Proteome-wide analysis of protein carboxy termini: C terminomics

Nature Methods 7, 508–511 (1 July 2010) | doi:10.1038/nmeth.1467

As proteome-wide C-terminal sequence analysis has been largely intractable, we developed a polymer-based enrichment approach to profile protein C-terminal peptides by mass spectrometry and identified hundreds of C-terminal peptides in the Escherichia coli proteome. We isotopically labeled GluC protease–digested and undigested samples and identified GluC substrates and their cleavage sites by quantification of neo–C-terminal peptides. 

Simultaneous intracellular chloride and pH measurements using a GFP-based sensor

Nature Methods 7, 516–518 (1 July 2010) | doi:10.1038/nmeth.1471

Chloride and protons perform important closely related roles in many cellular responses. Here we developed a ratiometric biosensor, ClopHensor, based on a highly chloride-sensitive Aequorea victoria GFP variant that is suited for the combined real-time optical detection of pH changes and chloride fluxes in live cells. 

Estimating prion concentration in fluids and tissues by quantitative PMCA

Nature Methods 7, 519–520 (1 July 2010) | doi:10.1038/nmeth.1465

Prions, the proteinaceous infectious agent responsible for prion diseases, can be detected with high sensitivity by protein misfolding cyclic amplification (PMCA) technology. Here we describe a quantitative PMCA procedure to calculate the concentration of very low levels of prions in biological samples.

Linking promoters to functional transcripts in small samples with nanoCAGE and CAGEscan

Nature Methods 7, 528–534 (1 July 2010) | doi:10.1038/nmeth.1470

Large-scale sequencing projects have revealed an unexpected complexity in the origins, structures and functions of mammalian transcripts. Many loci are known to produce overlapping coding and noncoding RNAs with capped 5|[prime]| ends that vary in size. 

Methods to identify the 5|[prime]| ends of transcripts will facilitate the discovery of new promoters and 5|[prime]| ends derived from secondary capping events. Such methods often require high input amounts of RNA not obtainable from highly refined samples such as tissue microdissections and subcellular fractions. Therefore, we developed nano–cap analysis of gene expression (nanoCAGE), a method that captures the 5|[prime]| ends of transcripts from as little as 10 ng of total RNA, and CAGEscan, a mate-pair adaptation of nanoCAGE that captures the transcript 5|[prime]| ends linked to a downstream region. Both of these methods allow further annotation-agnostic studies of the complex human transcriptome. 

Two-photon calcium imaging from head-fixed Drosophila during optomotor walking behavior

Nature Methods 7, 535–540 (1 July 2010) | doi:10.1038/nmeth.1468

Drosophila melanogaster is a model organism rich in genetic tools to manipulate and identify neural circuits involved in specific behaviors. Here we present a technique for two-photon calcium imaging in the central brain of head-fixed Drosophila walking on an air-supported ball. The ball's motion is tracked at high resolution and can be treated as a proxy for the fly's own movements. We used the genetically encoded calcium sensor, GCaMP3.0, to record from important elements of the motion-processing pathway, the horizontal-system lobula plate tangential cells (LPTCs) in the fly optic lobe. We presented motion stimuli to the tethered fly and found that calcium transients in horizontal-system neurons correlated with robust optomotor behavior during walking. Our technique allows both behavior and physiology in identified neurons to be monitored in a genetic model organism with an extensive repertoire of walking behaviors. 

Enhanced neuronal RNAi in C. elegans using SID-1

Nature Methods 7, 554–559 (1 July 2010) | doi:10.1038/nmeth.1463

We expressed SID-1, a transmembrane protein from Caenorhabditis elegans that is required for systemic RNA interference (RNAi), in C. elegans neurons.

This expression increased the response of neurons to double-stranded (ds)RNA delivered by feeding. 

Mutations in the lin-15b and lin-35 genes enhanced this effect. Worms expressing neuronal SID-1 showed RNAi phenotypes when fed with bacteria expressing dsRNA for known neuronal genes and for uncharacterized genes with no previously known neuronal phenotypes.

Neuronal expression of sid-1 decreased nonneuronal RNAi, suggesting that neurons expressing transgenic sid-1(+) served as a sink for dsRNA. This effect, or a sid-1(–) background, can be used to uncover neuronal defects for lethal genes. Expression of sid-1(+) from cell-specific promoters in sid-1 mutants results in cell-specific feeding RNAi. We used these strains to identify a role for integrin signaling genes in mechanosensation. 

Probabilistic density maps to study global endomembrane organization

Nature Methods 7, 560–566 (1 July 2010) | doi:10.1038/nmeth.1462

We developed a computational imaging approach that describes the three-dimensional spatial organization of endomembranes from micromanipulation-normalized mammalian cells with probabilistic density maps.

Applied to several well-known marker proteins, this approach revealed the average steady-state organization of early endosomes, multivesicular bodies or lysosomes, endoplasmic reticulum exit sites, the Golgi apparatus and Golgi-derived transport carriers in crossbow-shaped cells.

The steady-state organization of each tested endomembranous population was well-defined, unique and in some cases depended on the cellular adhesion geometry.

Density maps of all endomembrane populations became stable when pooling several tens of cells only and were reproducible in independent experiments, allowing construction of a standardized cell model. We detected subtle changes in steady-state organization induced by disruption of the cellular cytoskeleton, with statistical significance observed for just 20 cells. Thus, combining micropatterning with construction of endomembrane density maps allows the systematic study of intracellular trafficking determinants.

Insulin-like growth factors and risk of kidney cancer in men

British Journal of Cancer 103, 132-135 (29 June 2010) | doi:10.1038/sj.bjc.6605722

Background:

Insulin-like growth factor-I (IGF-I) has been shown to increase kidney growth, glomerular filtration rate, and renal function.

Methods:

In the prospective Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) study of 29 133 Finnish male smokers aged 50–69 years, serum concentrations of IGF were measured in samples collected in 1985–1988. A total of 100 men with kidney cancer diagnosed 5 years after blood collection through 1997 were compared with a subcohort of 400 men; logistic regression models were used to estimate the risk of developing kidney cancer.

Results:

Men with IGF-I levels >113 ng ml⁻¹ were 59% less likely to develop kidney cancer than men with levels 113 ng ml⁻¹ (odds ratio=0.41; 95% confidence interval=0.23–0.75). The IGF binding protein-3 (IGFBP-3) levels did not alter the association. No association was observed between IGFBP-3, or molar ratio of IGF-I/IGFBP-3, and kidney cancer.

Conclusions:

Low serum IGF-I levels in this cohort of older middle-aged male smokers are associated with increased kidney cancer risk, independent of IGFBP-3.

The association of diabetes with colorectal cancer risk: the Multiethnic Cohort

British Journal of Cancer (2010) 103, 120–126. doi:10.1038/sj.bjc.6605721 www.bjcancer.com  Published online 8 June 2010

Background:

  

Diabetics have been found to have a greater risk of colorectal cancer than non-diabetics.

Methods:

  

We examined whether this relationship differed by ethnic group, cancer site or tumour stage in a population-based prospective cohort, including 3549 incident colorectal cancer cases identified over a 13-year period (1993–2006) among 199 143 European American, African American, Native Hawaiian, Japanese American and Latino men and women in the Multiethnic Cohort.

Results:

  

Diabetics overall had a significantly greater risk of colorectal cancer than did non-diabetics (relative risk (RR)=1.19, 95% confidence interval (CI)=1.09–1.29, P-value (P)<0.001).

