Injury to the peritoneum during surgery is followed by a healing process that frequently results in the attachment of adjacent organs by a fibrous mass, referred commonly as adhesions. Because injuries to the peritoneum during surgery are inevitable, it is imperative that we understand the mechanisms of adhesion formation to prevent its occurrence. This requires thorough understanding of the molecular sequence that results in the attachment of injured peritoneum and the development of fibrous tissue. Recent data show that fibroblasts from the injured peritoneum may play a critical role in the formation of adhesion tissues. Therefore, identifying changes in gene expression pattern in the peritoneal fibroblasts during the process may provide clues to the mechanisms by which adhesion develop.In this study, we compared expression patterns of larger number of genes in the fibroblasts isolated from adhesion and normal human peritoneum using gene filters. Contributions of TGF-beta1 and hypoxia in the altered expression of specific genes were also examined using a semiquantitative RT-PCR technique.Results show that several genes are differentially expressed between fibroblasts of normal and adhesion peritoneum and that the peritoneal fibroblast may acquire a different phenotype during adhesion formation. Genes that are differentially expressed between normal and adhesion fibroblasts encode molecules involved in cell adhesion, proliferation, differentiation, migration and factors regulating cytokines, transcription, translation and protein/vesicle trafficking.Our data substantiate that adhesion formation is a multigenic phenomenon and not all changes in gene expression pattern between normal and adhesion fibroblasts are the function of TGF-beta1 and hypoxia that are known to influence adhesion formation. Analysis of the gene expression data in the perspective of known functions of genes connote to additional targets that may be manipulated to inhibit adhesion development. Peritoneal adhesions resulting from surgical injury are often associated with pelvic pain, bowel obstruction and infertility . EpidemiParietal and visceral peritoneum that surfaces the intraperitoneal organs is covered by a layer of squamous epithelial cells, the mesothelium. The submesothelial layer consists of fibroblasts, macrophages and blood vessels. Surgical abrasion to the peritoneum releases mesothelial cells, macrophages, fibroblasts, and blood containing cytokines and several cell types at the site of injury. Coagulation of blood creates a fibrinous mass between injured surfaces. In some patients fibrinolysis of clot followed by proliferation of mesothelial cells covers the wound. In others, failure of fibrinolysis followed by proliferation and migration of fibroblasts into the proteinous mass generates fibrous tissues of adhesion. Consequently, the process of adhesion formation include inflammatory response, fibrin deposition, cell-proliferation, -differentiation, -migration, -death, angiogenesis, extra cellular matrix (ECM) turnover regulated by cytokines, hypoxia, genetic and epigenetic factors .Recent studies illustrate roles of peritoneal fibroblasts in adhesion development -10. It iTissues were collected at the initiation of surgery and after the entry into the abdominal cavity of female patients (25–50 years) undergoing laparatomy for pelvic pain as described earlier . All pat2) for 3 days in DMEM (Life Tech.) supplemented with 10% fetal bovine serum (Life Tech.) and antibiotics . The monolayer of cells were passaged in trypsin-EDTA solution (Life Tech.). Cells at 3–5 passages were cultured in serum free medium in 75 cm2 flasks (Fisher Scientific, Pittsburgh, PA) to 75% confluency prior to studies.The primary cultures were maintained in a humidified incubator . Radiolabeled cDNA was separated from the free nucleotides using Bio-Spin 6 chromatography column (Bio-Rad Laboratories). Gene filters were labeled as adhesion or normal fibroblasts and washed with 0.5% sodium dodecyl sulfate (SDS) prior to prehybridization. Individual membrane was transferred to separate roller tubes of the hybridization oven (Fisher Scientific, Inc., Pittsburg, PA), each containing MicroHyb hybridization solution (Research Genetics) supplemented with Human Cot-1 DNA (Life Technology) and Poly dA (Research Genetics). The membranes were rotated at 10 RPM and at 42°C for 2 h. Radio-labeled cDNA prepared from adhesion and normal fibroblasts total RNA was denatured by heating in a boiling water bath for 3 min. The denatured probes were then injected into the prehybridization solution containing respective membrane. The membranes were hybridized with respective probe for 18 h at 42°C. The hybridization solution was then replaced with washing solution (2 × SSC containing 1% SDS). The temperature of the oven was raised to 50°C and RPM of rotors was increased to 15. Membranes were washed for 20 minutes when washing solution was replaced with a batch of fresh and prewarmed (50°C) washing solution. Washing was continued for additional 20 min. A third wash was performed with 0.5 × SSC solution containing 1% SDS at 55°C for 15 minutes. Membranes with cDNA spots facing up were covered with Saran wrap and exposed to phosphor screen (Kodak) for overnight. The screen was scanned with a Phosphor Imager . After acquisition of signal intensities from the normal and adhesion fibroblasts of one patient, filters were stripped according to protocol and subjected to gene filter experiments with the RNA samples from a second patient and images were scanned. Tiff images obtained from normal and adhesion fibroblasts of two patients were analyzed using Pathway 4 software (Research Genetics) for the identification of differentially expressed genes between the normal and adhesion fibroblasts of each patient.Total RNA was isolated from monolayer of fibroblasts at 12 h of culture in serum free medium using Trizol reagent (Invitrogen Inc.). Human Gene Filters  containing 4325 known human cDNA spots were used for the identification of differentially expressed genes between adhesion and normal fibroblasts from human peritoneum. Method suggested by the manufacturer was strictly followed. In brief, 10 μg of total RNA from monolayer cultures of fibroblasts were subjected to cDNA synthesis in presence of 10 μl 2, 0.2 mM dNTP, 0.5 U Taq Polymerase  and 1 μM each of sense and antisense primers. Primer sequences were determined using Primer3 software from the Internet . The control primers (sense 5'-ggaggttcgaagacgatcag-3' and antisense 5'-cgctgagccagtcagtgtag-3') were expected to provide an amplicon of 509 bp from human 18S ribosomal subunit cDNA (gi: 337376). Accession numbers of genes of interests are provided in Table Steady-state levels of mRNA of selected genes that are known to have a role in cellular adhesion, proliferation, migration, apoptosis and demonstrating different expression levels between adhesion and normal fibroblasts in the gene filter experiments were verified further by a previously described semiquantitative RT-PCR method . Total R. Band intensities were plotted to determine the linearity of PCR reactions for the amplification of target transcripts. Target cDNA were amplified by PCR from normal and peritoneal fibroblasts at specific PCR cycle within its linear range of amplification. Total RNA samples from normal and adhesion fibroblasts of 4 patients  were used for the RT-PCR experiments. Optical densities obtained from amplicons of 4 patients  were used to derive mean ± standard error of mean values representing relative levels of each mRNA species in normal and adhesion fibroblasts.Initially cDNA of interests were amplified from normal peritoneal fibroblasts at different (25 to 35) PCR cycles. PCR products were subjected to agarose gel electrophoresis. Molecular weight marker  were also loaded in adjacent lanes. DNA in the gel were stained with 1:10,000 dilution of SYBR Green I dye (Molecular Probes, Inc., Eugene, OR) and photographed using a DC 120 Kodak digital camera (Eastman Kodak, Rochester, NY) for the verification of size and analysis of band intensity using Image J software 1  or hypoxia (2% Oxygen) for 24 h. Control plates were cultured for the same duration in absence of TGF-β1 or hypoxia. Total RNA was isolated from the control, TGF-β1 and hypoxia treated cells and subjected to RT-PCR reactions as described above to determine relative levels of 18S ribosomal subunit and gene specific transcripts in the control and treated cells. RT-PCR experiments were conducted twice with the normal peritoneal fibroblasts isolated from 3 patients to have six control, six TGF-β1 treated and six hypoxia exposed amplicons. This included normal fibroblasts from one new patient and two patients that were used exclusively for RT-PCR experiments for the confirmation of gene array data. Optical densities of amplicons from six control or treated cells per mRNA species were used to derive the mean ± standard error of mean values for comparison.Effects of TGF-β1 or hypoxic conditions on the steady state levels of specific gene transcripts in the normal peritoneal fibroblasts were also studied to examine the possibility of adhesion causing factors potentiating the gene expression pattern in the normal fibroblasts similar to adhesion fibroblasts. Normal peritoneal fibroblasts were cultured in six well culture plates (FALCON). When confluent, monolayer of cells in culture were exposed to 1 ng/ml TGF-βBand intensity value of each RT-PCR experiment  was used to derive Mean ± Standard error of Mean using Statview 4.5 software (Abacus Concepts, Berkley, CA). Differences between Means were tested for significance by one-way analysis of variance with the specific post hoc test using the same software to compare differences in the steady state levels of different mRNA species.Hybridization intensities of radio labeled cDNA from normal or adhesion fibroblasts from both the patients were different when analyzed using Pathway software. Comparison of hybridization intensities from individual gene spots between normal and adhesion fibroblast RNA