diseases [Anti-TNF-diseases .α as a major player in disease pathology, there are likely to be alterations in TNF-α regulation that can distinguish between different disease manifestations and respond to different types of TNF-α blocking strategies. Furthermore, studies have reported elevated levels of TNF-α in patients treated with TNF-α blockers and have speculated about the possible consequences thereof [α regulation in patients with AS and RA being treated with infliximab and etanercept, we determined the serum concentration of TNF-α as well as the levels of the soluble TNF-receptors 1 and 2. The measurement of interleukin 6 and C-reactive protein in the serum samples was used to further assess the inflammatory activity in these patients. To follow the course of the disease, we calculated the Bath ankylosing spondylitis disease activity index (BASDAI) and the disease activity score 28 (DAS28) for the AS and RA patients, respectively, at the start of the study and after 12 weeks into treatment.Although different autoimmune disorders share TNF- thereof –8. In orα therapy for the first time with either etanercept or infliximab. Twenty-two AS and 20 RA patients received etanercept 50 mg subcutaneously weekly during the study, and patients being treated with infliximab received IV infusions of 3 mg/kg body weight (28 RA patients) or 5 mg/kg body weight (28 AS patients) at 0, 2, and 6 and then every 8 weeks (RA patients) or 6 weeks (AS patients) into therapy. Patients treated with infliximab had been treated previously only with NSAIDs, whereas RA patients had received 2 or more DMARDs, including methotrexate, before infliximab or etanercept. RA patients treated with infliximab or etanercept received concomitant methotrexate combined with low-dose glucocorticoids with mean dosages of 15 mg/week and 5 mg/day, respectively. After obtaining written informed consent, serum samples were taken at different time points and stored at −80°C until assayed. Clinical and laboratory assessments were conducted prior to administration of the respective TNF-α blocker at baseline and at 2 and 12 weeks into therapy. The serum concentration of C-reactive protein (CRP) and the disease activity were determined. Assessment of the disease activity was performed before and after 12 weeks of therapy following the Bath ankylosing spondylitis disease activity index (BASDAI) for the AS patients and the disease activity score 28 (DAS28) was calculated for the RA patients.The study cohort consists of 45 healthy blood donors, 50 patients with ankylosing spondylitis (AS), and 48 patients with rheumatoid arthritis (RA). AS patients met the criteria of the European Spondyloarthropathy Study Group  and RA wThe study was approved by the local ethics committee of the University of Rostock.α, and IL-6 in serum samples was performed using ELISA-kits  following the manufacturer's protocol. The detection limit was 10 pg/mL for all determinations.Quantification of sTNF-R1, sTNF-R2, sTNF-t-test was used to analyze changes in the disease activity scores during the study period. Comparisons among groups and among time points are depicted as box and whiskers plots. The boxes represent the range from the 25th to the 75th percentile and the whiskers represent the maximum and minimum values. The horizontal line through the box indicates the median.Statistical analysis was performed using GraphPad Prism software . One way ANOVA was used to compare parameters measured in the different study groups and repeated measures ANOVA were used to compare values obtained from the same patients at the three time points. ANOVA analyses were followed by Tukey's multiple comparison tests. Paired P < 0.001 for both comparisons). The gender distribution within the study cohort shows a higher proportion of females in the control group (25 of 45) and the RA patients (35 of 48), whereas females are underrepresented among the AS patients (21 of 50).Comparing the three groups analyzed in the present study, the age of the healthy controls and the AS patients is very similar with an age (mean ± SD) of 45 ± 12 and 45 ± 13 years in both groups. With a mean age of 56 ± 12 years in the RA group, these patients are significantly older (α therapy led to a reduction of the inflammatory activity as seen by a significant drop of the CRP concentration after 2 and 12 weeks into therapy (P < 0.01 for both treatments). The RA patients show a trend towards decreasing CRP levels during both treatments, but this effect does not reach statistical significance. The proinflammatory cytokine IL-6 also tends to decrease during therapy in all treatment groups, but only reached statistical significance in the AS patients treated with etanercept and infliximab at the 2 week time point (both P < 0.05). All serum parameters determined before and during anti-TNF-α therapy are listed in In the AS patient groups treated either with etanercept or infliximab, the anti-TNF-α blocker and after 12 weeks into therapy (All four treatment groups showed a significant decrease in the disease activity scores (BASDAI for AS and DAS28 for RA) determined before the first administration of a TNF- therapy . This reα positive (>10 pg/mL). This is around twice as many as TNF-α positive AS patients prior to treatment  than in AS patients (2.4 ± 7.2 pg/mL) and in healthy participants (2.5 ± 7.9 pg/mL).Prior to treatment, 32% (infliximab group) and 20% (etanercept group) of RA patients were found to be TNF-α blockade, no significant change in the level or in the percentage of TNF-α positive study subjects could be observed in the AS and the RA patients being treated with infliximab. Administration of etanercept led to a significant increase of the mean serum TNF-α concentrations in the AS patients  as well as in the RA patients  after 12 weeks into therapy  was found [α that is measured with the ELISA kits that we employed can be affected by either the presence of etanercept or infliximab in the sample. In order to do so, we mixed and incubated (30 minutes at room temperature) various concentrations of TNF-α with physiologically relevant amounts of either TNF-α blocker, and measured the amount of TNF-α using our anti-TNF-α ELISA assay. We found that both agents reduced the measured amount of TNF-α, with the effect by infliximab being much larger than that of etanercept  and P< 0.001 (RA versus control and RA versus AS). No significant differences in the serum TNF-R2 concentration could be detected when comparing the healthy control subjects with the two patient groups at the 0 week time point. The mean concentrations were 2.86 ± 1.80 ng/mL in the healthy subjects and 2.79 ± 1.33 and 3.22 ± 1.49 ng/mL in the AS and RA patient group, respectively. These results are in partial agreement with previously published data on RA patients, which showed elevated levels of both TNF-α receptors when compared with healthy controls [Serum TNF-R1 showed elevated