Positive associations were observed for colon cancer, cancers of both the right and left colon, and cancers diagnosed at a localised and regional/distant stage. The association with colorectal cancer risk was significantly modified by smoking status (P_Interaction=0.0044), with the RR being higher in never smokers (RR=1.32, 95% CI=1.15–1.53, P<0.001) than past (RR=1.19, 95% CI=1.05–1.34, P=0.007) and current smokers (RR=0.90, 95% CI=0.70–1.15, P=0.40).

Conclusion:

  

These findings provide strong support for the hypothesis that diabetes is a risk factor for colorectal cancer.

Skin cancers associated with autoimmune conditions among elderly adults

British Journal of Cancer 103, 112-114 (29 June 2010) | doi:10.1038/sj.bjc.6605733

Background:

Immunosuppression is a risk factor for certain skin cancers. Autoimmune conditions can involve the skin, and may involve immunosuppressive therapies.

Methods:

We conducted a population-based case–control study among elderly US adults using Surveillance, Epidemiology, and End Results-Medicare-linked data of 44 613 skin cancer cases and 178 452 frequency-matched controls. Medicare claims identified autoimmune conditions. Adjusted odds ratios (ORs) compared prevalence in cases and controls.

Results:

The most frequent autoimmune condition was rheumatoid arthritis (2.29%), which was associated with slightly increased risk of Merkel cell carcinoma (N=1977; OR (95%CI): 1.39 (1.10–1.74)). Risk of cutaneous non-Hodgkin's lymphoma (N=2652) was increased with psoriasis (OR (95%CI): 3.20 (2.62–3.92)). Risk of Kaposi's sarcoma (N=773) was elevated with ulcerative colitis (OR (95%CI): 2.76 (1.42–5.39)), and risk of other sarcomas (N=1324) was elevated with Graves disease (2.62 (1.30–5.31)).

Conclusions:

These findings suggest that immune disturbances in the skin, arising from autoimmune conditions or their treatment, promote development of skin cancer.

γ-Glutamyl transferase and breast cancer risk

British Journal of Cancer 103, 90-93 (29 June 2010) | doi:10.1038/sj.bjc.6605719

Background:

It has been reported that there is an increased risk of cancer in individuals with elevated levels of serum γ-glutamyl transferase (GGT).

Methods:

In the Guernsey Breast Cancer Cohort Study, GGT was measured in sera from 1803 normal women. Among these women, 251 subsequently developed cancer, of whom 96 developed breast cancer.

Results:

After adjustment for age at entry, height, weight, age at menarche and first birth with nulliparity, there was a highly significant relationship between elevated GGT and breast cancer risk. 

In the highest quartile, the hazard ratio (HR) was 2.17 (95% confidence interval (CI): 1.19, 3.93). When subdivided by menopausal status, there was a reduced non-significant effect in postmenopausal women, whereas for premenopausal women in the highest quartile, HR was 3.81 (95% CI: 1.37, 10.59). Premenopausal women with serum GGT levels above the normal range had a significantly elevated HR of 4.90 (95% CI: 1.86, 12.94).

Conclusions:

These results suggest that premenopausal women with high normal (above median) serum GGT or elevated levels (40 IU l⁻¹) are at increased risk of breast cancer and might benefit from close surveillance, possibly with breast magnetic resonance imaging scans. Serum GGT may mark previous exposure to carcinogens and lead to the identification of DNA adducts involved in mammary carcinogenesis.

Placental growth factor (PlGF) enhances breast cancer cell motility by mobilising ERK1/2 phosphorylation and cytoskeletal rearrangement

British Journal of Cancer (2010) 103, 82–89. doi:10.1038/sj.bjc.6605746 www.bjcancer.com Published online 15 June 2010

Background:

During metastasis, cancer cells migrate away from the primary tumour and invade the circulatory system and distal tissues. The stimulatory effect of growth factors has been implicated in the migration process. Placental growth factor (PlGF), expressed by 30–50% of primary breast cancers, stimulates measurable breast cancer cell motility in vitro within 3 h. This implies that PlGF activates intracellular signalling kinases and cytoskeletal remodelling necessary for cellular migration. The PlGF-mediated motility is prevented by an Flt-1-antagonising peptide, BP-1, and anti-PlGF antibody. The purpose of this study was to determine the intracellular effects of PlGF and the inhibiting peptide, BP-1.

Methods:

  

Anti-PlGF receptor (anti-Flt-1) antibody and inhibitors of intracellular kinases were used for analysis of PlGF-delivered intracellular signals that result in motility. The effects of PlGF and BP-1 on kinase activation, intermediate filament (IF) protein stability, and the actin cytoskeleton were determined by immunohistochemistry, cellular migration assays, and immunoblots.

Results:

  

Placental growth factor stimulated phosphorylation of extracellular-regulated kinase (ERK)1/2 (pERK) in breast cancer cell lines that also increased motility. 

In the presence of PlGF, BP-1 decreased cellular motility, reversed ERK1/2 phosphorylation, and decreased nuclear and peripheral pERK1/2. 

ERK1/2 kinases are associated with rearrangements of the actin and IF components of the cellular cytoskeleton. The PlGF caused rearrangements of the actin cytoskeleton, which were blocked by BP-1. 

The PlGF also stabilised cytokeratin 19 and vimentin expression in MDA-MB-231 human breast cancer cells in the absence of de novo transcription and translation.

Conclusions:

  

The PlGF activates ERK1/2 kinases, which are associated with cellular motility, in breast cancer cells. Several of these activating events are blocked by BP-1, which may explain its anti-tumour activity.

Osteosarcoma is characterised by reduced expression of markers of osteoclastogenesis and antigen presentation compared with normal bone

British Journal of Cancer 103, 73-81 (29 June 2010) | doi:10.1038/sj.bjc.6605723

Background:

Osteosarcoma (OS) is the most common primary bone tumour in children and adolescents. Patients who respond poorly to chemotherapy have a higher risk of metastatic disease and 5-year survival rates of only 10–20%. Therefore, identifying molecular targets that are specific for OS, or more specifically, metastatic OS, will be critical to the development of new treatment strategies to improve patient outcomes.

Methods:

We performed a transcriptomic analysis of chemo-naive OS biopsies and non-malignant bone biopsies to identify differentially expressed genes specific to OS, which could provide insight into OS biology and chemoresistance.

Results:

Statistical analysis of the OS transcriptomes found differential expression of several metallothionein family members, as well as deregulation of genes involved in antigen presentation. Tumours also exhibited significantly increased expression of ID1 and profound down-regulation of S100A8, highlighting their potential as therapeutic targets for OS. Finally, we found a significant correlation between OS and impaired osteoclastogenesis and antigen-presenting activity. The reduced osteoclastogenesis and antigen-presenting activity were more profound in the chemoresistant OS samples.

Conclusion:

Our results indicate that OS displays gene signatures consistent with decreased antigen-presenting activity, enhanced chemoresistance, and impaired osteoclastogenesis. Moreover, these alterations are more pronounced in chemoresistant OS tumour samples.

Metronomic gemcitabine suppresses tumour growth, improves perfusion, and reduces hypoxia in human pancreatic ductal adenocarcinoma

British Journal of Cancer 103, 52-60 (29 June 2010) | doi:10.1038/sj.bjc.6605727

Background:

The current standard of care for pancreatic cancer is weekly gemcitabine administered for 3 of 4 weeks with a 1-week break between treatment cycles. Maximum tolerated dose (MTD)-driven regimens as such are often associated with toxicities. Recent studies demonstrated that frequent dosing of chemotherapeutic drugs at relatively lower doses in metronomic regimens also confers anti-tumour activity but with fewer side effects.

Methods:

Herein, we evaluated the anti-tumour efficacy of metronomic vs MTD gemcitabine, and investigated their effects on the tumour microenvironment in two human pancreatic cancer xenografts established from two different patients.