Figure  demonstrGene filter data from two patients showed similar expression pattern of collagen type 1 alpha 2), Collagen type III , fibronectin 1, Matrix metalloproteinase-1 (MMP-1), Transforming Growth Factor beta-1 (TGF-β1), TGF-β2 and tissue plasminogen activator as reported earlier using multiplex PCR technique  interacts with laminin1 P1, collagen I, collagen IV, perlecan and fibulin-2 in the extracellular space and stabilizes the basement membrane. It also interacts with α6β1 and α3β1 integrin receptors on cells . RelativTM4SF molecules (tetraspanins) play important roles in cell migration and in the generation of complexes with integrins functionally relevant for cell motility, tumor progression and wound healing . It is pVersicans (CSPG2) are also known to influence α4β1 and α2β1 integrin mediated invasion of melanoma cells . Higher Because MT-1e transcripts are detected in cell types that have undergone myoepithelial differentiation , signifiContrary to increase in the above mentioned mRNA species in the adhesion fibroblasts, steady state levels of IGFBP3 precursor transcript were found to be lower. Because IGFBP-3 is known to inhibit cell growth by sequestering IGF, its decreased level may enhance proliferation of adhesion fibroblasts . ReducedOur attempts to examine the regulatory roles of TGF-β1 and hypoxia, factors known to promote adhesion development , on the 2 (PGE2) is shown to inhibit TGF-β1 induced expression of α-SMA, production of Collagen I and the transformation of fibroblasts to myofibroblasts via EP2 signaling [P311 (PTZ17) and Interferon γ treatments, which inhibits TGF-β1 induced myofibroblast transformation [It is known that a new phenotype of fibroblasts is induced during wound healing. These fibroblasts, termed- myofibroblasts, express higher levels of α-smooth muscle actin and vinculin-containing fibronexus adhesion complexes . Fibroblignaling . Additioormation ,47.During the course of normal wound healing, myofibroblasts disappear, possibly by apoptosis . In cont2, EP2 blockers, interferon γ, P311 and applying measures to induce apoptosis in the peritoneal fibroblast at the site of injury. The obvious question – "how to maintain apoptosis at a desired level for normal peritoneal healing?" however, remains to be answered.It is evident from our study that steady state levels of several genes are different between adhesion and normal peritoneal fibroblasts in human and that adhesion development may be a function of several genes. Changes in the functional interdependence of these genes at the site of injury may transform normal peritoneal fibroblast into cell type/s with altered phenotype. These cells- designated as adhesion fibroblasts may mimic previously known myofibroblasts and are highly proliferative. These cells resist apoptosis and secrete ECM molecules to renovate basement membrane. With changed expression pattern of cell surface molecules these cells may respond to intracellular signaling for migration over the fibrin clot. This altered nature of adhesion fibroblasts therefore may play a major role in the formation of the fibrous mass of adhesion-tissue that bridges adjacent and injured peritoneum. Blocking changes in the expression or function of genes necessary for this transformation of normal peritoneal fibroblasts may curtail adhesion formation. This could be achieved by the application of PGECollagen type IV  chain (COL4A2), Nidogen 2 (NID2), Chondroitin sulfate proteoglycan 2 (CSPG2), S100 Calcium binding protein A10 (S100A10), Transmembrane 4 superfamily member 1 (TM4SF1), Metallothioneine (hMT-Ie), Insulin-like growth factor binding protein 3 precursor (IGFBP3), Transforming growth factor (TGF), Prostaglandin E2 (PGE2), Urokinase Plasminogen activator receptor (uPAR), Annexin 2 and S100A10 complex (AIIt), tissue Plasminogen Activator (tPA), Plasminogen Activator Inhibitor (PAI), Cyclooxigenase (COX), Matrix metalloproteinase (MMP), Tissue Inhibitor of Metalloproteinase (TIMP), Interferon γ (IFN-γ), IL (Interleukin).GMS and MPD were responsible for the isolation of peritoneal fibroblasts from normal peritoneum and adhesion tissues as well as establishing hypoxia chambers. MPD provided patient information and valuable suggestions during writing the manuscript. UKR performed microarray and semiquantitative RT-PCR experiments, analyzed the data and wrote the manuscript.


Histone acetyltransferases can be classified into two distinct groups (type A and B) on the basis of cellular localization and substrate specificity. Type B histone acetyltransferases, originally defined as cytoplasmic enzymes that acetylate free histones, have been proposed to play a role in the assembly of chromatin through the acetylation of newly synthesized histones H3 and H4. To date, the only type B histone acetyltransferase activities identified are specific for histone H4.The acetylation of the core histone NHTo better understand the role of histone acetylation in the assembly of chromatin structure, we have identified additional type B histone acetyltransferase activities specific for histone H3. One such activity, termed HatB3.1, acetylated histone H3 with a strong preference for free histones relative to chromatin substrates. Deletion of the GCN5 and ADA3 genes resulted in the loss of HatB3.1 activity while deletion of ADA2 had no effect. In addition, Gcn5p and Ada3p co-fractionated with partially purified HatB3.1 activity while Ada2p did not.Yeast extracts contain several histone acetyltransferase activities that show a strong preference for free histone H3. One such activity, termed HatB3.1, appears to be a novel Gcn5p-containing complex which does not depend on the presence of Ada2p. Histones H3 and H4 are among the most evolutionarily conserved proteins (>90% identity from yeast→humans) . Octamer2-terminal tails of the histones. These domains, which protrude from the core of the nucleosome, are free to interact with, and be acted upon by, the nuclear environment. The past several years has seen the identification of numerous enzymes that are capable of modifying the histones. These enzymes are generally found in large, multi-subunit complexes and have activities that are not only specific for a given histone but are specific for particular amino acid residues within the histone [The physical characteristics of the histones are precisely regulated in the cell by an elaborate network of post-translational modifications that include acetylation, methylation, phosphorylation, ubiquitination and ADP-ribosylation -4. These histone ,6.2-terminal tails. Historically, these enzymes have been classified as either type A or type B, based upon substrate specificity and cellular localization [The most well characterized histone modifying enzymes are the histone acetyltransferases (HATs). HATs catalyze the transfer of an acetyl moiety from acetyl-coenzyme A to the ε-amino group of lysine residues in the histone NHlization . Found ilization -22. Theslization ,24.de novo assembly of chromatin is a complex, multi-step process that occurs most prominently during DNA replication  [2-terminal tail domains [Type B HATs were initially described as cytoplasmic enzymes that acetylate free histones in conjunction with chromatin assembly . The de nthesis) ,26. Foll domains . For new domains ,29. For  domains ,30,31. T domains . Followi domains ,34.In contrast to the type A HATs, only one type B HAT has been characterized to date, Hat1p. Hat1p is an evolutionarily conserved enzyme that specifically acetylates free histone H4 -38. Cons2-terminal tail are viable, concomitant deletion of both NH2-termini  results in a defect in nucleosome assembly and cell death [HAT1 gene produces no observable phenotype, combining a deletion of HAT1 with specific lys→arg mutations in the NH2-terminus of histone H3 generates defects in both telomeric silencing and DNA damage repair [Evidence has accumulated indicating that the acetylation of newly synthesized histones H3 and H4 play over-lapping roles in chromatin assembly. While yeast strains carrying a deletion of either the H3 or H4 NHll death ,44. In ae repair ,46. HoweTo identify potential histone H3-specific type B HATs, we have systematically surveyed yeast extracts for candidate activities. Here we detail one such activity, termed HatB3.1. We provide evidence that this is a novel complex that utilizes Gcn5p as its catalytic subunit. Intriguingly, unlike previously identified Gcn5p-containing HAT complexes, HatB3.1 contains Ada3p, but not Ada2p.The highly selective activity of the native Hat1p/Hat2p complex for free versus nucleosomal histone H4 is the primary characteristic that distinguishes this enzyme from the type A histone acetyltransferases ,36. Ther3H-acetyl Coenzyme A and equivalent amounts of either free histones or chromatin as substrate. Histones were then resolved by SDS-PAGE and acetylated species visualized by fluorography.It is difficult to reliably detect histone acetyltransferase activities in the relatively crude cytosolic and nuclear extracts. Therefore, to evaluate the intrinsic HAT activities present in each of the extracts, they were fractionated by anion and cation exchange chromatography. Fractions were assayed for HAT activity using Fractionation of the cytosolic extract on a DEAE column is shown Figure The cytosolic extract also contained at least two additional HAT activities. The first showed a clear peak that was centered on fraction 14 and acetylated free histones H3, H2B and H4. The activity of this HAT on chromatin was more difficult to determine as the H3 and H4 labeling seen in these fractions does not show a marked peak in fraction 14 and may be due to the leading edge of a HAT activity eluting at higher salt. Therefore, this activity may be a candidate type B HAT. There was also a distinct peak of HAT activity at fractions 18–20. With free histone substrates, this activity primarily acetylated histone H3. However, there was also a coincident peak of chromatin H3 and H4 acetylating activity in these fractions suggesting this activity is likely to be a type A HAT.DEAE