concentrations in both the AS and RA patient groups prior to anti-TNF-controls . The meacontrols , 16.P < 0.01 and P < 0.001 in the infliximab and the etanercept groups, resp.). In the RA patient groups, that displayed the highest initial receptor levels, neither of the TNF-α blockers used affected the serum TNF-R1 concentrations during the study period  when compared to the RA patients .No significant change in TNF-R2 was observed during infliximab treatment of the AS and RA patients. In the presence of etanercept, the measurement of serum TNF-R2 was interfered with by the TNF-R2 portion of the drug, and this resulted in values that were out of the range of the assay. We analyzed serial dilutions of etanercept using our TNF-R2 ELISA and found that the reactivity of etanercept in this immunoassay was about 10 to 15% when compared to the recombinant TNF-R2 standard calibrators . We therpatients . To testα were not detected in these patients. Interestingly, a recent study found that the levels of etanercept at 3 months into treatment can predict response to therapy at 6 months, suggesting that the monitoring of etanercept during treatment might be of prognostic value [As both AS and RA are treated by self-administration of etanercept, both at the same dosage and time interval, our measurements indicate that distinct differences in drug metabolism between our patient groups exist. This is in contrast to a study where no significant change in etanercept pharmacokinetics could be detected when comparing AS patients with RA patients . It is uic value .α frequently observed upon etanercept treatment likely reflects biochemical and physiological properties in the regulation of TNF-α and was not identified as being a risk factor for triggering further inflammatory reactions in our study. In AS patients, the measurement of TNF-α receptor 1 might be useful as a biological indicator of TNF-α blocker activity. The determination of serum etanercept might be useful in the monitoring of medication compliance and to allow for a quantification of the steady state concentration of these compounds in individual patients.The increase in measurable TNF-


Cytotoxic T lymphocytes (CTLs) are thought to exert immunologic selection pressure of escape mutation. Previous reports have showen that some overlapping peptide epitopes were presented by same HLA molecules. However, the abilities and properties of those CTLs to selection of same escape mutation are not well studied.CTL clones were established by stimulation of PBMC with a synthetic peptide (Nef138-8: RYPLTFGW or Nef138-10: RYPLTFGWCF) by limiting dilution method. Cytotoxic activity toward peptide-loaded cells was performed by 51Cr releasing assay. CTL suppression ability was tested by HIV-1 replication assay toward primary CD4+ cell. In vitro selection of escape mutation was performed by competitive HIV-1 replication assay. The frequency of tetramer positive cells in PBMC of HLA-A*24:02+ patients was detected by flow cytometry analysis.Both 8-mer and 10-mer epitopes specific CTLs were established from PBMC of the patients. The ability of Nef138-10-specific CTLs to suppression HIV-1 replication in vitro was much higher than that of Nef138-8-specific CTLs. In addition, at the early stage of infection, Nef138-10-specific CTLs was predominantly elicited in the patients more than the latter ones. Cross-reactive Nef138-8-specific CTLs recognizing both WT and 2F epitopes were detected in some patients. Moreover, in vitro competitive HIV-1 assay showed that both CTLs can select escape mutants, though the ability of Nef138-10-specific CTLs was stronger than that of Nef138-8-specific ones.The present study demonstrated that Nef138-10-specific CTLs play a major role in the selection of the escape mutation, and that Nef138-8-specific CTLs also have ability to select it. We showed selection of the same escape mutants by CTL specific for same HLA-restricted overlapping epitopes.


T. cruzi infection and/or with the development of the cardiac form in chronic chagasic patients. Genomic DNA of 300 subjects from an endemic area was extracted and analyzed by PCR using specific primers. TNF-alpha was assayed in culture supernatants by ELISA. An association was observed between the absence of the TNF-238A allele and negative serology. Furthermore, seropositive individuals carrying the TNF-238A allele produced significantly higher TNF-alpha levels without stimulation (p = 0.04) and after stimulation with LPS (p = 0.007) and T. cruzi antigens (p = 0.004). The present results suggest that the polymorphism at position -238 influences susceptibility to infection and that this allele is associated with higher TNF-alpha production in seropositive individuals.TNF-alpha plays an important role in trypanocidal mechanisms and is related to tissue injury. This cytokine has been detected in the heart of human chagasic patients where it is associated with tissue damage. This study investigated whether TNF-alpha levels and the presence of genetic polymorphisms are associated with the presence of  + T lymphocytes and macrophages are the main cells producing TNF-alpha. This study is an important contribution to explain the pathogenesis of Chagas disease.Chagas disease is an important parasitic disease that has no cure. The pathogenesis of the disease is still not completely understood. Studies using candidate genes are important to better understand the differences between individuals that lead to such heterogenous disease. TNF-alpha is a cytokine involved in the control of parasitemia during the acute phase and in cardiac injury during the chronic phase. The TNF-alpha gene is located in an important region of the MHC and its polymorphisms are associated with many parasitic and infectious diseases such as cerebral malaria and leishmaniasis. These studies are important since they were conducted in the same regions and involved populations leaving in the same conditions. The present study shows that patients with the indeterminate form produce higher levels of TNF-alpha than cardiac patients. The data suggest a control mechanism between inflammatory and regulatory cytokines. In addition, the presence of the TNF-238G allele contributes to the development of negative serology. We show that CD8 Trypanosoma cruzi. The disease continues to be a major public health problem in most Latin America countries, affecting around 9 million people T. cruzi in serum, a normal 12-lead electrocardiogram (ECG), and normal chest, esophagus and colon exams. The remaining 30–40% of patients will subsequently develop a determinate form of chronic disease: cardiac, digestive (megaesophagus and megacolon), or cardiodigestive Chagas disease is an important chronic infection caused by the protozoan Geographic variations exist in the severity and prevalence of the clinical forms of Chagas disease, but the reasons for this clinical and epidemiological