Results:

Metronomic and MTD gemcitabine significantly reduced tumour volume in both xenografts. However, K^trans values were higher in metronomic gemcitabine-treated tumours than in their MTD-treated counterparts, suggesting better tissue perfusion in the former. These data were further supported by tumour-mapping studies showing prominent decreases in hypoxia after metronomic gemcitabine treatment. Metronomic gemcitabine also significantly increased apoptosis in cancer-associated fibroblasts and induced greater reductions in the tumour levels of multiple pro-angiogenic factors, including EGF, IL-1α, IL-8, ICAM-1, and VCAM-1.

Conclusion:

Metronomic dosing of gemcitabine is active in pancreatic cancer and is accompanied by pronounced changes in the tumour microenvironment.

Betulinic acid induces apoptosis and inhibits hedgehog signalling in rhabdomyosarcoma

British Journal of Cancer 103, 43-51 (29 June 2010) | doi:10.1038/sj.bjc.6605715

Background:

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in childhood with the ability to resist apoptosis by the activation of survival promoting and anti-apoptotic proteins.

Methods:

Efficacy of the apoptosis-inducing agent betulinic acid (BA) was determined in RMS cell cultures and in vivo by measuring cell viability, survival, apoptosis, hedgehog signalling activity, and neovascularisation.

Results:

Betulinic acid had a strong cytotoxic effect on RMS cells in a dose-dependent manner. The BA treatment caused a massive induction of apoptosis mediated by the intrinsic mitochondrial pathway, which could be inhibited by the broad-range caspase inhibitor zVAD.fmk. Exposure of hedgehog-activated RMS-13 cells to BA resulted in a strong decrease in GLI1, GLI2, PTCH1, and IGF2 expression as well as hedgehog-responsive luciferase activity. Intraperitoneal injection of 20 mg BA per kg per day significantly retarded growth of RMS-13 xenografts in association with markedly higher counts of apoptotic cells and down-regulation of GLI1 expression compared with control tumours, while leaving microvascular density, cell proliferation, and myogenic differentiation unaffected.

Conclusion:

Our data show that induction of apoptosis and inhibition of hedgehog signalling are important features of the anti-tumourigenic effect of BA in RMS and advices this compound for the use in a multimodal therapy of this highly aggressive paediatric tumour.

Marked anti-tumour activity of the combination of YM155, a novel survivin suppressant, and platinum-based drugs

British Journal of Cancer 103, 36-42 (29 June 2010) | doi:10.1038/sj.bjc.6605713

Background:

Survivin, a member of the inhibitor of apoptosis protein family, is an attractive target for cancer therapy. We have now investigated the effects of the combination of YM155, a novel small-molecule inhibitor of survivin expression, and platinum compounds (cisplatin and carboplatin) on human non-small cell lung cancer (NSCLC) cell lines.

Methods:

The anti-cancer efficacy of YM155 in combination with platinum compounds was evaluated on the basis of cell death and progression of tumour xenografts. Platinum compound-induced DNA damage was evaluated by immunofluorescence analysis of histone γ-H2AX.

Results:

Immunofluorescence analysis of histone γ-H2AX showed that YM155 delayed the repair of double-strand breaks induced in nuclear DNA by platinum compounds. The combination of YM155 and platinum compounds also induced synergistic increases both in the number of apoptotic cells and in the activity of caspase-3. Finally, combination therapy with YM155 and platinum compounds delayed the growth of NSCLC tumour xenografts in nude mice to an extent greater than that apparent with either treatment modality alone.

Conclusion:

These results suggest that YM155 sensitises tumour cells to platinum compounds both in vitro and in vivo, and that this effect is likely attributable to the inhibition of DNA repair and consequent enhancement of apoptosis.

Novel compounds with antiangiogenic and antiproliferative potency for growth control of testicular germ cell tumours

British Journal of Cancer 103, 18-28 (29 June 2010) | doi:10.1038/sj.bjc.6605725

Backround:

Testicular germ cell tumour (TGCT) is the most common cause of death from solid tumours in young men and especially for platinum-refractory patients novel treatment approaches are urgently needed. 

Using an in silico screening approach for the detection of novel cancer drugs with inhibitory effects on the tyrosine kinase activity of growth factors (e.g., VEGFR, PDGFR), we identified two compounds (HP-2 and HP-14) with antiangiogenic and antiproliferative potency, which were evaluated in endothelial cell models and TGCT cells.

Results:

HP-2 and HP-14 effectively inhibited the growth of VEGFR-2-expressing TGCT cell lines (Tera-1, Tera-2 and 2102EP) and endothelial cell models, while they failed to supress the growth of VEGFR-2-lacking tumour cells.

cDNA-microarrays revealed an inhibition of the expression of several growth factor receptors and related signal transduction molecules. Vascular endothelial growth factor (VEGF)-induced cell migration was also potently inhibited. 

Cell cycle-regulating proteins such as p21 and p27 were upregulated, leading to an S-phase arrest. Additional in vivo evaluations confirmed the antiangiogenic potency and good tolerability of the novel substances.

Conclusion:

Our data show that the identified novel compounds inhibit the growth of TGCT cells and decrease angiogenic microvessel formation. The mode of action involves cell cycle arresting effects and changes in the expression pattern of several angiogenic genes. The novel compounds may qualify as new candidates for targeted treatment of TGCT and merit further evaluation.

PHD2 in tumour angiogenesis

British Journal of Cancer (2010) 103, 1–5. doi:10.1038/sj.bjc.6605682 www.bjcancer.com Published online 11 May 2010

 Originally identified as the enzymes responsible for catalysing the oxidation of specific, conserved proline residues within hypoxia-inducible factor-1α (HIF-1α), the additional roles for the prolyl hydroxylase domain (PHD) proteins have remained elusive.

Of the four identified PHD enzymes, PHD2 is considered to be the key oxygen sensor, as knockdown of PHD2 results in elevated HIF protein.

Several recent studies have highlighted the importance of PHD2 in tumourigenesis. However, there is conflicting evidence as to the exact role of PHD2 in tumour angiogenesis.

The divergence seems to be because of the contribution of stromal-derived PHD2, and in particular the involvement of endothelial cells, vs tumour-derived PHD2. This review summarises our current understanding of PHD2 and tumour angiogenesis, focusing on the influences of PHD2 on vascular normalisation and neovascularisation.

Molecular and clinical dissection of CD24 antibody specificity by a comprehensive comparative analysis

Laboratory Investigation 90, 1102-1116 (July 2010) | doi:10.1038/labinvest.2010.70

CD24 is a small, highly glycosylated cell surface protein that is linked to the membrane through a glycosyl-phosphatidylinositol anchor.

It is overexpressed in many human carcinomas and its expression is linked to bad prognosis. Lately, lack or low expression of CD24 was used to identify tumor stem cells resulting in conflicting data on the usefulness of this marker.

In many immunohistochemical studies, the mAb SN3b was used but the epitope and specificity of this antibody have never been thoroughly investigated.

In other studies based mainly on cytofluorographic analysis, the mAb ML-5 was applied.

In this study, we compared the epitope of mAb SN3b to the CD24 mAbs SWA-11 and ML-5 that both bind to the core protein of CD24.

Using tissue microarrays and affinity-purified CD24 glycoforms, we observed only a partial overlap of SN3b and SWA11 reactivity.

The mAb SN3b recognizes sialic acid most likely on O-linked glycans that can occur independently of the CD24 protein backbone. The SN3b epitope was not related to common sialylated cancer-associated glycan structures.