fractionation of the nuclear extract also revealed several distinct HAT activities Figure . There wUnbound material from the initial DEAE fractionation of the cytosolic and nuclear extracts was analyzed by cation exchange chromatography (carboxymethyl sepharose (CM)). While this fraction from the cytosolic extract appeared inactive, there were several additional HAT activities resolved from the nuclear extract  were grown and protein extracts prepared exactly as for the wild type strain. Nuclear extracts were again fractionated via DEAE column chromatography and fractions of equivalent conductivity assayed for HAT activity as described above.To gain insight into the identity of the catalytic subunit of HatB3.1, we constructed null mutants for each of the yeast HAT's that have demonstrated histone H3 activity as well as the known type B HAT, sas2 and Δsas3 strains, the robust H3 acetylation attributed to the HatB3.1 activity appeared unaffected by deletion of these enzymes  activities in this region of the gradient confirmed that HatB3.1 was a chromatographically distinct HAT activity exhibiting specificity for free histone H3.Parallel comparison of the HAT activity profiles from each strain provided biochemical evidence for the dependency of specific histone acetyltransferase activities on the presence of a particular HAT catalytic subunit . As with the Hat1p-dependent activity in cytosolic extracts, this activity also appeared to be specific for free histone H4. This result confirmed previous observations indicating that Hat1p is localized to both the cytoplasm and the nucleus [Analysis of the activity profile from nuclear extracts derived from a Δ nucleus ,42. In aada2 and Δada3 strains and the status of the HatB3.1 activity determined by DEAE chromatography. As shown in Figure ADA2 did not affect either the HatB3.1 or HatB3.2 activity but did cause a substantial increase in the free histone H4 specific activity that eluted late in the DEAE gradient. However, the HAT activity profile of the Δada3 extracts was strikingly similar to that seen for the gcn5 extracts with both the HatB3.1 and HatB3.2 activities absent. These results indicated that the HatB3.1 activity was dependent on ADA3 and that Ada2p is either not a component of the HatB3.1 activity or is not required for its stability.There are two proteins, Ada2p and Ada3p, that are components of all known Gcn5p-containing HAT complexes and that are required for the activity of these complexes -11,13,14To further characterize HatB3.1, this activity was purified through several chromatographic steps. The purification scheme is diagramed in Figure The proteins that flowed through the CM sepharose column were applied to a Mono-Q column and then eluted with a linear salt gradient. Fractions containing free histone H3 activity were pooled and concentrated by precipitation with 75% ammonium sulfate. The sample was then fractionated by size exclusion chromatography using a Superose 6 column. As seen in Figure The absence of HatB3.1 activity in extracts from a Δgcn5 strain indicated that HatB3.1 was dependent on Gcn5p, either indirectly via Gcn5p-mediated transcriptional regulation, or directly, as its catalytic subunit. While the HatB3.1 activity was highly purified relative to the initial nuclear extract, the peak Superose 6 fractions were still too complex to allow the definitive identification of specific bands that co-purified with the activity Figure . ExtensiDuplicate blots were probed with anti-Ada2p and anti-Ada3p antibodies to determine whether these proteins also co-fractionated with the HatB3.1 complex. As expected, both Ada2p and Ada3p are present in the nuclear extracts Figure . Howeverde novo assembly of chromatin [Considerable genetic and biochemical evidence indicates that, in most organisms, newly synthesized histone H3 is acetylated and that this acetylation plays a role in the hromatin ,31,43-482+-NTA agarose fractionated whole cell extracts, as these enzymes fortuitously bind to this resin [There have been at least a dozen distinct HAT complexes identified in yeast -22,36,42is resin ,14,17,22Most histone acetyltransferases have substrate specificities that direct the acetylation of specific residues within one or more of the core histones . Howeversas2 strain, there is a dramatic increase in an activity present in nuclear extracts that acetylates free histone H4 and which elutes from a DEAE column at a salt concentration similar to that of the nuclear form of Hat1p  ,14,31,62histones ,63-68. HSeveral properties of HatB3.1 indicate that it is distinct from previously identified Gcn5p-containing complexes. First, HatB3.1 is the only native Gcn5p-containing complex that does not have detectable activity on nucleosomal substrates. Second, the apparent molecular weight of HatB3.1 (~500 kDa), as determined by size exclusion chromatography, is much lower than that of the SAGA, ADA, SALSA and SLIK complexes but is similar to that reported for the A2 complex ,13,14,64gcn5 Δsas3 mutants, deletions of the GCN5, ADA2 and ADA3 genes have similar in vivo consequences [gcn5 and Δada3 mutants may be the result of the complex functional redundancies observed in the assembly of chromatin. For example, Δhat1 and Δhat2 mutants only display phenotypes when combined with mutations in multiple lysine residues in the histone H3 NH2-terminal tail [Ada2p, Ada3p and Gcn5p form a module that provides the catalytic activity to their associated type A HAT complexes ,67,69,70equences ,59,71,72nal tail ,46. UncoGCN5 and ADA3 but that is independent of ADA2.In conclusion, we have fractionated yeast cytoplasmic and nuclear extracts and resolved several putative histone H3-specific type B histone acetyltransferase activities. One of these activities, HatB3.1, is highly specific for histone H3 that is free in solution. A combination of genetic and biochemical evidence indicates that HatB3.1 is a novel complex that depends on GCN5, SAS3, SAS2, ADA2, ADA3 and HAT1 were constructed using PCR-mediated gene disruption with the HIS3 reporter gene [UCC1111 was used as the wild type yeast strain that serves as the genetic background for all deletion strains . Null muter gene .2, 10 mM HEPES [pH 6.0]). Supernatant from a 1500 × g, 15' spin at 4°C was retained as a cytosolic extract. Pelleted material was washed once with Buffer A then resuspended in DN(1000) (DN buffers contain 25 mM Tris [pH 7.5], 10% glycerol, 0.1 mM EDTA and mM [NaCl] listed in parentheses). Supernatant from another 1500 × g spin as above yielded the nuclear extract. This extract was dialyzed O/N at 4°C into DN(0) to a conductivity similar to that of the cytosolic extract. Extracts were cleared by high speed centrifugation (~30,000 × g) prior to their chromatographic fractionation.Cells were grown to mid-log phase in 1% yeast extract, 2% peptone, 2% glucose and 50 μg/mL ampicillin at 30°C. Cells were harvested at 4000 × g, 10', 4°C and total grams of cells recorded. All buffers contain 1.0 mM PMSF. Spheroplasts were prepared essentially as described previously using 0.25 mg of Zymolyase  per gram of cells for spheroplasting . Spheroppurifier – Pharmacia) was employed for all column runs.All columns were equilibrated with and run using DN Buffers. HPLC (ÄKTA DEAE – Cleared extracts were loaded onto a HiPrep 16/10 DEAE FF column (Pharmacia). Following a 5 C.V. wash with DN(50), proteins were eluted with a linear, 20 C.V., salt gradient from 50 mM to 1.0 M NaCl. A flow rate of 1.0 mL/min. was used and 3.0 mL fractions were collected.CM – Either pooled peak fractions, dialyzed into DN(0) until at similar conductivity as DN(50) start buffer, or Flowthrough from the DEAE were loaded onto a HiPrep 16/10 CM FF column (Pharmacia). The column was washed and proteins eluted as described above.Mono Q – The flowthrough fraction from the CM column was loaded onto a Mono Q HR 5/5 column (Pharmacia). Following a 5 C.V. wash with DN(50), a 20 C.V., linear, salt gradient was employed as above and 0.5 mL fractions were collected.4)2SO4 (0.516 g/mL) over 30' at 4°C. Following an additional 30' equilibration period at 4°C, precipitated protein was pelleted (10,000 × g, 10', 4°C) and resuspended in 300 μL cold, DN(0).Peak fractions of HatB3.1 activity from the Mono Q column were pooled and brought to 75% . The column was equilibrated with and run in DN(350) at a flow rate of 0.3 mL/min. and 0.25 mL fractions were collected. Molecular weight standards (Sigma, MW-GF-1000) were run using the same parameters and 24 μL aliquots of every other fraction run on a 10% SDS-polyacrylamide gel. The elution profile of the MW standards was determined by protein visualization via Coomassie blue staining.3H-Acetyl Coenzyme A (5.50 Ci/mmol, Pharmacia) and ~1.0 mg/mL core histones or chromatin in a final volume of 100 μL at 1X [DN(75)]. 50 μL of each reaction was analyzed for HAT activity via liquid scintillation counting. The remaining assay mixture was brought to 1X [SDS Load Dye] to stop the reaction. In general, aliquots (24 μL) of these remaining assay mixtures were run on 18% SDS-polyacrylamide gels to resolve the histones. Gels were incubated in Autofluor , dried down and acetylated histone species visualized via fluorography.Chicken erythrocyte core histones and chromatin were isolated as previously described ,76. TypiSuperose 6 fractions exhibiting HAT B3 activity, as determined above, were run on 10% SDS-polyacrylamide gels and proteins were either visualized by silver staining or transferred to nitrocellulose using a semi-dry transfer apparatus (Biorad). Blots were processed following standard procedures. Goat, polyclonal antibodies against Gcn5p, Ada2p and Ada3p (Santa Cruz Biotechnology, Inc.) were used at 1:100 dilutions in 5% Milk/TBS-T. Donkey, HRP-labeled Anti-Goat IgG secondary antibody (Santa Cruz Biotechnology, Inc.) was used at 1:2500 dilution followed by detection with ECL+Plus (Pharmacia) and visualization via phosphoimager (STORM 860, Pharmacia).A.R.S. performed all of the experiments presented here and drafted the manuscript. M.R.P. directed the project and edited the manuscript.