heterogeneity are unknown The genes for tumor necrosis factor-alpha  and lymphotoxin-alpha, which are located in the MHC III region on chromosome 6, are closely linked to the HLA class I and class II genes T. cruzi infection and/or the development of the cardiac clinical form in chronic chagasic patients.Recent studies have investigated the role of TNF-alpha gene polymorphisms in Chagas disease T. cruzi that started in 1975. Epidemiological and entomological data demonstrated the interruption of vector transmission of the parasite to humans in 1999, and an international commission certified Brazil to be free of transmission in 2005 The study was performed in the municipality of Água Comprida , situated in the Vale do Rio Grande, southern region of Triângulo Mineiro, Minas Gerais State, Brazil. The town was endemic for Chagas disease and was included in the first National Campaign against T. cruzi antibodies showed that 25.6% of these subjects were positive and 74.5% were negative A total of 300 unrelated individuals agreed to participate in the study. Only subjects who were 25 years or older were included , since they corresponded to the youngest seropositive individuals of the sample studied. Serological screening for anti-The study was approved by the Research Ethics Committee of Universidade Federal do Triângulo Mineiro, Brazil (protocol 343). All individuals provided written informed consent.T. cruzi infection was evaluated by passive hemagglutination , enzyme-linked immunosorbent assay  and indirect immunofluorescence using fluorescein isothiocyanate (FITC)-conjugated to rabbit anti-human IgG (Sigma). The assays were performed according to manufacturer instructions and the results are expressed quantitatively. A subject who presented at least two positive tests was defined as positive The presence or absence of T. cruzi were submitted to clinical examination, electrocardiography and chest, esophagus and colon contrast X-ray exams for classification into the cardiac, digestive, mixed, or indeterminate form Patients infected with 6 cells/well) were cultured in the presence of 5 µg/mL T. cruzi (strain Y) antigens, 2 µg/mL Salmonella typhimurium lipopolysaccharide (LPS) (Sigma), and 5 µg/mL phytohemagglutinin (PHA) (Sigma). The plates were incubated at 37°C in a 5% CO2 atmosphere for 48 h. The culture supernatants were collected and stored at −70°C.Peripheral blood samples (20 mL) were collected with a vacuum system using heparin as anticoagulant. Peripheral blood mononuclear cells (PBMC) were separated by density centrifugation on a Ficoll-Hypaque gradient (Pharmacia) according to manufacturer recommendations. After separation, the cells were washed by centrifugation in RPMI medium (Gibco) and resuspended in RPMI medium supplemented with 5% fetal bovine serum (Gibco), 2 mM L-glutamine (Gibco), 50 mM 2-mercaptoethanol (Merck), and 40 µg/mL gentamicin. PBMC (2×10p-nitrophenyl phosphate (Sigma). Absorbance was read at 405 nm in a microplate reader (BioRad). The sensitivity limit of the test was 10 pg/mL.For TNF-alpha titration, microplates (Nunc) were sensitized overnight with anti-TNF-alpha mAb (Pharmingen). Nonspecific binding was prevented by incubating the plates with 2% BSA (Sigma) in PBS. The plates were incubated overnight with 100 µL of the culture supernatants in PBS diluted 1∶2, 2% BSA, and recombinant human TNF-alpha (Pharmingen). The plates were then washed four times with PBS and 0.05% Tween 20 and incubated with biotinylated anti-TNF-alpha mAb (Pharmingen) for 2 h, followed by washing and incubation for 2 h with streptavidin-conjugated alkaline phosphatase. Finally, the plates were washed four times and enzymatic activity was developed by incubating the plates with g for 15 min at controlled room temperature (approximately 27°C).The mass of red and white blood cells was submitted to osmotic lysis using Tris-EDTA lysis buffer (20∶5) consisting of 1 M Tris-HCl and 0.5 M EDTA, pH 8. The samples were centrifuged at least three times at 1334×For DNA extraction, the samples were treated by two different techniques depending on the amount of red blood cells remaining in the leukocyte pellet to guarantee the best quality DNA. A commercially available kit  was used for samples containing few red blood cells and phenol-chloroform extraction was used for samples containing higher amounts of red blood cells. The samples were resuspended in water and DNA was analyzed by 1% agarose gel electrophoresis.2 (Gibco), 1.0 µL 50 mM MgCl2 (Gibco), 3.0 µL 2 mM dNTPs (Invitrogen), 3.0 µL of each primer  (Gibco), 0.30 µL  Taq DNA polymerase (Gibco), and 5.0 µL genomic DNA at a concentration of 20 µg/mL. The following PCR conditions were used: 5 min at 95°C for initial denaturation and 35 cycles at 95°C for 1 min (denaturation), 55°C for 45 s , and 72°C for 45 s (extension), followed by a final extension step at 72°C for 3 min. Next, RFLP was performed using 0.1 µL AvaII  (New England Biolabs), 1.0 µL NE 4 buffer provided with the restriction enzyme, 0.4 µL sterile and filtered Milli-Q water, and 8.5 µL of the PCR product in a final volume of 10 µL. The samples were incubated for approximately 18 h at controlled room temperature (approximately 27°C). The digestion products were analyzed on 12% polyacrylamide gel (37∶5∶1) stained with 1% silver nitrate. Digestion of the PCR products from patients homozygous for the TNF-238A allele (-238A/A) generated only one 71-base pairs (bp) fragment, whereas those from patients homozygous for the TNF-238G allele (-238G/G) were completely digested (51 and 20 bp). All three fragments  were present in heterozygous patients.Polymorphisms in the promoter region of the TNF-alpha gene were determined by PCR amplification, followed by digestion with appropriate restriction enzymes. For TNF-238G/A 2 (Gibco), 1.0 µL 50 mM MgCl2 (Gibco), 3.0 µL 2 mM dNTPs (Invitrogen), and 3.0 µL of each primer  (Gibco), 0.30 µL  Taq DNA polymerase (Gibco), and 5.0 µL genomic DNA at a concentration of 20 µg/mL in a final volume of 30 µL. The following PCR conditions were used: 5 min at 95°C for initial denaturation, followed by 35 cycles at 95°C for 1 min (denaturation), 52°C for 45 s , and 72°C for 45 s (extension), followed by a final extension step at 72°C for 3 min. For RFLP, the mixture contained 0.1 µL NcoI (10 U/µL) (Gibco), 1.5 µL REACT 3 buffer provided with the restriction enzyme, 10.5 µL sterile and filtered Milli-Q water, and 3.0 µL of the PCR product in a final volume of 12.1 µL. The samples were incubated for approximately 18 h at 37°C. The digestion products were analyzed on 10% polyacrylamide gel (37∶5∶1) stained with 1% silver nitrate. Digestion of the PCR products from patients homozygous for the TNF-308A allele generated only one 107-bp fragment, whereas those from patients homozygous for the TNF-308G allele were completely digested (87 and 20 bp). All three fragments  were present in heterozygous patients.For