Both SN3b epitope positive or negative CD24 glycoforms supported the binding of P-selectin and Siglec-5.

In breast cancer, the SN3b reactivity was associated with bad prognosis, whereas SWA11 was not.

In renal cell cancer, the SN3b epitope was completely absent but SWA11 reactivity was a prognostic factor.

Our results shed new light on the tumorbiological role of CD24 and resolve discrepancies in the literature related to the use of different CD24 mAbs.

DNA methylation of HOXD3 as a marker of prostate cancer progression

Laboratory Investigation 90, 1060-1067 (July 2010) | doi:10.1038/labinvest.2010.57

DNA methylation in gene promoters causes gene silencing and is a common event in cancer development and progression. 

The ability of aberrant methylation events to serve as diagnostic and prognostic markers is being appreciated for many cancers, including prostate cancer. 

Using quantitative MethyLight technology, we evaluated the relationship between HOXD3 methylation and clinicopathological parameters including biochemical recurrence, pathological stage, Gleason score (GS), and Gleason pattern in a series of 232 radical prostatectomies performed between 1998 and 2001. 

HOXD3 methylation was significantly greater in GS 7 cancers vs GS≤6 cancers (P-value <0.001) as well as pT3/pT4 vs pT2 cancers (P-value <0.001). The proportion of cases with high methylation in GS 7 vs ≤GS 6 and pT3/pT4 vs pT2 were also significantly different (P-values=0.002 and 0.005, respectively). 

There were also significant increases in methylation from Gleason pattern 2–3 and from pattern 3 to 4/5 (paired t-test P-values=0.01 and <0.001, respectively), whereas methylation from lymph node metastases was decreased when compared with matched tumor tissue (P-value=0.029). HOXD3 methylation was associated with biochemical recurrence in univariate analysis (P-value=0.043) and showed evidence for interaction with pathological stage as a predictor variable in Cox regression analysis (P-value=0.028). 

The results indicate that HOXD3 methylation distinguishes low-grade prostate cancers from intermediate and high-grade ones and may also have prognostic value when considered together with pathological stage.

Fibroblast-derived HB-EGF promotes Cdx2 expression in esophageal squamous cells

Laboratory Investigation 90, 1033-1048 (July 2010) | doi:10.1038/labinvest.2010.71

The molecular basis of attaining columnar phenotype in Barrett's esophagus is poorly understood. One hypothesis states that factors locally produced by cells of mesenchymal origin in chronic reflux esophagitis induce metaplastic transformation.

This study was performed to elucidate the factors secreted from fibroblasts that cause columnar phenotype in adjacent squamous epithelium. Human fibroblast cells were exposed to acidified medium for 20 min, followed by medium neutralization for 2 h, and then total RNA was hybridized to Sentrix Human-6 Expression BeadChips. Furthermore, esophageal mucosal biopsy specimens from reflux esophagitis patients were examined for HB-EGF expression using immunohistochemistry. In addition, cells from the human esophageal squamous epithelial cell line HET1A were treated with recombinant HB-EGF, and changes in expressions of Cdx2 and columnar markers were analyzed.

The gene expression profile revealed significant upregulation of a variety of growth factors and inflammatory chemokines in response to acid exposure. Among them, HB-EGF was upregulated more than 10-fold. Biopsy specimens from reflux esophagitis patients showed a strong expression of HB-EGF in fibroblast cells underlying the damaged epithelium. Furthermore, in vitro stimulation of HET1A cells with HB-EGF increased Cdx2 in dose-dependent manners.

Functional analysis of human Cdx2 promoter also revealed its upregulation by HB-EGF stimulation, showing the role of potential responsive elements (AP-1 and NF-κB) for its transcriptional activation. Moreover, the columnar markers cytokeratin 7 and villin were also upregulated by HB-EGF stimulation. HB-EGF induces several genes characteristics of columnar phenotypes of esophageal squamous epithelium in a paracrine manner.

Flagellin delays spontaneous human neutrophil apoptosis

Laboratory Investigation 90, 1049-1059 (July 2010) | doi:10.1038/labinvest.2010.77

Neutrophils are short-lived cells that rapidly undergo apoptosis. However, their survival can be regulated by signals from the environment. Flagellin, the primary component of the bacterial flagella, is known to induce neutrophil activation.

In this study we examined the ability of flagellin to modulate neutrophil apoptosis. Neutrophils cultured for 12 and 24 h in the presence of flagellin from Salmonella thyphimurim at concentrations found in pathological situations underwent a marked prevention of apoptosis.

In contrast, Helicobacter pylori flagellin did not affect neutrophil survival, suggesting that Salmonella flagellin exerts the antiapoptotic effect by interacting with TLR5.

The delaying in apoptosis mediated by Salmonella flagellin was coupled to higher expression levels of the antiapoptotic protein Mcl-1 and lower levels of activated caspase-3.

Analysis of the signaling pathways indicated that Salmonella flagellin induced the activation of the p38 and ERK1/2 MAPK pathways as well as the PI3K/Akt pathway.

Furthermore, it also stimulated IκBα degradation and the phosphorylation of the p65 subunit, suggesting that Salmonella flagellin also triggers NF-κB activation.

Moreover, the pharmacological inhibition of ERK1/2 pathway and NF-κB activation partially prevented the antiapoptotic effects exerted by flagellin. Finally, the apoptotic delaying effect exerted by flagellin was also evidenced when neutrophils were cultured with whole heat-killed S. thyphimurim. Both a wild-type and an aflagellate mutant S. thyphimurim strain promoted neutrophil survival; however, when cultured in low bacteria/neutrophil ratios, the flagellate bacteria showed a higher capacity to inhibit neutrophil apoptosis, although both strains showed a similar ability to induce neutrophil activation.

Taken together, our results indicate that flagellin delays neutrophil apoptosis by a mechanism partially dependent on the activation of ERK1/2 MAPK and NF-κB. The ability of flagellin to delay neutrophil apoptosis could contribute to perpetuate the inflammation during infections with flagellated bacteria.

Intraperitoneal LPS amplifies portal hypertension in rat liver fibrosis

Laboratory Investigation 90, 1024-1032 (July 2010) | doi:10.1038/labinvest.2010.60

Recent studies have shown that the risk of variceal bleeding in patients with liver cirrhosis increases with infections such as spontaneous bacterial peritonitis (SBP).

In this study, we hypothesized that pretreatment with intraperitoneal LPS may escalate portal hypertension.

In fibrotic livers (4 weeks after bile duct ligation, BDL), the activation of Kupffer cells (KCs) by zymosan (150 μg/ml) in the isolated non-recirculating liver perfusion system resulted in a transient increase in portal perfusion pressure.

Pretreatment with intraperitoneal LPS (1 mg/kg body weight (b.w.) for 3 h) increased basal portal perfusion pressure, and prolonged the zymosan-induced increase from transient to a long-lasting increase that was sustained until the end of the experiments in BDL but not in sham-operated animals.

Pretreatment with gadolinium chloride (10 mg/kg b.w.), MK-886 (0.6 mg/kg b.w.), Ly171883 (20 μM) or BM 13.177 (20 μM) reduced the maximal and long-lasting pressure increase in BDL animals by approximately 50–60%. The change in portal perfusion pressure was paralleled by a long-lasting production of cysteinyl leukotriene (Cys-LT) and thromboxane (TX) after LPS pretreatment. However, the response to vasoconstrictors was not altered by intraperitoneal LPS.

Western blot analyses showed an increased Toll-like receptor (TLR)4 and MyD88 expression after LPS pretreatment. In vivo experiments confirmed that intraperitoneal LPS increased basal portal pressure, and extended the portal pressure increase produced by intraportal zymosan or by LPS infusion.