The rapid progress in the field of genomics is increasing our knowledge of multi-gene diseases. However, any realistic hope of gene therapy treatment for those diseases needs first to address the problem of co-ordinately co-expressing several transgenes. Currently, the use of internal ribosomal entry sites (IRESs) is the strategy chosen by many researchers to ensure co-expression. The large sizes of the IRESs (~0.5 kb), and the difficulties of ensuring a well-balanced co-expression, have prompted several researchers to imitate a co-expression strategy used by many viruses: to express several proteins as a polyprotein. A small peptide of 18 amino acids (2A) from the foot-and-mouth disease virus (FMDV) is being used to avoid the need of proteinases to process the polyprotein. FMDV 2A is introduced as a linker between two proteins to allow autonomous intra-ribosomal self-processing of polyproteins. Recent reports have shown that this sequence is compatible with different sub-cellular targeting signals and can be used to co-express up to four proteins from a single retroviral vector. This short peptide provides a tool to allow the co-expression of multiple proteins from a single vector, a useful technology for those working with heteromultimeric proteins, biochemical pathways or combined/synergistic phenomena. For the last 20 years, the gene therapy field has centred many of its efforts on finding ways to deliver a therapeutic gene to certain target cells in order to produce a therapeutic result. It was soon clear that it was necessary to deliver at least two genes, because a reporter/marker gene was needed in order to track the expression of the therapeutic gene . There has been a large increase in vector development during these years, with the appearance of many new viral and non-viral vectors. However, since the late 1980s, few improvements have been made 'inside' those vectors. The linkage of the two genes of interest (therapeutic and reporter) has remained the same. The different strategies known for co-expression were reported during the 1980s -splicing, multiple promoters, fusions, reinitiation and IRESs-, at the same time that the first gene therapy experiments were being performed . Not surprisingly, this strategy is indeed used by cells, although not very often, in particular for the co-ordinated secretion of different proteins and peptides. Recently, several groups have been trying to utilize this co-expression strategy. One of the possibilities is to introduce the target site for a cellular proteinase between two cistrons cloned in frame forming a single open reading frame . In thicis-acting hydrolase element). From a biotechnological standpoint, all that is needed is to clone the coding sequence of 2A, followed by the codon for the first amino acid of the next FMDV protein (2B), in frame between the two genes one wishes to co-express. The synthesis of the peptide bond between the last amino acid (Gly) of 2A and the first (Pro) of 2B is skipped, producing an upstream protein with a C-terminal tail of 18aa (2A) and a downstream protein with a Pro at the N-terminus. The extra sequences have minimal effect on the activity of most proteins and none on their stability. In fact, the 2A peptide has been used as an efficient tag for immunoprecipitation and Western blotting, although commercial antibodies are not yet available. Interestingly, additional CHYSEL sequences have been found in viruses other than FMDV  underwent a rapid co-translational self-processing. It was soon realised that the key was a small 18aa peptide (2A) that directed its own separation from the growing polyprotein. During the last decade, this mechanism has been studied in detail, resulting in a simple model: the small 2A peptide, during its translation, interacts with the exit tunnel of the ribosome to induce the "skipping" of the last peptide bond at the C-terminus of 2A. The crucial point is that the ribosome is able to continue translating the downstream gene, after releasing the first protein fused in its C-terminus to 2A (reviewed in ). This tces, see ).Herpes simplex virus-1 thymidine kinase, HSV1TK; interleukin-12, IL-12; viral antigens, etc.) in transient transduced or stable cells lines and in animals. A full list of publications using 2A is available on the web [The initial publications using this strategy have shown that 2A skipping can be used in the typical viral vectors used for gene therapy (retrovirus and adeno-associated virus) to reliably co-express many reporter proteins  and therapeutic proteins  ,9. Not oam genes ,9.The CHYSEL strategy of co-expression is also compatible with the most disparate sub-cellular localisations -10. ProtThe results reported in reference  should bΔP/ΔP × CD3ζ -/- mice  were transplanted with bone marrow from wt C57BL/6 mice or CD3εΔP/ΔP × CD3ζ -/- mice transduced with a retrovirus encoding the four CD3 subunits, and in both cases TCR surface expression was detected and the T cells proliferated normally after immune stimulation. Bone marrow from CD3εΔP/ΔP × CD3ζ -/- mice without CD3 transduction did not restore T-cell development. T cells were also reconstituted in sub-lethally irradiated RAG-1-/- mice (lacking mature T and B lymphocytes) in which bone marrow from CD3εΔP/ΔP mice (lacking CDε and with a severe inhibition of CD3γ and CD3δ), transduced with a retrovirus encoding these three subunits (via two 2A sequences), was used for a transplant into the RAG-1-/- mice. The same experiment using three vectors encoding the CD3 subunits separately was unsuccessful.Lethally irradiated CD3εThe development of FMDV 2A as a cloning tool is an example of how dangerous pathogenic viruses can be harnessed by biotechnology for human benefit. Their molecular "tricks" (as IRES or CHYSEL sequences) are gradually becoming part of the biotechnologists' toolbox. The development of the polycistronic vectors here discussed is a big step forward, a decade and a half after the launching of the very first gene therapy trial with the aim of introducing in blood cells just a single therapeutic gene, adenosine deaminase (ADA), and the NEO marker . These rNone declared.


Functional decline threatens independent living and is common among individuals diagnosed with cancer, especially those who are elderly. The purpose of this study was to explore whether dietary and exercise practices are associated with physical function status among older cancer survivors.Mailed surveys were used to ascertain data on physical function, dietary fat, fruit and vegetable (F&V) consumption, and exercise among elderly diagnosed with early stage (I-II) breast (N = 286) or prostate cancer (N = 402) within the past 18 months.Sixty-one percent of respondents reported diets with <30% of energy from fat, 20.4% reported F&V intakes of 5+ daily servings, and 44.6% reported regular vigorous exercise. Significant, independent associations were found between physical functioning and reported dietary fat intake, F&V consumption, and exercise. A simultaneous multiple regression model controlled for age, race, gender, time since diagnosis and concurrent health behaviors yielded the following estimates: (1) 0.2 increase in the SF-36 physical function subscale (PFS) score with each reported 1% decrease in percent energy from fat (p < .0001); (2) 0.9 increase in the SF-36 PFS score for each reported serving of F&V/day (p = .0049); and (3) 15.4 increase in the SF-36 PFS score with a positive response for regular vigorous exercise (p < .0001).Results of this cross-sectional survey suggest that regular vigorous exercise and consumption of diets low in fat and rich in F&Vs are associated with higher levels of physical functioning among older cancer survivors. Interventions that promote healthful lifestyle change may deliver considerable benefit within this ever increasing and vulnerable population. There are 9.6 M cancer survivors in the US today, and 61% are age 65 or older . UnfortuWe explored associations between lifestyle factors and physical functioning among elderly cancer survivors who were screened for Project LEAD (Leading the Way in Exercise And Diet), a home-based, diet and exercise intervention trial . BrieflyT-tests and chi-square analyses were performed to determine if respondents differed from non-respondents on age, time since diagnosis, race and gender, and to test for differences between male and female respondents. Pearson correlation coefficients indicated associations between F&V intake and percent dietary fat, and physical function scores. T-tests were used to explore associations between exercise and function. Linear regression analyses permitted an examination of associations between physical function and health behaviors controlling for several likely confounders including gender, age, race, time since diagnosis and concurrent health behaviors. Given the homogeneity in stage at diagnosis, stage information was omitted from this analysis.Between August 2000 and May 2003, 2,431 cases were identified and physician approval for contact was granted for 2,034 cases. Incomplete address information existed for 24 of these cases, yielding 2,010 viable posted surveys, of which 688 complete surveys were returned (34% response rate). Characteristics of respondents and non-respondents are provided in Table Data regarding physical function and lifestyle behaviors are provided in Table Modest correlations were obtained between the indicators of exercise and diet, with positive agreement noted between F&V intake and exercise (Pearson ρ = .12/p = .003) as well as F&V intake and dietary fat (ρ = .42/p < .0001). An inverse association was noted between dietary fat and exercise (ρ = -.08/p = .05). In bivariate associations, all three behaviors were significantly associated with SF-36 physical functioning scores (p < .05): F&V intake (ρ = .09), dietary fat (ρ = -.10), and vigorous exercise (ρ = .37). In multivariable linear regression analyses, with the SF-36 PFS score serving as the dependent variable and controlling for age, race, gender, time since diagnosis, and concurrent health behaviors, the simultaneous associations of the three indicators remained statistically significant (independent) and yielded the following estimates: (1) a 0.2 increase in the SF-36 PFS score with each reported 1% decrease in percent energy from fat (p < .0001); (2) a 0.9 increase in the SF-36 PFS score for each reported serving of F&V/day (p = .0049); and (3) a 15.4 increase in the SF-36 PFS score with a positive response for regular vigorous exercise (p < .0001).In comparing these cross-sectional data on lifestyle behaviors of older cancer survivors to normative data reported on healthy elders, as well as to previous data reported on general populations of breast and prostate cancer survivors (where a dearth of data have been reported on older cancer survivors per se), we find both differences and similarities. Like data that exist on healthy elders (age 65+) responding to the 2000 Behavioral Risk Factor Surveillance System (BRFSS) survey , or findA potential explanation for the higher physical function scores exhibited within our sample may relate to the higher prevalence of healthful lifestyle practices which in turn may increase physical function. This links back to the primary thrust of this study, which was to determine evidence for a link between functional status and health behavior.Our results suggest that regular vigorous exercise and a diet rich in F&Vs and low in fat is associated with higher levels of physical functioning among elderly recently diagnosed with cancer. Although the association between exercise and improved function has been reported repeatedly in other studies among elders ,23, thisFurthermore, only one study to date has reported the link between diet composition and physical function. Ortega et al. found that diets low in fat and high in F&Vs were associated with higher levels of physical functioning among a sample of upper socio-economic, elderly male Spaniards at risk for cardiovascular disease . This prThe primary limitations of this study relate to respondent bias and cross-sectional design. Our response rate of 34% is indeed less than the 59% we have achieved in previous mailed surveys among similar populations . Given tFindings of this study support the recent American Cancer Society diet and exercise guidelines for survivors , which cF&V: Fruits and VegetablesSF-36: Short Form-36 Health SurveyPFS: Physical Function SubscaleThe author(s) declare that they have no competing interests.WDW, ECC, MCM, CFP and HJC conceived of the study, and participated in its design. DCS participation in the coordination of the study, as well as data cleaning and interpretation of findings. RS performed the statistical analysis. All authors read and approved the final manuscript.