TNF-308G/A T. cruzi antigens or medium alone. The cells were transferred (2×105 cells/tube) to 5 mL polystyrene tubes  and washed once with cold buffer (PBS-5% BSA) by centrifugation at 400×g for 10 min at 20°C. Cell pellets were resuspended in 100 µL PBS-BSA buffer and reacted with FITC anti-CD8 plus PE-Cy5 anti-CD4 mAb or FITC anti-CD14 for surface labeling. After 30 min of incubation in the dark at 4°C, the samples were washed three times in buffer by centrifugation at 300×g for 5 min at 20°C and fixed and permeabilized with freshly prepared Fixation/Permeabilization Working Solution (Pharmingen) for 30 min at 4°C, followed by two washed in 2 mL buffer. After the last wash, the cell pellets were resuspended in 100 µL 1X Permeabilization Buffer and reacted with PE anti-TNF-alpha at 4°C in the dark. After 30 min of incubation, the cells were washed twice with 2 mL 1X Permeabilization Buffer and then resuspended in 1% paraformaldehyde in Dulbecco's PBS (Sigma) for analysis. A total of 20,000 events/tube were acquired using a FACScalibur® flow cytometer (Becton Dickson). The Cell Quest™ software provided by the manufacturer was used for data acquisition and analysis.The following antibodies purchased from BD Pharmingen were used: FITC anti-CD8, FITC anti-CD14, and PE-Cy5 anti-CD4. PE anti-TNF-alpha and appropriate isotype controls were used. All of these antibodies were used according to manufacturer instructions. PBMC were recovered from 48 h cell cultures in the presence of Genotype and allele frequencies were analyzed statistically by the chi-square test. The strength of association was estimated using odds ratios and 95% confidence intervals (CI). The Mann-Whitney or Kruskal-Wallis test was used to analyze the association of TNF-alpha levels with genotypes, allele frequency, serology, and clinical forms. The unpaired t-test was used to analyze the association of intracellular TNF-alpha expression with the cardiac or indeterminate form. Analysis was performed using the StatView software, version 4.57 . The level of significance was set at 5% (p<0.05).T. cruzi and 132 (44%) with negative serology. All subjects were from the same endemic area. A total of 214 subjects were genotyped to position -238. Of these, 100 individuals were seropositive for T. cruzi and 114 were seronegative. All 300 individuals were genotyped to position -308. Of these, 168 individuals were seropositive for T. cruzi and 132 were seronegative.A total of 300 individuals were included in the study, 168 (56%) with positive serology for T. cruzi. Sixty-six (55.46%) had the cardiac form and 53 (44.54%) had the indeterminate form. Only 39 and 66 patients with the cardiac form were genotyped to positions -238 and -308, respectively. Among the indeterminate patients, 36 were genotyped to position -238 and 53 to position -308. Clinical manifestations were only compared between patients with the cardiac and indeterminate forms.Clinical classification was possible in 119 of the 168 subjects infected with T. cruzi antigen   and seropositive individuals produced more TNF-alpha than seronegative individuals after PHA stimulation  . When the TNF-238AA and TNF-238GA genotypes were grouped as allele A presence and the genotype TNF-238GG was considered to be allele A absence, the data verified that allele A absence was more frequent among seronegative individuals   Table 2.2X, TNF-238G/A p = 0.28; TNF-308G/A p = 0.64).T. cruzi antigen , and LPS stimulation  . After stimulation with T. cruzi antigens, 28.47% and 38.80% of CD8+ cells were positive for TNF-alpha in patients with the cardiac and indeterminate forms, respectively. In CD4+ cells, positivity for TNF-alpha was 2.78% for cardiac patients and 6.54% for indeterminate patients (+ cells, low and high levels. In cardiac patients, 63.36% and 3.76% of CD14+ cells were classified as low and high producers, respectively. These percentages were 29.96% and 0.70% in patients with the indeterminate form (The presence of intracellular TNF-alpha was analyzed by flow cytometry in 48-h cultured cells. In the absence of stimulation, 1.18% and 1.83% of CD8patients Figure 4T. cruzi infection. TNF-alpha is an important cytokine involved in parasite control during the acute phase The immune response plays an important role in the control of T. cruzi, possibly by modulating the expression of iNOS and adhesion molecules involved in rolling, adhesion and extravasation during inflammatory events in response to T. cruzi invasion Higher TNF-alpha levels were produced by seronegative individuals without stimulation, indicating that individuals who did not acquire the infection are able to produce TNF-alpha spontaneously. High basal levels of TNF-alpha may improve the host defense against T. cruzi-induced chronic cardiomyopathy Analyzing the development of the clinical form, TNF-alpha levels were significantly higher in patients with the indeterminate form than in those with the cardiac form. Comparison of cardiac patients with the control group showed higher levels of TNF-α in the cardiac group without stimulation and after LPS stimulation. Furthermore, indeterminate patients produced higher level of the cytokine after PHA stimulation. This fact suggests that TNF-alpha production by PBMC might be more important for the ability of the cytokine to control parasite growth than for promoting tissue damage or the development of heart lesions. It is important to point out that in the present study most patients classified as having the cardiac form did not exhibit severe heart involvement such as heart failure. Severe and end-stage Chagas disease has been associated with high levels of TNF-alpha Regarding TNF-alpha gene polymorphisms, this study provided evidence of an association between the absence of allele A at position -238 and seronegativity. We observed that individuals carrying the TNF-238A allele produce higher levels of TNF-α than those without the allele. This result suggests that the TNF-alpha polymorphism affects gene expression and that this effect depends on the cell population and on the strength of the stimulus since no differences were observed after PHA stimulation. Moreover, infected individuals carrying the TNF-238A allele produced higher levels of TNF-alpha than those without allele A. This result suggests that the -238 polymorphism exerts its potential function in infected subjects, probably as a result of clonal expansion and immunoregulatory mechanisms established during infection. The effect of the polymorphism might be more pronounced under these conditions. No significant association was established for the -308 polymorphism.Chlamydia trachomatis, although these findings were not statistically significant A higher proportion of the TNF-238A allele among patients when compared to controls was demonstrated for various other infectious diseases such as infection with T. cruzi antibodies from an endemic area in