In conclusion, upregulation of TLR4 and MyD88 expression in fibrotic livers confers hypersensitivity to LPS. This may lead to escalation of portal hypertension by production of TX and Cys-LT after endotoxin-induced KC activation. Therefore, LT inhibitors may represent a promising treatment option in addition to early administration of antibiotics in SBP.

Primary cilia organization reflects polarity in the growth plate and implies loss of polarity and mosaicism in osteochondroma

Laboratory Investigation 90, 1091-1101 (July 2010) | doi:10.1038/labinvest.2010.81

Primary cilia are specialized cell surface projections found on most cell types. Involved in several signaling pathways, primary cilia have been reported to modulate cell and tissue organization. Although they have been implicated in regulating cartilage and bone growth, little is known about the organization of primary cilia in the growth plate cartilage and osteochondroma. 

 Osteochondromas are bone tumors formed along the growth plate, and they are caused by mutations in EXT1 or EXT2 genes. In this study, we show the organization of primary cilia within and between the zones of the growth plate and osteochondroma. Using confocal and electron microscopy, we found that in both tissues, primary cilia have a similar formation but a distinct organization. The shortest ciliary length is associated with the proliferative state of the cells, as confirmed by Ki-67 immunostaining.

Primary cilia organization in the growth plate showed that non-polarized chondrocytes (resting zone) are becoming polarized (proliferating and hypertrophic zones), orienting the primary cilia parallel to the longitudinal axis of the bone. The alignment of primary cilia forms one virtual axis that crosses the center of the columns of chondrocytes reflecting the polarity axis of the growth plate. We also show that primary cilia in osteochondromas are found randomly located on the cell surface. Strikingly, the growth plate-like polarity was retained in sub-populations of osteochondroma cells that were organized into small columns. Based on this, we propose the existence of a mixture (‘mosaic’) of normal lining (EXT^+/− or EXT^wt/wt) and EXT^−/− cells in the cartilaginous cap of osteochondromas.

Novel guggulsterone derivative GG-52 inhibits NF-κB signaling in intestinal epithelial cells and attenuates acute murine colitis

Laboratory Investigation (2010) 90, 1004–1015; doi:10.1038/labinvest.2010.54; published online 1 March 2010

 We already showed that the plant sterol guggulsterone has been reported to inhibit nuclear factor-κB (NF-κB) signaling in intestinal epithelial cells (IECs) and to attenuate dextran sulfate sodium (DSS)-induced colitis.

This study investigates the anti-inflammatory effects of novel guggulsterone derivatives on IEC and preventive and therapeutic murine models of DSS-induced colitis. Novel guggulsterone derivates with high lipophilicity were designed and four derivates, including GG-46, GG-50B, GG-52, and GG-53, were synthesized.

Two guggulsterone derivatives, GG-50B and GG-52, significantly inhibited the activated NF-κB signals and the upregulated expression of interleukin-8 (IL-8) in COLO 205 cells stimulated with tumor necrosis factor-α (TNF-α).

Pretreatment with GG-50B and GG-52 attenuated the increased IκB kinase (IKK) and IκBα phsophorylation induced by TNF-α. In preventive and therapeutic models of murine colitis, administration of GG-52 significantly reduced the severity of DSS-induced colitis, as assessed by disease activity index, colon length, and histology.

In contrast, GG-50B did not show a significant reduction in the colitis severity. Moreover, the efficacy on attenuating colitis by GG-52 was comparable to that by sulfasalazine or prednisolone. These results indicate that the novel guggulsterone derivative GG-52 blocks NF-κB activation in IEC and ameliorates DSS-induced acute murine colitis, which suggests that GG-52 is a potential therapeutic agent for the treatment of inflammatory bowel diseases.

Effects of Arkadia on airway remodeling through enhancing TGF-β signaling in allergic rats

Laboratory Investigation 90, 997-1003 (July 2010) |doi:10.1038/labinvest.2010.78

Upregulation of transforming growth factor-β (TGF-β) signaling is interrelated with the development of airway remodeling.
In this study, we examined the role of two E3 ubiquitin ligases, Arkadia and Smurf2, which are critically required for TGF-β signaling in airway remodeling. Rats were immunized with ovalbumin (OVA) and then challenged with an OVA aerosol.
In in vitro experiments, normal human bronchial epithelial cells were stimulated with TGF-β₁ with or without the preincubation of Arkadia/Smurf2 small interfering RNA (siRNA) or lactacystin (an inhibitor of proteasomal degradation).

In the lungs of OVA-treated rats, a large number of inflammatory cells were present near the airways. An increased subepithelial collagen deposition was associated with high expression levels of Smad7, SnoN and Ski mRNAs, Arkadia, Smurf2, and TGF-β type I receptor (TβRI), but low expression levels of Smad7, SnoN and Ski proteins. Smad7, SnoN and Ski interacted with both Arkadia and Smurf2 while TβRI only interacted with Smurf2 but not with Arkadia. In in vitro experiments, the inhibitory effect of TGF-β₁ on the expression of Smad7, SnoN and Ski was reversed by Arkadia siRNA and lactacystin, whereas the stimulatory effect of TGF-β₁ on the expression of TβRI protein and Smad7/SnoN/Ski mRNAs was not affected. In contrast, Smurf2 siRNA did not influence the effects of TGF-β₁ on the expression of the above proteins.

Our results suggest that Arkadia may contribute to the pathogenesis of airway remodeling through enhancing TGF-β signaling by inducing the reduction of Smad7, SnoN and Ski proteins in OVA-sensitized and -challenged rats.

Modified multipotent stromal cells with epidermal growth factor restore vasculogenesis and blood flow in ischemic hind-limb of type II diabetic mice

Laboratory Investigation (2010) 90, 985–996; doi:10.1038/labinvest.2010.86; published online 3 May 2010

Diabetes is increasing in the world and causes severe cardiovascular complications. Diabetes-induced limb ischemia leads to foot amputation and therapeutic remedies are urgently needed. Here we report that local injection of mesenchymal stem cells (MSCs) prestimulated with epidermal growth factor (EGF) restored blood flow and vasculogenesis in the ischemic hind-limb of type II diabetic (db⁻/db⁻) mice. Bone marrow cells from db⁻/db⁻ mice are altered as evidenced by increased oxidative stress and reduced Akt and adhesion molecules when compared with control (db⁻/db⁺). Femoral artery ligation-induced ischemia was performed in the hind-limb of db⁻/db⁻ and db⁻/db⁺ mice for 28 days. Enhanced green fluorescent protein (EGFP)-MSCs stimulated±exogenous EGF for 24 h were injected locally into the ischemic muscle. Blood flow measured with MoorLDI-Laser and microangiography assessed with X-ray showed 100% recovery in db⁻/db⁺ compared to 50% recovery in db⁻/db⁻ mice. Interestingly, db⁻/db⁻ mice had 60 and 96% blood flow recovery and 61 and 98% of vasculogenesis when treated with MSCs alone or MSCs modified with EGF, respectively. Western blot analysis of hind-limb muscles revealed an increase in Akt and vascular endothelial growth factor receptor phosphorylation and hypoxia-inducible factor) expression in db⁻/db⁻ mice injected with MSCs or MSCs+EGF compared to db⁻/db⁻ mice. Fluorescent microscopic images show that EGFP-MSCs differentiate into new microvessels. Adhesion and migration of MSCs on cultured endothelial cells were ICAM1-, VCAM1- and Akt-dependent mechanism and elevated when MSCs were prestimulated with EGF compared with nonstimulated MSCs. Our novel study data provide evidence that in type II diabetes, stimulated MSCs with EGF enhance the recovery of blood flow and angiogenesis.