Several previous epidemiological studies have shown a relation between drinking water quality and death in cardiovascular disease whereas others have not found such a relationship. An intervention study was undertaken to evaluate the effect of water with added magnesium and natural mineral water on blood pressure.A group of 70 subjects with borderline hypertension was recruited and consumed 1) a water low in minerals, 2) magnesium enriched water or 3) natural mineral water, in a random, double blind fashion during four weeks.Among persons with an initial low excretion of magnesium or calcium in the urine, the urinary excretion of magnesium was increased in the groups consuming the two waters containing magnesium after 4 weeks. A significant decrease in blood pressure was found in the group consuming mineral water at 2 and 4 weeks.The results suggest that minerals taken in water are significant for the body burden and that an intake of mineral water among persons with a low urinary excretion of magnesium or calcium may decrease the blood pressure. Further studies should investigate the extent of mineral deficiency in different populations and the efficiency of different vehicles for supplying minerals, particularly magnesium and calcium. A relation between mortality from ischaemic heart disease (IHD) and drinking water characteristics was first shown in Japan in 1957  and reviIn a case-control study, an inverse relation was found between the amount of magnesium in drinking water and death from acute myocardial infarction and for females also between the amount of calcium and death . Diets rRegarding individual minerals, several studies have been reported where hypertensive subjects were treated orally with nutritional doses of magnesium . The resEpidemiological studies on cardiovascular disease suggest that drinking water is an important vehicle for the supply of minerals . This isThe present intervention study was undertaken to determine the effect of minerals in water on one of the major risk factors for cardiovascular disease – blood pressure. Subjects with slightly elevated blood pressure consumed water with different levels of minerals. Serum and urinary levels of minerals were measured as a marker of intervention and blood pressure was measured before and after the intervention.Female and male subjects, aged 45 – 64 years (n = 70) were recruited by advertising in local newspapers. Inclusion criteria were living in an area with low magnesium content in the drinking water, systolic pressure 15 mm above normal values for their age, diastolic pressure above 90 mm Hg, and within 20% of ideal body weight. Exclusion criteria were hypertension target organ damage, chronic diseases (heart, liver, kidney, diabetes mellitus), pregnancy, and taking oral contraceptives or regular intake of mineral supplements. Subjects with a diastolic pressure above 100 mm Hg were advised to consult a physician for treatment. A few persons decided not to seek a physician's advice and choose to participate anyway. The Ethical committee at the Medical faculty, University of Gothenburg, approved the study.Blood pressure was measured using standardized techniques before the intervention, at 2 weeks and at the end at 4 weeks. Two separate recordings were made (diastolic pressure as Korothoff phase 5) after 5 minutes of supine rest. The blood pressure is reported as the average of these recordings.idem). Magnesium and calcium levels in urine were expressed as the creatinine ratio.Blood samples were taken before and after the intervention to determine the serum concentration of magnesium, calcium, sodium, creatinine and potassium . Before and after the intervention period, 24 hours urine samples were collected and the amounts of magnesium, calcium, and creatinine were determined . Table Table In spite of the random allocation to the different waters, it was found that the group consuming water C comprised a larger number of persons with a high initial systolic pressure. In the groups receiving waters A and B, none of the subjects had systolic blood pressures above 170 before the intervention. The subjects drinking water C were divided into those with an initial systolic pressure above and below 170 mm. For the group with the higher pressure (n = 6), there was a decrease in the systolic pressure before and at 4 weeks p = 0.023) but no difference at 2 weeks or for diastolic pressure. The results from the remainder of the group are also shown in Table  but no dThe study is of exploratory character, based on a relatively small number of subjects and should be interpreted with care. There is also a lack of some data that retrospectively would have been of interest such as sodium in the urine and the effect of water with only calcium added. We do not think, however, that this has any influence on the major conclusions from the study.The intervention with the two waters with added magnesium influenced the body burden in terms of an increased excretion of magnesium in urine. This is consistent with findings from previous intervention studies ,17 althoThe intervention with water containing high amounts of several minerals decreased the blood pressure significantly in contrast to water with magnesium only where no significant effect was detected. This does not exclude that an effect could have been found with the latter water, had the intervention time been longer. On the other hand, the finding supports the concept that interventions should be performed under conditions similar to the ones present in normal environments, rather than with one specific agent. This could also explain the lack of an effect in previous studies where single minerals have been given as reviewed in the introduction.In summary, the results suggest that waterborne minerals constitute a supply for the body burden, that the urinary excretion can be used as a physiologically relevant indicator of the body burden of magnesium and calcium, and that the supplementation of magnesium together with other minerals may reduce blood pressure among persons with a low body burden of magnesium and calcium, either due to an insufficient intake through food or water, or through some metabolic/clinical disturbance. Additional studies are needed to explore this further.The study was supported by an unconditional grant to Gothenburg University from the Nestlé Water Institute, Vittel, France. The authors had full freedom for data analysis, manuscript preparation and submittance to a journal. RR has not received any fees or salaries for this work, including article processing charges, nor does he own any shares or has any other financial interest in the company. MA was an employee at the Water Institute at the time of the study.RR and MA jointly developed the research plan. RR conducted the field study. RR and MA jointly analyzed the data and wrote the manuscript.The pre-publication history for this paper can be accessed here:


Chronic obstructive pulmonary disease and emphysema are a frequent result of long-term smoking, but the exact mechanisms, specifically which types of cells are associated with the lung destruction, are unclear.+ and CD8+ T lymphocytes that expressed chemokine receptors CCR5 and CXCR3 (both markers of T helper 1 cells), but not CCR3 or CCR4 (markers of T helper 2 cells). Lung lymphocytes in patients with chronic obstructive pulmonary disease and emphysema secrete more interferon gamma—often associated with T helper 1 cells—and interferon-inducible protein 10 and monokine induced by interferon, both of which bind to CXCR3 and are involved in attracting T helper 1 cells. In response to interferon-inducible protein 10 and monokine induced by interferon, but not interferon gamma, lung macrophages secreted macrophage metalloelastase , a potent elastin-degrading enzyme that causes tissue destruction and which has been linked to emphysema.We studied different subsets of lymphocytes taken from portions of human lungs removed surgically to find out which lymphocytes were the most frequent, which cell-surface markers these lymphocytes expressed, and whether the lymphocytes secreted any specific factors that could be associated with disease. We found that loss of lung function in patients with chronic obstructive pulmonary disease and emphysema was associated with a high percentage of CD4These data suggest that Th1 lymphoctytes in the lungs of people with smoking-related damage drive progression of emphysema through CXCR3 ligands, interferon-inducible protein 10, and monokine induced by interferon. The underlying changes that cause smoking-related damage are unclear. This paper suggests that there is a skew towards increased T helper 1 lymphocytes, which may drive progression of this damage A rho smoke ,9,10,11.ho smoke . Furtherho smoke . It has ho smoke .Chemokines, their receptors, and cell adhesion molecules regulate migration of immune cells into inflamed tissue ,16,17,18In this study we determined the dominant T helper phenotype in lung samples from ex-smoker individuals with moderate to severe COPD and emphysema and control individuals with no evidence of smoking-related lung disease. Analysis of chemokine receptor expression on isolated peripheral lung lymphocytes from ex-smokers with COPD/emphysema indicated that both CD4 and CD8 T helper cells are strongly polarized to the Th1 phenotype compared to T cells isolated from lung tissue of normal individuals or