Peru. No significant differences were observed between patients and controls or between asymptomatic individuals and patients with cardiomyopathy for either polymorphic region Other studies on Chagas disease were conducted in Peru, Mexico and Brazil. The first study analyzed 87 healthy controls and 85 individuals seropositive for anti-T. cruzi antigens and in the absence of any stimulus.The present study differs from previous investigations because all individuals were from a region of high endemicity, with a greater probability of a more homogenous condition of exposure and accurate clinical analysis after some years of infection, permitting a sufficient time interval for the development of the clinical forms of the disease. This is advantageous in relation to the study's characteristics, though it represents a disadvantage due to the possible death of patients with more severe forms of the disease. Furthermore, in the present study, functional analysis showed that PBMC from seropositive individuals carrying the TNF-238A allele produced significantly higher levels of TNF-alpha when stimulated with LPS and Other investigators observed higher TNF-alpha production by LPS-stimulated PBMC in individuals homozygous for the TNF-308A allele + and CD8+ cells are the major source of TNF-alpha after antigen stimulation. The number of CD14+ T lymphocytes expressing TNF-alpha was significantly higher in cardiac patients, whereas the number of CD4+ lymphocytes producing this cytokine was high in patients with the indeterminate form. The stimulation of CD8+ T lymphocytes with exogenous antigens, including T cruzi lysate antigens, under similar culture conditions has been demonstrated previously Flow cytometry analysis indicated that CD14The importance of TNF-alpha as an activator of the mechanisms involved in parasite elimination In conclusion, the present results suggest that the TNF-238A allele exerts a significant effect on human susceptibility to infection. Furthermore, seropositive individuals carrying the TNF-238A allele produce higher levels of TNF-alpha after antigen and polyclonal stimulation, suggesting that the presence of the allele is associated with higher TNF-alphaproductionIn addition to its trypanocidal activity, TNF-alpha seems to trigger regulatory networks, such as the induction of IL-10


Psoriasis is a common inflammatory skin disease with a multifactorial genetic basis. A dysregulated interplay between keratinocytes and infiltrating immune cells underlies the cutaneous inflammation in psoriasis. Keratinocytes are important producers of antimicrobial peptides such as hBD-2 and LL37 and cytokines such as TNF-alpha, which are essential elements in this process of cell-cell communication . RecentlAn optimized cytokine mix was used to induce a psoriatic phenotype starting from normal human keratinocytes. Complexes of siRNA or anti-miRNA and SECosomes were made and validated prior to transfection. 24h post-tranfection, qPCR analysis was performed to evaluate mRNA expression levels.Transfection experiments with the complexes showed a stable knockdown efficiency of more than 80% of hBD-2, LL37, TNF-alpha and miR-203 mRNA.in vitro work we prepared and characterized siRNA and anti-miRNA complexes with SECosomes against hBD-2, LL37, TNF-alpha and miR-203 respectively. These complexes efficiently knock-down the targeted genes with concomitant downregulation of the associated proteins. Hereafter we will test the therapeutic applicability of our complexes in xenografted psoriatic skin by topical application.In this


Current treatment for children with polyarticular forms of Juvenile Idiopathic Arthritis (Poly JIA) results in approximately 50% of the patients demonstrating clinically inactive disease (CID)  while on treatment. Over 40% of the children with Poly JIA are treated with a TNF antagonist biologic often started ≤ 6 months (mos) of disease onset. Currently we are unable to accurately predict which children demonstrating CID while on an anti-TNF therapy will or will not have a flare of the JIA upon discontinuation of the anti-TNF agent. Anti-TNF therapy has known short- and medium-term toxicities in children with JIA; the long-term toxicities are unknown and the treatment is expensive. The ultimate goal of this project is to validate biomarkers that are predictive. These interim results demonstrate clinical aspects of assessing CID and stopping anti-TNF therapy.In 12 pediatric rheumatology centers, 120 children with Poly JIA demonstrating CID while on anti-TNF therapy will be enrolled and followed for up to 14 mos. In those subjects who demonstrate CID for the first 6 mos of the study, the anti-TNF therapy will be stopped. The primary outcome variable (POV) is disease flare within the next 8 months using a validated definition of disease flare .Forty eight subjects were enrolled by 12/20/2010. At the time of enrollment, the mean (median) age was 11.5 yrs (12.4) and disease duration 5.9 yrs (4.4). JIA subtype was Extended Oligo in 5, Poly RF negative in 38, and Poly RF positive in 5. 20 subjects were ANA positive, 27 ANA negative and 1 unknown. There were 35 females and 14 males; 45 were Caucasian and 3 African-American; 3 were Hispanic. Thirty eight (79%) were taking etanercept, 7 (15%) adalimumab and 3 (6%) infliximab. Sixteen (33%) were taking background methotrexate, while 32 (66%) were on anti-TNF therapy monotherapy. At the baseline study visit, the mean (median) duration of anti-TNF therapy was 2.2 yrs (1.5). All 48 subjects met the criteria for CID at the base line visit. At the time of analysis, 7/48 (14%) failed to demonstrate continued CID for the first 6 months of the study. Eighteen subjects demonstrated CID for the first 6 mos of the study and stopped anti-TNF therapy at the month 6 study visit. One subject discontinued the study due to logistical reasons (relocation of the family). In the 18 subjects stopping anti-TNF therapy, 6 (33%) have flared at a mean (median) of 56 days (28) after stopping the anti-TNF therapy. In the 12 subjects not demonstrating flare the mean (median) time off the anti-TNF agent is 143 days (158). This study is ongoing and the other 23 subjects have not yet completed the first 6 mos of the study.In children with one of the polyarticular forms of JIA on stable anti-TNF therapy, having achieved CID at one visit, approximately 14% will not remain in CID for the next 6 months. In those who stop the anti-TNF therapy, 66% have remained off the anti-TNF therapy without demonstrating flare to date.Daniel J. Lovell: Abbott Immunology Pharmaceuticals, 5, Amgen Inc., 5, Centocor, Inc., 5, UCB, Inc., 5; Steven P. Spalding: None; Karen Onel: None; Beth S. Gottlieb: None; Hermine I. Brunner: Abbott Laboratories, 5, Amgen Inc., 5, Centocor, Inc., 5, UCB, Inc., 5; Yukiko Kimura: None; Paula W. Morris: None; Judyann C. Olson: Abbott Laboratories, 2; Anne Johnson: None; Edward H. Giannini: None.