Dendritic cells and their role in atherogenesis

Laboratory Investigation 90, 970-984 (July 2010) | doi:10.1038/labinvest.2010.94

Dendritic cells (DCs) are the most potent professional antigen-presenting cells with the unique ability of primary immune response initiation. DCs originate from bone marrow progenitors, which circulate in the peripheral blood and subsequently penetrate peripheral tissues, where they give rise to immature DCs. In peripheral tissues, DCs continuously monitor the microenvironment and, when the cells encounter ‘danger’ signals, DCs undergo differentiation and maturation. Maturing DCs usually migrate to lymphatic tissues, where they form contacts with T cells to initiate a primary immune response. DCs were identified in arteries in 1995 and since then, further knowledge has been gained about the peculiarities of vascular-associated DCs and their role in atherosclerosis.

Immune reactions toward modified lipoproteins and other factors ignited by resident vascular DCs as well as by newly arrived DCs, which originate from blood monocytes, are believed to destabilize arterial homeostasis from very earlier stages of atherogenesis. There is a remarkable heterogeneity of DCs in atherosclerotic lesions. Some DCs mature and become capable of forming clusters with T cells directly within the arterial wall.

The predictive value of the numbers of circulating DC precursors in coronary artery disease and in atherosclerosis has been assessed, and it has been shown that DCs have a role in plaque destabilization. Over recent decades, DCs have proven to be a valuable instrument in immunotherapy approaches against cancer and various autoimmune diseases, and this explains the demand that the accumulated knowledge be applied to the field of atherosclerosis immunotherapy.

Iranians find new strains of tuberculosis

Iranian researchers have discovered two new strains of tubercles bacillus, which are believed to be resistant to the existing treatments.

Ali Akbar Velayati, the head of Tuberculosis and Respiratory Disorders Research Center, told IRIB News Agency that neither of the existing medications is capable of treating the newly-discovered strains of Mycobacterium tuberculosis.

He added that the two strains were discovered by Dr Farnian from Tuberculosis and Respiratory Disorders Research Center in two separate joint studies conducted in collaboration with Malaysian and Belarus researchers.

Velayati went on to say that Iranian scientists have also succeeded in producing nano-crystals which can penetrate the membrane of the drug-resistant tubercles bacillus, adding that this finding can pave the way for the development of new anti-TB medication.

The head of Tuberculosis and Respiratory Disorders Research Center added that drug-resistant tuberculosis has become a global concern, turning tuberculosis into a fatal disease.

From:- Press TV
http://tinyurl.com/2btnw65

Turning over the Chance card on MS susceptibility

Nature Immunology 11, 570 - 572 (2010) doi:10.1038/ni0710-570

CD8⁺ T cells expressing two distinct T cell antigen receptors fail to be tolerized and can induce autoimmunity.

In this issue of Nature Immunology, Goverman and colleagues present an intricate series of experiments whose results identify a previously unknown mechanism by which T cell antigen receptor (TCR)-transgenic CD8⁺ anti-myelin T cells can be induced to break tolerance and cause severe demyelination¹. Although the model relies on cutting-edge technology and an engineered immune system, the underlying scheme shown here may be surprisingly relevant for the pathogenesis of the most prevalent human demyelinating disease, multiple sclerosis.

Not a split decision for human hematopoiesis

Nature Immunology 11, 569 - 570 (2010) doi:10.1038/ni0710-569

Hematopoietic lineage schemes commonly show two distinct lymphoid and myeloid branches arising from the hematopoietic stem cell early during blood cell development. A new study of human hematopoiesis demonstrates that, similar to findings in mice, this split is not as dichotomous as is often presented.

Hematopoiesis is a remarkable developmental process in which mature lymphoid and myeloid cells with diverse functions are generated from a single hematopoietic stem cell (HSC). Seminar presentations on this topic frequently start with a slide indicating that the first step in HSC differentiation results in the generation of multipotent progenitors committed to either the lymphoid lineage or the myeloid lineage

Antimicrobial activity of mucosal-associated invariant T cells

Nature Immunology Published online: 27 June 2010 | doi:10.1038/ni.1890


Mucosal-associated invariant T lymphocytes (MAIT lymphocytes) are characterized by two evolutionarily conserved features: an invariant T cell antigen receptor (TCR) α-chain and restriction by the major histocompatibility complex (MHC)-related protein MR1. Here we show that MAIT cells were activated by cells infected with various strains of bacteria and yeast, but not cells infected with virus, in both humans and mice. This activation required cognate interaction between the invariant TCR and MR1, which can present a bacteria-derived ligand. 


In humans, we observed considerably fewer MAIT cells in blood from patients with bacterial infections such as tuberculosis. In the mouse, MAIT cells protected against infection by Mycobacterium abscessus orEscherichia coli. Thus, MAIT cells are evolutionarily conserved innate-like lymphocytes that sense and help fight off microbial infection.

The transcription factor MafB antagonizes antiviral responses by blocking recruitment of coactivators to the transcription factor IRF3

Nature Immunology Published online: 27 June 2010 | doi:10.1038/ni.1897


Viral infection induces type I interferons (IFN-α and IFN-β) that recruit unexposed cells in a self-amplifying response. We report that the transcription factor MafB thwarts auto-amplification by a metastable switch activity. MafB acted as a weak positive basal regulator of transcription at the IFNB1 promoter through activity at transcription factor AP-1–like sites. Interferon elicitors recruited the transcription factor IRF3 to the promoter, whereupon MafB acted as a transcriptional antagonist, impairing the interaction of coactivators with IRF3. Mathematical modeling supported the view that prepositioning of MafB on the promoter allows the system to respond rapidly to fluctuations in IRF3 activity. Higher expression of MafB in human pancreatic islet beta cells might increase cellular vulnerability to viral infections associated with the etiology of type 1 diabetes.

An immunoglobulin-like receptor, Allergin-1, inhibits immunoglobulin E–mediated immediate hypersensitivity reactions

Nature Immunology 11, 601 - 607 (2010) 


Anaphylaxis is a life-threatening immediate hypersensitivity reaction triggered by antigen capture by immunoglobulin E (IgE) bound to the high-affinity IgE receptor (FcεRI) on mast cells. However, the regulatory mechanism of mast cell activation is not completely understood. Here we identify an immunoglobulin-like receptor, Allergin-1, that contains an immunoreceptor tyrosine-based inhibitory motif (ITIM)-like domain, and show it was preferentially expressed on mast cells. Mouse Allergin-1 recruited the tyrosine phosphatases SHP-1 and SHP-2 and the inositol phosphatase SHIP. Coligation of Allergin-1 and FcεRI suppressed IgE-mediated degranulation of bone marrow–derived cultured mast cells. Moreover, mice deficient in Allergin-1 developed enhanced passive systemic and cutaneous anaphylaxis. Thus, Allergin-1 suppresses IgE-mediated, mast cell–dependent anaphylaxis in mice.

Transposable elements have rewired the core regulatory network of human embryonic stem cells

Nature Genetics 42, 631 - 634 (2010) 

Detection of new genomic control elements is critical in understanding transcriptional regulatory networks in their entirety. We studied the genome-wide binding locations of three key regulatory proteins (POU5F1, also known as OCT4; NANOG; and CTCF) in human and mouse embryonic stem cells. In contrast to CTCF, we found that the binding profiles of OCT4 and NANOG are markedly different, with only ~5% of the regions being homologously occupied. We show that transposable elements contributed up to 25% of the bound sites in humans and mice and have wired new genes into the core regulatory network of embryonic stem cells. These data indicate that species-specific transposable elements have substantially altered the transcriptional circuitry of pluripotent stem cells.