individuals with non-smoking-related obstructive lung disease. The same cells spontaneously secreted more IFN-γ and CXCR3 receptor ligands MIG and IP-10 in the COPD and emphysema group than in the group without emphysema. Further, IP-10 and MIG, but not IFN-γ, upregulated macrophage metalloelastase  from isolated lung macrophages. Together, our findings reveal the strong association between COPD/emphysema- and Th1-driven adaptive immunity, suggesting a link to lung destruction mediated by IFN-γ, MIG, and IP-10.Twenty-eight non-atopic ex-smoker individuals see  undergoiAll participants were recruited from the surgical clinic at the Michael E. DeBakey Veterans Affairs Medical Center and the Methodist Hospital, and were undergoing lung resection for diagnostic or therapeutic purposes . Study pHigh-resolution CT (two in emphysema group and two in control group) or conventional CT analysis was used to detect emphysema, characterized by the presence of areas of low attenuation contrasted with surrounding normal lung parenchyma ,33. CT s7 cells for future analysis.Lung lymphocytes were isolated by modifying established protocols, using a combination of mechanical fragmentation, enzyme digestion, and centrifugation procedures described previously ,36,37. VThe following monoclonal antibodies were purchased from BD Biosciences Pharmingen : FITC-, Cy5-, and PE-conjugated anti-CD4, -CD8, -CD3, -CD14, -CD69, -CXCR3, -CCR3, -CCR4, and -CCR5. For enzyme-linked immunosorbent assay studies, anti-human antibodies to IFN-γ, IL-4, IP-10, MIG, I-TAC, and the appropriate secondary reagents were purchased from R&D Systems (Minneapolis, Minnesota, United States).7 cells/ml, and 50 μl of cells was incubated with antibodies to CD3 and CD4 or CD8.Phenotypic characterization of T cells was done by two-color flow cytometry  using combinations of the following monclonal antibodies: FITC-conjugated anti-CD4, -CD8, and -CD14; PE- and Cy5-conjugated anti-CCR4, -CCR3, -CCR5, and -CXCR3. Freshly isolated lung lymphocytes were resuspended to 1 × 10Lung lymphocytes were cultured in the presence or absence of phorbol myristate acetate (PMA)/ionomycin and brefeldin A for 12 h. Cells were harvested, fixed with formaldehyde, permeabilized with saponin, and intracellularly labeled for IFN-γ and IL-4, in addition to staining for surface CD69, CD4, and CD8 according to the manufacturer's recommendations (Fastimmune, BD Biosciences Pharmingen).Lung lymphocytes were isolated from surgical tissue and cultured in vitro in triplicate for 4 d. Supernatants were collected and stored at –80 °C for future analysis. Standard antibody-based enzyme-linked immunosorbent assay was used to measure supernatant concentrations of IP-10, MIG, IL-4, and IFN-γ according to the manufacturer's instructions (R&D Systems and BD Biosciences Pharmingen).Peripheral blood mononuclear cells and lung macrophages were isolated by positive selection using immunomagnetic beads conjugated with anti-CD14, and cultured in serum-free medium (RPMI, L-glutamine, and Pen/Strep) prior to overnight stimulation with 0, 50, 250, or 500 ng/ml of IFN-γ, IL-4, MIG, I-TAC, and IP-10. Supernatants were collected, and MMP12 was detected using anti-human MMP12 (R&D Systems) by Western blotting according to the manufacturer's instructions.+ lung macrophages stimulated overnight with rIP-10 (500 ng/ml) in the presence or absence of blocking anti-CXCR3 antibodies (5 μg/ml, R&D Systems). Two-step real-time reverse transcription PCR was used to determine the relative expression of mRNA using the ABI Perkin Elmer Prism 5700 Sequence Detection System  as described previously [Total cellular RNA was extracted from CD14eviously .Paraffin-embedded, and fresh-frozen lung sections (5 μm) were immunostained using monoclonal antibodies against human MMP12 (R&D Systems) or non-immune antisera by an immunoperoxidase protocol  and counterstained with hematoxylin as recommended by the manufacturer.p < 0.05 was considered statistically significant.The Mann-Whitney test (non-parametric, two-tailed) and Student's T test (two-tailed) were used to compare differences between the two groups of subjects. n = 10) and emphysema (n = 18) groups, and did not discriminate between these populations . Together, these data indicate that a strong type 1 bias is characteristic of the T cells isolated from the peripheral lung of participants with COPD and emphysema and that this immune phenotype correlates with the lung destruction that is characteristic of this disease. Further, we have shown for the first time, to our knowledge, that CXCR3 expression, a marker of Th1 inflammation, extends to lung monocytes and macrophages.Although human lung macrophages are not known to express CXCR3, we suspected based on the immunohistochemical localization of this chemokine receptor that CD14or CXCR3 . In addi+/CD8+ lung lymphocytes. Surface staining for CD4 was not feasible with this protocol; however, the percentage of CD8−/IFN-γ+ cells was approximately equal to that of CD8+/IFN-γ+ cells . This information confirmed our finding that the predominant Th1 bias in COPD/emphysema reflects the microenvironment unique to the lungs of ex-smoker individuals. The prevalence of asthma among people who smoke is currently not known, but in order to study COPD/emphysema in a population without other confounding variables, people who might have had asthma were excluded, and thus our findings are restricted to non-asthmatic individuals with emphysema.+ and CCR8+ T cells express IL-4, and CCR4 expression was prominent in people with severe atopic dermatitis, which decreased upon abatement of disease activity [+/CXCR3+ T cells in the airway epithelium and submucosa [+ T cells in emphysema patients and the functional interplay between Th1-related chemokines and elastolytic MMPs.Our use of T cell chemokine receptor expression analysis to determine recruitment of lung T cells is not without precedent. Analysis by immunohistochemistry of airway mucosa of people with atopic asthma after antigen challenge revealed that large numbers of CCR4activity ,43. Immuubmucosa . We exteIn addition to detailing surface chemokine receptor expression, we have functionally confirmed the marked Th1 bias of peripheral lung T cells, demonstrating that either at rest or following stimulation, these cells secrete IFN-γ and not IL-4. Our findings therefore confirm the utility of chemokine receptor expression patterns in the initial assessment of T cell effector phenotype.Destruction of lung parenchyma in emphysema is thought to occur through excessive proteolysis mediated by the elastin-degrading enzymes MMP2, MMP9, and MMP12 from the MMP family, and by neutrophil elastase from the serine proteinase family ,46. CytoAlthough this was an entirely human study, our findings show remarkable parallels with studies performed in mice. MMP12 deficiency has been shown to protect mice against emphysema after chronic exposure to cigarette smoke, implying that MMP12 may be the key proteinase in the development of emphysema in this species ,50. StudMany people develop long-term lung problems after smoking, including a condition called emphysema. At the very end of the airways are tiny air sacs. In healthy people, the air sacs stretch and relax easily on breathing in and out. But in people with emphysema, the air sacs fill up with air but can't empty out properly, so air gets trapped, making breathing difficult. While the symptoms of emphysema can be treated, there are no treatments that can reverse the damage to the lung.The researchers studied two groups of patients, all ex-smokers who had been admitted to a hospital to have part of their lung removed—some because of cancer, some for other reasons. The researchers studied the lung samples and looked to see exactly what type of immune cells the patients with emphysema had in their lungs and found that most of the immune cells were of one particular type. The researchers also showed that the immune cells could tell other lung cells to produce chemicals that can damage the lung.Lung damage in emphysema may not be caused directly by toxins in cigarette smoke. Instead, if you have emphysema, your body may react to the toxins and produce a special kind of immune cell that is key in causing the lung damage. So perhaps if doctors can find a way to change how this cell behaves, it might be possible to reduce or limit the lung damage. Obviously, not smoking, or stopping smoking, is the best way to prevent COPD or emphysema.The study is quite small, which means that the results may not be completely accurate; in particular, the study did not include detailed information from patients who had never smoked. So it is too soon to say for sure whether these special immune cells really are the link between smoking and lung damage in emphysema. Researchers will need to study many more patients with emphysema as well as people who have never smoked.Two places to start are the patient Web pages of the following professional organizations.http://www.yourlunghealth.org/diseases_conditions/copd/American Association for Respiratory Care: http://www.brit-thoracic.org.uk/public_content.asp?pageid=9&catid=21&subcatid=177The British Thoracic Society:


Osteosarcoma is a highly malignant bone neoplasm of children and young adults. It is characterized by extremely complex karyotypes and high frequency of chromosomal amplifications. Currently, only the histological response (degree of necrosis) to therapy represent gold standard for predicting the outcome in a patient with non-metastatic osteosarcoma at the time of definitive surgery. Patients with lower degree of necrosis have a higher risk of relapse and poor outcome even after chemotherapy and complete resection of the primary tumor. Therefore, a better understanding of the underlying molecular genetic events leading to tumor initiation and progression could result in the identification of potential diagnostic and therapeutic targets.We used a genome-wide screening method – array based comparative genomic hybridization (array-CGH) to identify DNA copy number changes in 48 patients with osteosarcoma. We applied fluorescence in situ hybridization (FISH) to validate some of amplified clones in this study.PRDM16), 6p21.1 (CDC5L, HSPCB, NFKBIE), 8q24, 12q14.3 (IFNG), 16p13 (MGRN1), and 17p11.2 (PMP22 MYCD, SOX1,ELAC27). We validated some of the amplified clones by FISH from 6p12-p21, 8q23-q24, and 17p11.2 amplicons. Homozygous deletions were noted for 32 clones and only 7 clones showed in more than one case. These 7 clones were mapped to 1q25.1 (4 cases), 3p14.1 (4 cases), 13q12.2 (2 cases), 4p15.1 (2 cases), 6q12 (2 cases), 6q12 (2 cases) and 6q16.3 (2 cases).Clones showing gains (79%) were more frequent than losses (66%). High-level amplifications and homozygous deletions constitute 28.6% and 3.8% of tumor genome respectively. High-level amplifications were present in 238 clones, of which about 37% of them showed recurrent amplification. Most frequently amplified clones were mapped to 1p36.32 (This study clearly demonstrates the utility of array CGH in defining high-resolution DNA copy number changes and refining amplifications. The resolution of array CGH technology combined with human genome database suggested the possible target genes present in the gained or lost clones. Osteosarcoma (OS) is a primary malignant tumor of bone arising from primitive bone-forming mesenchymal cells and it accounts for approximately 60% of malignant bone tumors in the first two decades of life . These tChromosomal aberrations in osteosarcoma are highly complex and characterized by high frequency of amplifications. These amplifications may result in the overexpression of genes and contribute to the genomic instability in osteosarcoma. The identification of genes within the amplified sites is crucial for understanding the biology and clinical behavior of osteosarcoma. Until, recently gene amplification has been detected by PCR, southern blot analysis or FISH-based approach using gene specific probes. These techniques are inherently restricted to the previously known amplified genes in the genome. In contrast, genome-wide screening of amplified chromosomal regions with CGH has become an important tool for the detection of amplified regions in the tumor genome. So far published chromosomal CGH studies in osteosarcoma have identified several high-level chromosomal amplifications at 1p22, 1p31, 1q21, 1q23, 2q24, 3p25, 3q26, 6q24.3, 4q12, 5p14-p15, 5q33, 6p12-p21, 6q24.3, 7p21-p22, 8q12-q23, 10p21, 10q11.1, 10q22, 11q13, 11q23, 12p13, 12q12-q15, 17p11.2, 17q21, 18q22, 19p13.1 and 20p11.2 [A total of 48 tumors from 42 patients  were collected from the Texas Children's Cancer Center, Houston, TX (tumors 193, 196, 204, 209, 221, 226, 248, 274, 295, 311, 326, 341, 345, 360, 400, 464, 481, 501, 527, 591 and 606) and Memorial Sloan Kettering Cancer Center, New York (tumors 06, 15, 24, 25, 27, 29, 32, 34, 40, 48, 68, 76, 78, 79, 80, 82, 83, 85, 88, 95, 98, 99, 102, 123, 423, 425, and 474). All tissues in this study were obtained after IRB approved informed consents were signed. The age at diagnosis ranged from 5 years to 71 years at diagnosis. The histological information of 42 patients is presented in Table The array used in this study consists of 967 human BACs, which were spaced approximately 3 megabase across the whole genome. These arrays were obtained from Spectral Genomics, Houston, TX. The experiments were performed according to the manufacturer's protocol. Arrays were pre-hybridized with human Cot-I DNA (GIBCO Invitrogen, Carlsbad, CA) and salmon testes DNA to block the repetitive sequences on BACs. One microgram of normal DNA (reference) and tumor DNA (test) was labeled with cy5-dUTP and cy3-dUTP respectively, by random priming. To avoid dye bias, we performed dye swap experiments for each sample. The probe mixture is dissolved in hybridization mixture, denatured, cooled, and mounted with 22 × 60 mm coverslip. Hybridizations were performed in sealed chambers for 16–20 hours at 60°C. After post hybridization washes, arrays were rinsed, dried with compressed air, and scanned into two 16-bit TIFF image files using Gene Pix 4000A two-color fluorescent scanner (Axon Instruments, Inc., Union City, CA) and quantitated using GenePix software (Axon Instruments, Union City, CA).After scanning of the slide, the fluorescent intensities of the green and red channels were background subtracted. The resulting values were normalized by intensity based local weighted regression method (Lowess) to correct for systematic bias in dye labeling and fluorescent intensity . Then thp-values of the each clone by comparing to the clone with median variance using chi-square distribution . The clones that have p-value greater than preset cutoff 0.9 were considered as invariant clone set, i.e. clones that do not vary significantly in all experiments. Then the mean and standard deviation of the log ratios of these invariant clones in each experiment were calculated. The clones with log ratios that exceed mean +/- 2 × SD of the invariant set were considered gains and losses, respectively. For amplification and homozygous deletions, clones were defined to have at least 2 fold of the upper bound threshold and 4-fold of lower bound threshold, respectively. The gene(s) present in the clones were identified using UCSC browser  by downloading gene table (refFlat) from human gene assembly, July 2003. We search the candidate genes based on linear mapping position, which include 100 kb up and downstream from the clone center position. The supplemental data for this article is available at: We developed a new analytical method called invariant analysis to define the significant copy number changes. This method is designed to: i) increase the power of the analysis by combining all the cases in our dataset to define an invariant population (unchanged population); and, ii) to address the signal to noise differences among individual cases due to sample and hybridization variability. Our goal is to define a set of unchanged clones that can be used to calculate the upper and lower bound thresholds of the log ratios for the unchanged population in each experiment. First, we calculated the variance of each clone from all the experiments. We computed the . The experiments in each of the two groups, amplification and normal, used for comparison were defined based on the invariant analysis (see above). The clones that have p <0.001 were considered as significant. We chose a stringent cutoff to minimize the multiple testing problem.Significant clones in 6p, 8q, 12q and 17p amplicons were calculated using 2-sample t-test with randomized variance model FISH was performed to validate and quantify chromosomal amplicons using clones from 6p12-p21 (RP11-91E11, AL391415, RP11-81F7, RP11-79I2, RP11-90H17 and RP11-79F13), 8q24.3 (RP11-89K10), and 17p11.2-p12  clones, and centromeric clone from 6 (pEDZ6) were labeled with Spectrum Red or Spectrum Green (Vysis, Downers Grove, IL) by nick translation. Hybridization and FISH analysis was performed as described previously .To define the gains and losses in our experiments, we used invariant analysis for the first time to describe genomic changes by array CGH. In this method, we defined an invariant clone set that has low variance of log ratios among all the array experiments. After the mean and standard deviation of the log ratios in the invariant set of each experiment were calculated, clones that have higher or lower log ratios than the mean +/- 2SD of the invariant set (upper bound and lower bound) were used to define gains and losses. We chose to use this method because it addresses some of the shortcomings of the modeling method, such as using all information provided in a set of experiment to determine the unchanged population instead of using one experiment at a time. However, the variation of each experiment is accounted for because the thresholds are calculated using the invariant set from each experiment. It also does not require a separate reference set for comparison. Finally, it provides an adjustable cutoff to optimize the thresholds to the training data, if provided.The amplified and homozygously deleted clones were defined to have at least 2 fold of the upper bound and 4-fold of lower bound, respectively. Figure Copy number changes excluding clones from sex chromosomes were involved in a significant fraction of most tumor genome. The estimated average genomic distance between clones was ~3–4 Mb. The frequency of clones showing gains (79%) was greater than losses (66%). High-level amplifications and homozygous deletions constitute 28.6% and 3.8% of tumor genome respectively. The most frequently deleted clones were identified from the chromosomal regions 2q31.1, 3p14.1, 4p16.2, 6q12, 6q21, 6q27, 7q35, 10p15.1, 10q22-q23, 10q25-q26, 11q25, 13q12.2, 