Candidatus Liberibacter asiaticus’ (Las), and redesignated them as LasAI and LasAII in lieu of the previous names HyvI and HyvII. As a phloem-limited, intracellular bacterial pathogen, Las has a significantly reduced genome and causes huanglongbing (HLB), a devastating disease of citrus worldwide. Bioinformatic analyses revealed that LasAI and LasAII share the structural features of an autotransporter family containing large repeats of a passenger domain and a unique C-terminal translocator domain. When fused to the GFP gene and expressed in E. coli, the LasAI C-terminus and the full length LasAII were localized to the bacterial poles, similar to other members of autotransporter family. Despite the absence of a typical signal peptide, LasAI was found to localize at the cell surface by immuno-dot blot using a monoclonal antibody against the partial LasAI protein. Its surface localization was also confirmed by the removal of the LasAI antigen using a proteinase K treatment of the intact bacterial cells. When co-inoculated with a P19 gene silencing suppressor and transiently expressed in tobacco leaves, the GFP-LasAI translocator targeted to the mitochondria. This is the first report that Las encodes novel autotransporters that target to mitochondria when expressed in the plants. These findings may lead to a better understanding of the pathogenesis of this intracellular bacterium.Proteins secreted by the type V secretion system (T5SS), known as autotransporters, are large extracellular virulence proteins localized to the bacterial poles. In this study, we characterized two novel autotransporter proteins of ‘ Yersinia [Haemophilus [Haemophilus [Shigella flexneri, AIDA-I of Escherichia coli, and BrkA of Bordetella pertussis, are localized to the bacterial poles [Autotransporters are large multi-domain virulence factors encoded by genomes of diverse gram-negative bacteria. A typical autotransporter consists of three functional domains: a Sec-dependent N-terminal signal peptide, a secreted passenger domain (α-domain) and a conserved C-terminal translocator domain (β-domain) . The cenersinia , Hia of ophilus , and Hapophilus . Compareophilus ,9. Deletophilus . It has al poles . Recentlal poles .Candidatus Liberibacter asiaticus’ is a Gram-negative, fastidious alpha-Proteobacterium, causing huanglongbing (HLB), a devastating disease of citrus worldwide. HLB causes rapid decline and shortens the life span of infected trees [Diaphorinacitri [I and HyvII) were identified that contained multiple, nearly-identical, leucine-rich repeats (LRRs). The diversity and plasticity of these two genes may have implications for how these intracellular bacteria adapt to their host ecological niches [‘ed trees . Having citri ,15. Intrcitri . Within l niches .I and LasAII). We determined that LasAI and LasAII are polar and surface localized in bacteria in addition to being targeted to the mitochondria when expressed in plant cells. Previously, we demonstrated that the Las bacterium may act as an “energy parasite” by encoding a functional ATP translocase for direct ATP/ADP importation from their host cells [Prophages in many bacterial genomes are associated with bacterial pathogenicity and biofilm formation . In the st cells . TogethelasAI and lasAII, previously reported as hyvI and hyvII, are located in two prophage regions in the Las Psy62 genome [lasAI encodes a 919 amino-acid hypothetical protein with a predicted molecular mass of 103.5 kDa. LasAI contains 12 full nearly identical leucine-rich repeats (LRRs) and 4 partial LRRs. The repeated sequence of the motifs is LEQIDLSKLEQIDLSEMAVLTQKMNIIDGIVNNLATQTKDVGRK.  The 12 full repeats within the lasAI gene share 93-100% identity at the nucleic acid level and 84-100% identity at the protein level. lasAII contains 1026 bp and encodes a 341 amino-acid hypothetical protein with a predicted molecular mass of 38.9 kDa. LasAII has only one partial LRR. The translocator domains of LasAI and LasAII share 80% identity at the amino acid level while the passenger domains share 50% identity at the amino acid level. Using the SignalP signal peptide prediction software, no signal sequence was identified in LasAI or LasAII and little information about the function of LasAI or LasAII was obtained from a BLAST search of the NCBI protein database. The LasAI and LasAII passenger domains share low level (about 25%) amino acid sequence similarities with the LRR protein of Colwellia psychrerythraea 34H (GenBank accession number: AAZ26055) and the cell wall associated biofilm protein of Staphylococcus epidermidis (ZP_06614153). Surprisingly, the passenger domains of LasAI and LasAII share similar LRR repeat structures with the Toll-like receptors (TLRs) that function as sentinels of the innate immune system by binding a variety of ligands, including lipopolysaccharide, flagellin and dsRNA, through a LRR ligand-binding domain [I and LasAII translocator domains were predicted to contain ten and twelve β-stranded secondary structures respectively by the YASPIN Secondary Structure Prediction program. However, the 3D structure predicted by the I-TASSER program did not form the typical β-barrel structure, which was reported for the translocator domain of the autotransporter NalP from Neisseriameningitidis [I and LasAII possess architectural features of the autotransporter family, including passenger domains with large repeated sequences that form coiled-coils and translocator domains containing β-stranded structures.The 2 genome . The 276g domain . LasAI agitidis . DespitelasAI was cloned into the pET102D-TOPO vector and protein expression was induced in E. coli BL21 (DE) cells . A protein of the expected size for LasAI was shown on SDS-PAGE and confirmed by Western blot . No signal was detected for the 40 kDa protein on the same Western blot , or the full length lasAII gene (pET102-gfp-lasAII), the expression of GFP was observed at the cell poles of E. coli by confocal laser scanning microscopy (CLSM)  was performed. The E. coli cells expressing the LasAI protein were not labeled by anti-LasAI antibody even though the isolated LasAI protein and the E. coli cells expressing the LasAI protein can be detected by Western blot and immuno-dot blot. The absence of surface labeling indicates that the LasAI protein produced in E. coli may not be secreted and folded properly.To further confirm the surface localization of LasAI and LasAII in plant cells and the role of the LasAI and LasAII translocator domains, full-length LasAI, full-length LasAII and the translocator domain of LasAI were cloned into the pGDY vector and transformed into Agrobacterium tumefaciens strain GV 2660. Transient expression results showed no detectable GFP in tobacco plants inoculated with these constructs, except with the pGDY vector alone. Co-inoculation of tobacco leaves with a P19 gene silencing suppressor and pGDY-lasAI-TD construct facilitated GFP expression (data not shown). Only a few cells had detectable GFP in the infiltrated zone when the full length lasAI or lasAII constructs were co-inoculated with the gene silencing suppressor. CLSM and propidium staining results showed that the expression of pGDY-lasAI-TD appeared to localize in the mitochondria. MitoTracker labeling and CLSM confirmed that GFP-lasAI-TD targeted to the mitochondria in tobacco leaves. As shown in lasAI-TD fusion protein. The autofluorescence of chloroplasts was not observed by MitoTracker detection using a 560nm low-pass filter as shown by differential interference contrast (DIC)  onto the bacterial cell surface. These findings reveal that LasAI and LasAII are unique