A global network of transcription factors, involving E2A, EBF1 and Foxo1, that orchestrates B cell fate

 Nature Immunology Received 8 March; accepted 19 May; published online 13 June 2010; doi:10.1038/ni.1891


It is now established that the transcription factors E2A, EBF1 and Foxo1 have critical roles in B cell development. Here we show that E2A and EBF1 bound regulatory elements present in the Foxo1 locus. E2A and EBF1, as well as E2A and Foxo1, in turn, were wired together by a vast spectrum of cis-regulatory sequences. These associations were dynamic during developmental progression. Occupancy by the E2A isoform E47 directly resulted in greater abundance, as well as a pattern of monomethylation of histone H3 at lysine 4 (H3K4) across putative enhancer regions. Finally, we divided the pro-B cell epigenome into clusters of loci with occupancy by E2A, EBF and Foxo1. From this analysis we constructed a global network consisting of transcriptional regulators, signaling and survival factors that we propose orchestrates B cell fate.

Genome-wide association study for vitiligo identifies susceptibility loci at 6q27 and the MHC

Nature Genetics

 

42,

 

614–618

 

(2010)

 

doi:10.1038/ng.603

We conducted a genome-wide association study of generalized vitiligo in the Chinese Han population by genotyping 1,117 cases and 1,429 controls. The 34 most promising SNPs were carried forward for replication in samples from individuals of the Chinese Han (5,910 cases and 9,916 controls) and Chinese Uygur (713 cases and 824 controls) populations. We identified two independent association signals within the major histocompatibility complex (MHC) region (rs11966200, P_combined = 1.48 × 10⁻⁴⁸, OR = 1.90; rs9468925,P_combined = 2.21 × 10⁻³³, OR = 0.74). Further analyses suggested that the strong association at rs11966200 might reflect the reported association of theHLA-A*3001, HLA-B*1302, HLA-C*0602 and HLA-DRB1*0701 alleles and that the association at rs9468925 might represent a previously unknown HLAsusceptibility allele. We also identified one previously undescribed risk locus at 6q27 (rs2236313, P_combined = 9.72 × 10⁻¹⁷, OR = 1.20), which contains three genes: RNASET2, FGFR1OP and CCR6. Our study provides new insights into the genetic basis of vitiligo.

New CDC Guidelines Prefer Use of Blood Tests

New CDC Guidelines Prefer Use of Blood Tests, Including QuantiFERON®-TB, to Diagnose Tuberculosis Infection in Certain Populations
TB Remains a Major Public Health Threat in Both Developing and Developed Countries

 Today the United States (U.S.) Centers for Disease Control and Prevention (CDC) issued new and important guidelines on the detection of Mycobacterium tuberculosis infections, the causative agent of tuberculosis (TB). In these landmark guidelines, CDC advises that Interferon Gamma Release Assay (IGRA) blood tests are now preferred over the 100+-year-old tuberculin skin test (TST) for diagnosing TB infection in certain populations, including people who typically do not return for the necessary reading of TST results, and those who have received Bacille Calmette-Guerin (BCG) as a vaccine or for cancer therapy. Typically the TST or IGRAs, such as QuantiFERON®-TB Gold (QFT), manufactured by Cellestis Limited, should be used as aids to diagnose infection with M. tuberculosis.
"In the U.S., up to 14 million Americans may be infected with TB bacteria and are at risk of developing full-blown, highly contagious TB. With these sobering numbers, complacency about TB's public health impact is not an option," said Antonino Catanzaro, M.D., professor of medicine, University of California San Diego, and Non-Executive Independent Director, Cellestis Limited. "These guidelines encapsulate the enormous body of clinical evidence on the performance of the QFT test and reflect the significant benefits this test is bringing to TB control worldwide."
The CDC report, "Updated Guidelines for Using Interferon Gamma Release Assays to Detect Mycobacterium tuberculosis Infection — United States, 2010" along with a companion implementation guide, appears in the June 25, 2010, Volume 59, No. RR-5 issue of the CDC's Morbidity & Mortality Weekly Report (MMWR). TST's drawbacks – which include a higher risk for false positives, especially in people who have been BCG-vaccinated; irritating TB-extract that must be injected under the skin; and the need for a second doctor's visit – were evaluated by the CDC and factored into their recommendations.
Approximately one person dies of TB every 17 seconds. Each infected person represents a potential yet preventable future outbreak. Convenient and trustworthy testing for TB infection is vital in order to efficiently identify the appropriate persons for treatment and thereby prevent its spread.
The populations specified by CDC in these guidelines, represent a majority of those being screened for TB infection. "With a specificity of more than 99 percent, QFT virtually eliminates false positive results and is simple to administer," said Tony Radford, chief executive officer, Cellestis Limited. "With more than 400 peer-reviewed, published clinical studies, QFT is a modern, scientifically–validated solution for reliable diagnosis of TB infection, and offers significant economic and public health advantages."
Specific highlights from the recommendations with regards to IGRAs include:
IGRAs are preferred over the TST for testing persons who have received BCG (as a vaccine or for cancer therapy).
IGRAs are preferred over the TST for diagnosing TB infection for persons from groups that historically have low rates of returning to have TSTs read.
IGRAs may be used in place of (not in addition to) TST in all situations in which CDC recommends testing, and is considered acceptable medical and public health practice.
IGRAs may be used in place of TST (without preference) to test recent contacts of persons with infectious tuberculosis.
IGRAs may be used in place of TST (without preference) for periodic screening to address occupational exposure to TB.
A TST is preferred for testing children aged <5 years. Use of an IGRA in conjunction with TST has been advocated by some experts to increase diagnostic sensitivity in this age group. Recommendations regarding use of IGRAs in children have also been published by the American Academy of Pediatrics.

About Tuberculosis
Tuberculosis (TB) is a contagious disease caused by a bacterium called Mycobacterium tuberculosis. TB bacteria usually attack the lungs, but can affect any part of the body such as the kidney, spine, and brain. If not treated properly, TB can be fatal. TB bacteria is spread through the air when a person with TB disease of the lungs or throat coughs, sneezes, speaks, or sings, which may lead people in close proximity to become infected.
According to the World Health Organization, about one person dies of TB every 17 seconds, causing nearly 2 million deaths annually. TB continues to be a contagious scourge in developing countries, and with the world shrinking rapidly due to global migration, it is a major public health threat in developed nations as well, including the United States. Each infected person represents a potential yet preventable future outbreak. Convenient and trustworthy testing for TB infection is necessary in order to quickly identify the appropriate persons for treatment and thereby prevent its spread.
About QuantiFERON®-TB Gold (QFT)
QuantiFERON®-TB Gold (QFT) is a simple blood test that accurately identifies people infected with Mycobacterium tuberculosis, the causative agent of Tuberculosis (TB). As a modern alternative to the 110 year old Tuberculin Skin Test (TST), also known as the Mantoux, QFT offers unmatched specificity, high sensitivity and simplicity. QFT enables focused TB therapy by providing clinicians with an accurate, reliable and convenient TB diagnostic tool. QFT is unaffected by previous BCG vaccination and most other environmental mycobacteria. Unlike the TST, it requires only one patient visit, is a controlled laboratory test and provides an objective, reproducible result that is unaffected by subjective interpretation. Results can be available within 24 hours.
QFT is available for use in all clinical settings in which TST is commonly used. Examples include contact tracing, regular employee testing, for example for health care workers, as well as screening programs for prisoners and immigrants. QFT's application in the screening of immunosuppressed patients prior to anti-TNF-alpha therapy initiation and in patients with HIV, cancer or organ transplants offers distinct advantages over the TST.
QFT® is sold directly in the U.S. by Cellestis Inc. and through Quest Diagnostics, Inc. and other commercial laboratories. In Europe QFT is provided by Cellestis GmbH (Germany); and in Australia/New Zealand by Cellestis International Pty. Ltd. (Australia). QFT is also available through Cellestis Commercial Partners in Japan, Europe, the Middle East, Africa, South America and Asia.
About Cellestis Limited
Cellestis Limited, a listed Australian biotechnology company founded in 2000 in Melbourne, Australia, develops and manufactures the QuantiFERON-TB Gold In-Tube (QFT) test, a breakthrough blood test for the detection and control of tuberculosis. The QuantiFERON technology is a patented method for detecting cell mediated immune (CMI) responses of T-cell lymphocytes using whole blood samples. In comparison to existing methods of measuring CMI, this unique technology provides accuracy and sensitivity along with major savings in operator time, labor and reagents. Using its patented QuantiFERON technology, Cellestis develops diagnostics tests that measure immune function for diseases with an unmet medical need.
Cellestis is proud to be exploring opportunities to enhance the global effort to eliminate TB. Cellestis is an industry partner of FIND (the Foundation for Innovative New Diagnostics) and the Stop-TB Partnership.
For more information, please visit www.cellestis.com.