autotransporters and the T5SS may play an important role in Las pathogenesis. Furthermore, using transient gene expression in tobacco leaves, we demonstrated that LasAI contains sufficient structural information for targeting the host mitochondria as do other members of the autotransporter family [We previously reported the genetic diversity and characteristics of two hypervariable proteins (HyvI and HyvII) from the Psy62 Las genome and global Las isolates that contain up to 12 nearly identical tandem repeats . In the Erwiniachrysanthemii [Xylella fastidiosa which is important for virulence and is also associated with bacterial autoaggregation and biofilm formation [Pseudomonasstutzeri A15 [Secreted proteins play a central role in the interactions of bacteria and their hosts. Gram-negative bacteria have evolved several specialized secretion systems to deliver effectors into their hosts, such as the Type Ш secretion system (T3SS) and the Type IV secretion system (T4SS). As an intracellular bacterium, Las does not have a T3SS or a T4SS, but may use the Sec secretion system ,22. Amonnthemii  and the ormation . In addieri A15 .I and LasAII proteins are unique autotransporters because they share no homology with any other members of the autotransporter family. Only coiled-coil domains were predicted and no signal peptides or anchor domains were identified in the LasAI and LasAII proteins when using the Trimeric Autotransporter Adhesins (TAAs) domain annotation tool [I and LasAII have the ability to export their native passenger domains and GFP fusion proteins to the E. coli cell surface, the C-terminal translocator domains of LasAI and LasAII may form β-barrel structures through which the passenger domain can pass [I and LasAII, the β-barrel structures of these translocator domains are atypical [Neisseriameningtidis, the crystal structure contains a 12-stranded β-barrel with a hydrophilic pore filled by an N-terminal α-helix [I and LasAII translocator domains are different from other translocators as there are no conserved amino acids between the Las translocator domains and the known translocator domains. The crystallized structures of LasAI and LasAII will be important for understanding the passenger domain export mechanism.The amino acid sequences of autotransporters are highly divergent except for the conserved translocator domain. LasAion tool . Since tcan pass . Howeveratypical . In othe α-helix . It is nI and LasAII localize at bacterial poles as do other reported autotransporter members, including IcsA and SepA of Shigella flexneri, AIDA-I of diffusely adherent E. coli and BrkA of Bordetella pertussis [N. meningitides and BrkA from B. pertussis localize at the pole of E. coli, suggesting that autotransporters contain information required for polar localization [S. flexneri two regions within the passenger domain were involved in pole targeting [I translocator domain alone has the ability to target the bacterial poles. Further investigation should identify the region(s) essential for LasAI and LasAII to localize to the bacterial poles.LasAertussis . It has lization . It is iargeting . In contI and LasAII were surface-localized autotransporters, LasAI was present in greater amounts in whole-cell lysates and outer membrane but undetectable in the culture supernatant, indicating either that itis inefficient cleavage in E. coli or that the cleaved passenger domain remains tightly associated with the translocator domain. The passenger domains of T5cSS, such as Hia from Hinfluenza. , are usually not cleaved and stay tightly associated with the cells [E. coli, BrkA is proteolytically cleaved at the bacterial surface, and the extracellular domain, though cleaved, remains tightly associated with the translocator domain [I and LasAII surface localization failed by IFA, although production of LasAI and GFP fusion proteins in E. coli was confirmed by immuno-dot blot and Western blot. The exported native passenger domains could not bind the primary antibody, indicating that they may not fold properly on the cell surface of E. coli. This was also observed with the IcsA of S. flexneri, which can be labeled at the surface of wild type S. flexneri but cannot be labeled in the E. coli cells expressing IcsA [I and LasAII surface localization by IFA and determine whether passenger domains are cleaved.Typically T5aSS autotransporters exposed at the cell surface are proteolytically cleaved at the junction of the passenger domain and the outer-membrane embedded translocation domain . Althoughe cells . In E. cr domain . Furthering IcsA . Once a lasAI and lasAII were constructed for Agrobacterium-mediated transient expression. Because no GFP expression was detected with the infiltrations containing our constructs, we used the gene silencing suppressor p19 to enhance the ectopic expression in plant leaves since post-transcriptional gene silencing (PTGS) is reported as a general feature in Agrobacterium-mediated transient expression [lasAI translocater domain (pGDY-lasAI-TD) was co-infiltrated with the p19 construct; while no GFP was detected when infiltrated with the lasAI translocater domain (pGDY-lasAI-TD) alone. However, only a few GFP expressing cells were detected from the leaves co-inoculated with p19 and the full gene constructs of either lasAI or lasAII . This result may be due to GFP-LasAI and GFP-LasAII fusion proteins improperly folded in the plant cells. Surprisingly, we observed the mitochondria aggregation in these infiltrated cells even though there was no detectable GFP expression. We speculate that LasAI and LasAII may have the ability to self-cleave and produce functional subunits targeted to the mitochondria similar to the autotransporter VacA from Helicobacter pylori [lasAI and lasAII from a Las prophage/phage  can act as inducers of PTGS in plant cells. To the best of our knowledge, this is the first evidence of bacterial prophage/phage gene inducing PTGS in plants. Further characterization of the PTGS conferred by LasAI and LasAII may shed light on the evolution and adaptation of the Las bacterium.To understand the function of these novel autotransporters, pression –31. As er pylori . FurtherI translocator domain-GFP fusion protein targeted to the mitochondria of tobacco leaf cells. Using the YASPIN secondary structure program, LasAI and LasAII were predicted to contain at least ten β-stranded structures in the translocator domain. It is worth noting that four β-strands in the YadA autotranspoter of Yersinia are sufficient for its mitochondrial localization in yeast [I and LasAII is not exceptional.With the aid of the gene silencing suppressor p19, we revealed that the LasAin yeast . Furtherin yeast , indicatlasAI and lasAII genes were reported in samples of distinct geographical origins and a single origin [I and LasAII passenger domains contain characteristics of the LRR family of proteins. The LRR proteins are important for immune responses, adhesion, invasion, signal transduction, and DNA/RNA processing [I translocator domain, which did not affect mitochondrial morphology, we observed mitochondrial aggregation in cells infiltrated with full length lasAI or lasAII. This phenomenon could be explained if the translocator domain was integrated into the mitochondrial outer membrane with the LRR passenger domain facing the cytosol, thus causing mitochondrial aggregation. By transmission electron microscopy, mitochondrial aggregation was observed in Las infected periwinkle, which agrees with our observation that mitochondrial aggregation is caused by LasAI and LasAII expression in tobacco. Most of the reported LRR