From:-PRNewswire

Experimental Vaccine Protects Monkeys from New Ebola Virus

1-New research has found that an experimental Ebola vaccine developed by researchers at the National Institutes of Health protects monkeys against not only the two most lethal Ebola virus species for which it was originally designed, both recognized in 1976, but also against a newer Ebola virus species that was identified in 2007. Nancy J. Sullivan, Ph.D., of the Vaccine Research Center at the National Institute of Allergy and Infectious Diseases (NIAID), NIH, led the study team, which included collaborators from the U.S. Army Medical

2-“The important work by Dr. Sullivan and her colleagues shows that it is possible to generate immunity to newly identified species of Ebola virus with a vaccine originally designed to protect against a different species,” says NIAID Director Anthony S. Fauci, M.D. “This finding will guide future vaccine design and may open an avenue for developing a single vaccine that works against both known and emerging Ebola virus species.” 3-The experimental Ebola vaccine being developed at NIAID has two components, a prime and a boost. The prime consists of a DNA vaccine containing a small piece of genetic material encoding surface proteins from Zaire ebolavirus and Sudan ebolavirus. The boost consists of a weakened cold virus that delivers the Zaire ebolavirus surface protein.
4-Previously, Dr. Sullivan and her collaborators demonstrated that the prime-boost strategy produces a strong antibody response in monkeys. More importantly, the experimental vaccine induces a robust reaction by the cellular arm of the immune system. The cellular arm includes T cells, which help orchestrate the overall immune response.
5-However, developing one vaccine to protect against multiple Ebola virus species poses a challenge, she says. To the antibody-producing arm of the immune system, each species looks different. Neutralizing antibodies that recognize one Ebola species cannot readily recognize, or cross-neutralize, the others. T cells, in contrast, can cross-react, even when the target viruses share only small pieces in common.
6-After the emergence of Bundibugyo ebolavirus (BEBOV) in 2007, Dr. Sullivan’s team decided to revisit the prime-boost vaccine regimen to see if the cellular immunity generated would confer protection against the new virus species.
7-Four cynomolgus macaques received the DNA prime vaccine.A year later, the animals were boosted with the vector vaccine. Shortly after the boost, the four vaccinated monkeys and four unvaccinated ones serving as controls were exposed to lethal levels of BEBOV. All the unvaccinated animals became ill, and three died. None of the vaccinated animals showed any sign of illness. Analysis showed that the vaccinated monkeys developed T-cell responses sufficient to prevent or control infection by the novel Ebola virus species, even though the vaccine did not contain material from BEBOV and no antibodies against BEBOV were produced. The animal study was conducted in maximum-level biosafety containment laboratories at U.S. Army Medical Research Institute of Infectious Diseases.
For more information on NIAID research on Ebola and other hemorrhagic fever viruses, visit NIAID's Ebola/Marburg portal.

Retina Transplants from Stem Cells

Human embryonic stem cells can be coaxed into three-dimensional structures of retinal cells

1-Scientists have created a three-dimensional, retina-like structure out of human embryonic stem cells that they hope could someday serve as a retinal transplant for people with macular degeneration and other diseases of the retina. Their method, published recently in Journal of Neuroscience Methods, offers a potential new source of cells for retinal transplants.

2-Hans Keirstead, lead author of the paper and a stem cell biologist at University of California, Irvine, says that the method is designed to provide an alternative to human fetal tissue transplants, which have been conducted on a small group of patients and have resulted in improved vision. Fetal cells are difficult to obtain and raise ethical issues. "We really wanted to build upon that technique by creating a renewable source of tissue," he says.

3-In this study, the researchers first created two types of cells from the human embryonic stem cells: early-stage retinal cells, and retinal pigment epithelium (RPE) cells, which provide nourishment to the cells responsible for vision in the retina. The researchers then grew these two types of cells together in a chamber designed to expose them to a gradient of concentrations of solutes and growth-promoting chemicals. The cells could form three-dimensional structures, a feat rarely achieved with stem cells.

4-Keirstead believes that the study points to two important strategies for creating retinal transplants: growing early retinal cells along with RPE cells, and bathing the cells in a gradually changing solution that encourages the development of three-dimensional layers of cells. His team found that this approach generated early-stage retinal cells that were on the path of differentiating into all of the various cell types in the retina.

5-Keirstead believes that a retinal transplant will work best when made of cells that have not fully developed. "The three-dimensional layer is purposefully young," he says. Previous studies have found that younger cells are more likely to integrate with existing tissue after transplantation, rather than die.

6-Robert Lanza, chief scientific officer at Advanced Cell Technologies, who was not involved in the study, says that his team discovered several years ago that, when turning human embryonic stem cells into RPE cells, other stem cells would spontaneously form layers, including patches of photoreceptors. "This paper shows that you can take advantage of this natural process, and for the first time use tissue engineering techniques to generate three-dimensional retina-like structures," he says.

7-But Lanza is skeptical about the clinical usefulness of such structures. "You can't just transplant a retina and restore sight," he says, because it requires making a series of complex connections with the brain. Although he says there could prove to be some advantage to using three constructs of cells, "for the moment, replacing individual cell types might be the best approach for helping patients suffering from eye disease."

8-Scientists have been working on several approaches to retinal transplants. One approach, led by Advanced Cell Technologies, is to turn human embryonic stem cells into RPE cells and transplant them into the retina. The therapy would work best in the early stages of degeneration to halt further progress, rather than to restore vision that is already lost. Another approach is to transplant stem cells that are in the early stages of becoming light-sensitive photoreceptors, which has demonstrated efficacy in mice.

9-Yet another strategy is to use young tissue instead of individual cells. Fetal tissue transplants have shown some success in animals as well as a small group of humans. A study published in 2008 found that seven out of 10 patients who received the transplants had improved vision. However, there has been debate about whether these transplants actually integrate into the existing tissue.

10- Keirstead has conducted a series of studies in animals that he says demonstrates that transplanted tissue is functioning in the eye. If so, the strategy could be useful for later-stage degeneration, when the existing retina has lost much of its function.

11-For Keirstead's team, the next step is to show that tissue derived from stem cells can function properly. His lab is currently transplanting the tissue into rats to determine whether the transplants can survive and incorporate into the eye, and whether they improve the animals' vision.

From:- MIT Technology Review