proteins contain an N-terminal signal peptide for secretion across the bacterial membrane and a C-terminal membrane attachment region followed by a hydrophobic transmembrane [I and LasAII lack the classical signal sequence but the translocator domain can export the LRR passenger domain across bacterial membranes. AdpC, a LRR protein lacking a signal peptide, was also reported to be located on the outer membrane surface when it was expressed in a heterologous E. coli host [I and LasAII passenger domains target mitochondria are critical to understanding the functions of these proteins.Proteins containing tandem repeats are associated with diverse functions, and the variable numbers of tandem repeats affect the pathogenicity or antigenicity of several human and animal pathogens . Deletioe origin . It is iocessing . The LRRocessing . Severalocessing . Comparemembrane . In contoli host . FurtherCa. Liberibacter asiaticus’ is an obligate, intracellular bacterium with a significantly reduced genome. We are the first to demonstrate that Las encodes two novel autotransporters (LasAI and LasAII) that target mitochondria when expressed in plant cells. Although the functions of these effectors remain to be elucidated, we hypothesize that Las encodes these autotransporters to modulate energy biosynthesis since Las may directly import ATP/ADP from the cytosol of host cell for its energy and biosynthesis [I and LasAII, including an investigation into whether LasAI and LasAII passenger domains target mitochondria, identification of the eukaryotic binding partners, and characterization of protein structures. These studies will lead to a better understanding of Las pathogenesis, and thereby yield a better control strategy for HLB.In conclusion, ‘ynthesis . On the ynthesis . Future Escherichia coli Top10  was used as a host for plasmid construction and E. coli BL21 (DE3) cells  for recombinant protein expression. E. coli was grown in Luria-Bertani (LB) medium at 37°C. Agrobacterium tumefaciens strain GV2660 was cultured at 28°C in LB and used to mediate transient expression in the leaves of Nicotianabenthamiana. Antibiotics were used at the following concentrations: carbencilin, 50 µg/mL; kanamycin, 50 µg/mL.Strains and plasmids used in this study are listed in Nbenthamiana.  seeds were stored at 4°C for 2 days prior to germination. Subsequently the seeds were germinated in chambers programmed for cycles of 16 h light and 8 h dark at 26°C. The seedlings were then transferred into FaFard 4P mix soil in plastic containers and grown in controlled greenhouse conditions.Agrobacterium-mediated transient expression, the full length lasAI gene, lasAII gene and translocator domain (TD) of lasAI were amplified using genomic DNA from infected plants using the primers listed in lasAI, pGDY-lasAII and pGDY-lasAI-TD. The recombinant plasmids were verified by sequencing and then transformed into A. tumefaciens GV2660 by electroporation.For lasAI, or the N-terminal GFP fusion with the full length lasAII from pGDY, pGDY-lasAI-TD and pGDY-lasAII plasmids, respectively . Selected clones were cultivated at 37°C in LB broth for plasmid isolation and sequence verification. The clones were designated as pET102-gfp, pET102-lasAI, pET102-gfp-lasAI-TD and pET102-gfp-lasAII.For protein expression, the GFP-pET-F and GFP-pET-R primers were used for PCR amplification of either GFP alone, the N-terminal GFP fusion with the translocator domain of lasAI consensus clone was transformed into E. coli BL21 (DE3) expression cells . LasAI expression was induced and the protein purified using ProBond™ purification system under hybrid conditions according to the manufacturer’s instructions . The purified protein was separated using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue. Membrane transfer was performed by iBlot according to the manufacturer’s instructions . Western blotting was performed with a primary antibody against purified partial LasAI  from an immunized mouse . Goat anti-mouse HRP-conjugated antibody was used as the secondary antibody and detected by chemiluminescence following the manufacturer’s instructions .The pET102-E. coli BL21 containing plasmid pET102-lasAI as previously described [°C. To obtain the outer membrane protein, the pellet was suspended in 20mM Tris buffer (pH7.4) containing 0.5% sarkosyl and centrifuged again at 28000 rpm for 1hr at 4°C. The final pellet was dissolve in SDS-PAGE buffer. To obtain the AD fraction, the protein secreted but remaining bound to the cell surface, and the cell pellets were suspended in PBS and N-hexadecane was added. The suspensions were vortexed and centrifuged, and the liquid phase was filtered. The proteins were precipitated by acetone and dissolved in SDS-PAGE buffer. Proteins were separated on SDS-PAGE and detected by Western blot as described above.Whole-cell protein lysates, culture supernatant protein (CS), outer membrane protein (OM) and surface associated protein (AD) were prepared from escribed ,41–43. F°, washed three times with PBS and adjusted to a final concentration of 0.45 as determined by measurements of the optical density at 660 nm. Three microliters of each serial dilution  was spotted onto a nitrocellulose membrane in three replicates. The membrane was air dried and the Western blot procedure was performed as described above. The proteinase K treatment of intact cells was performed in PBS with 10 mM MgCl2 [Immuno-dot blotting was performed as described previously . The promM MgCl2  and thenA. tumefaciens GV2660 strain containing different constructs was cultured over night in 2 mL LB medium with the addition of antibiotics, and the following day 50 µL of the cell cultures were inoculated into new 5 mL LB aliquots containing the same antibiotics. These cultures grew until the OD600 value reached approximately 1.0. The cultures were then centrifuged for 10 min at 3000 g, and re-suspended in 5 mL of Agromix . After the suspension stood at room temperature for at least 3 h, the OD600 value was adjusted to different optical densities with Agromix. The P19 suppressor was also cultured and treated in the same way [600 of 1.0 was mixed with the p19 suppressor and infiltrated into 4 week-old Nbenthamiana.  leaves with a 1 mL needleless syringe. The experiments were performed with ten independent replicates.The same way . The finAfter two days of infiltration, the infiltrated zone was excised and the epidermal layers were peeled for mitochondrial staining with MitoTracker Red CMXRos according to the manufacturer’s protocol  and imaged using a confocal laser scanning microscope (CLSM), Zeiss LSM 510. GFP was detected with a 505-530 nm BP filter and MitoTracker was detected with a 560 nm LP filter.E. coli BL21 (DE3) cells containing pET102-gfp, pET102-gfp-lasAI-TD and pET102-gfp-lasAII constructs were cultured and induced as described above. The collected cells were mounted in antifade solution  for CLSM. Images were taken with a 505 nm LP filter and 40Χ or 63Χ (oil) objectives.The Midribs were sampled from healthy and Las-infected periwinkle. The infiltrated zone was sliced from tobacco. The samples were fixed and sectioned for TEM micrographs as described previously .Figure S1A: E. coli containing the pET102-lasAI constructWestern blot of . Lane1: whole-cell lysate; lane 2: culture supernatant; lane 3: cell associated protein. B: E. coli containing GFP and GFP fusion proteinsWestern blot of . Lane 1: E. coli containing plasmid pET102-gfp; lane 2: E. coli containing plasmid pET102-gfp-lasAI-TD; lane 3: E. coli containing plasmid pET102-gfp-lasAII.(TIF)Click here for additional data file.