13q14.3, 13q22.1, 17p13.3 and 17q12 (Table Homozygous deletions were noted for 32 clones (3.8%). Recurrent homozygous deletions were noted for 7 clones that are were mapped to 1q25.1 (4 cases), 3p14.1 (4 cases), 13q12.2 (2 cases), 4p15.1 (2 cases), 6q12 (2 cases), 6q12 (2 cases) and 6q16.3 (2 cases). Figure ROR1), 1p36.1 (PRDM16), 1q21, 1q23 (TNFF6), 2q24, 3p25, 3q26.1, 4p16.3, 5p14, 5q33, 6p11.2-p21, 7p21, 8q12.1, 8q24.13, 10p21, 10q11.1, 10q22 (KCNMA1), 11q13, 11q23 (GRIK4), 12q12, 12q13-q15, 12q21-q21.33, 17p11.2-p12, 17q21 (NGFR), 18q22, and 19p13.1 (NFAT). Of these amplified sites, 6p11.2-p21, 8q12.1, 8q24.13, 12q12, 12q13-q15, 12q21-q21.33, 16p13 and 17p11.2-p12 were frequent.Previous studies using CGH have identified several chromosomal amplification sites in osteosarcoma. Because of the limitation of the method, it fails to pinpoint the precise site of amplicon. However, the present study by array CGH has identified 238 clones (28.6%) with high-level amplifications. Recurrent amplifications were noted in ~37% of the total amplified clones  cases analyzed. High-level amplification of the clones from same region was noted in 25% of the cases by array CGH. We found that most of the cases with amplification of 6p12-p21 displayed either increased or slightly varying degree of copy number increase across the 6p12-p21 region. The combined log ratios from all the cases defined the boundaries of amplification between RP3-329A5 and RP11-79F13. The amplicon spans approximately 9.4 Mb with amplification peak for clone RP11-81F7. Further, we used FISH to validate 6p amplicon on tumor metaphase and interphase cells from cases 274, 364, 426 and 527. Increased copy numbers for clones RP11-91E11, AL391415, RP11-81F7, RP11-79I2, RP11-90H17 and RP11-79F13 were noted in interphase cells with maximum copy number increase for clone RP11-81F7 Figure . This waMost cases with 8q gain, displayed varying degree of copy number increase predominantly from 8q12.1 (16.9%), 8q21.13 (29%), and 8q24.3 (35%). High-level amplifications were also noted from 8q12.1  tumors analyzed by array CGH. Three distinct amplicons – AMP1 (12q12), AMP2 (12q14.1) and AMP3 (12q21.33) were noted across the entire long arm of chromosome 12 Figure . Of thesAmplification of 17p11.2 was noted in 27% of the cases analyzed by array CGH. The amplicon was composed of three clones RP11-64B12 (p = 0.0000014), RP11-89K6 (p = 0.00000005) and RP11-189D22 (p = 0.0000001) and covering 3.7 Mb region on the short arm of chromosome 17 Figure . We usedThis study represents the first application of genome-wide copy number changes by array CGH in osteosarcoma. Recent studies in breast, renal and bladder cancer showed the potential assessment of this technology in detecting high-resolution copy number changes ,11,14. Tp53 significantly correlates with genome-wide DNA instability and seems to represent a major genetic factor contributing to the extremely high levels of genomic instability found in high-grade osteosarcomas [Gene amplification is an important genetic mechanism in human cancers, as it clearly associated with tumor progression and has a prognostic significance and has even provided a target for therapeutics ,18. Thessarcomas .CDC5L, HSPCB, and NFKBIE, and HGNC and MRPL14 are the target genes from 6p12-p21 amplicon. Of these genes, CDC5L may be an important gene in cancer because of its role as a positive cell cycle regulator for G2/M transition[HSPCB was shown recently by cDNA microarray studies on osteosarcoma [Our analysis have identified frequently amplified clones from 6p11.2-p21, 8q12.1, 8q24.13, 12q12, 12q13-q15, 12q21-q21.33, 16p13 and 17p11.2-p12. Amplification of clones from 6p12-p21 region was noted in 25% of the cases analyzed. This was consistent with the previously published results by CGH. By array CGH, we refined the 6p amplicon to 9.4 Mb with amplification peak for clone RP11-81F7. We recently demonstrated the origin of 6p amplicon as consequence of tandem duplication of clones RP11-81F7 and RP11-79F13 [ransition. ConsistNSE2 (breast cancer membrane protein 101 kDa) gene.High-level amplifications were also noted from 8q12.1 (RP11-550I15 – 6.3%), 8q21.13 (RP11-89H1 – 6.3%), 8q24.3 (RP11-89K10 – 6.3%) and RP11-637F16 (12.5%). There were no candidate genes present in clones RP11-550I15, RP11-89H1 and RP11-637F16, but clone RP11-89K10 contained GLI, CHOP, SAS, HMGI-C, CDK4, HDM2, and PRIM1 from 12q13-q15 region in osteosarcoma [IFNG from AMP2 , which is physically mapped close to the HDM2 oncogene locus[IFNG strongly suppresses osteoclastogenesis by interfering with the RANKL-RANK signaling pathway. IFNG induces rapid degradation of the RANK adaptor protein, TRAF6, resulting in strong inhibition of the RANKL-induced activation of the transcription factor NFKB and JNK [ELK3 and PCTAIRE protein kinase 2 (PCTK2). ELK3 is a member of the ETS-domain transcription factor family and the protein is activated by signal-induced phosphorylation [PCTK2 belongs to the cdc2/cdkx subfamily of the ser/thr family of protein kinases and play an important role in the regulation of the mammalian cell cycle [CCND2, ETV6, and KRAS2 from 12p region [High-level amplification of clones on 12q revealed three distinct sites of amplifications – AMP1 (12q12), AMP2 (12q14.1) and AMP3 (12q21.33). Pervious studies have shown the amplification osarcoma ,23. The ene locus. Previou and JNK . The AMPrylation . The proll cycle . High-lep region .PP3A, SMCR5, DRG2, FL11, MYCD, SOX 17, ELAC2, and PMP22). Recent studies have shown the amplification of some of the genes identified in the present study (PMP22, and TOP3A) from 17p11.2-p12 in high-grade OS by semi-quantitative PCR and cDNA microarrays [Amplification of 17p11.2 was noted in 27% of the cases analyzed by array CGH. Our array CGH analysis has identified three clones with high-level amplifications that spans ~3.7 Mb region on 17p11.2. Several candidate genes were identified within these clones  in an atypical myoproliferative disorder [NUFIP1P) and BAI3 gene (brain-specific angiogenesis inhibitor gene), which is to homologous to BAI1 and shown to suppress glioblastoma [The present array CGH analysis has identified seven recurrent clones exhibiting homozygous deletions from 1q25.1, 3p14.1, 13q12.2, 4p15.1, 6q12, 6q12 and 6q16.3. These chromosomal regions were consistent with previously reported studies by loss of heterozygosity (LOH) and CGH ,31. The disorder . Homozygblastoma .In summary, high resolution array-based CGH revealed large number of chromosomal aberrations previously identified in osteosarcoma by chromosomal CGH and conventional cytogenetic methods. The present study allowed precise identification of smaller DNA copy number alterations, which suggest the presence of specific target genes in osteosarcoma. Although this study suggested several possible target genes from amplified regions from 6p, 8q, 12q and 17p, but these genes should be validated by other molecular and immunohistochemical approaches on well-defined large patient samples. Further, interaction or association studies between small genomic losses and gains will facilitate the identification of new genetic pathways in the pathogenesis of osteosarcoma.None declared.TKM and KJ have contributed towards the data analysis. LP, ML, RG, and CL were assisted in sample collection and clinical information of the patients. X-YL has involved in array CGH experiments and data collection. CPH has involved in extracting the gene information from BAC clones. SS has provided the arrays used in this study. PHR was involved in the planning, and organization of the project.The pre-publication history for this paper can be accessed here:


Small-cell neuroendocrine carcinoma in the duodenum is an extremely rare neoplasm with poor prognosis.A 57-year-old man presented with sudden onset gastrointestinal bleeding and fainting attacks. Duodenoscopy and hypotonic duodenography revealed a 3 × 3 cm protruding tumor with ulcerations situated opposite the ampulla of Vater in the second part of the duodenum. Local excision of the tumor was performed, followed by adjuvant chemotherapy with 5-fluoro uracil and leucovorin. Examination of the tumor by immunohistochemistry and electron microscopy indicated it to be neuroendocrine in nature, expressing synaptophysin and AE1/AE3, and containing dense core granules. The patient showed no sign of recurrence and has been disease-free for more than 48 months after surgery.Most cases of small-cell neuroendocrine carcinoma in the duodenum show rapid progression of the disease, and even radical surgery with or without chemotherapy do not prevent death. We report a rare subtype of small-cell neuroendocrine carcinoma. This subtype appears to have a much better prognosis, and may be amenable to local excision, if the lesion is away from the ampulla of Vater. Duodenal Neuroendocrine tumors constitute 5% of all gastrointestinal neuroendocrine tumors ,2. Most A 57-year-old man presented with sudden gastrointestinal tract bleeding and episode of fainting. Duodenoscopy Figure  and hypoA laparotomy was performed. As there was no serosal invasion or regional lymphadenopathy wide local excision of the tumor was performed. On gross examination, the tumor showed two ulcerations and two different morphological components Figure  and 2b. 2) and leucovorin (20 mg/m2) were administered. The patient showed no sign of recurrence and is disease-free 48 months after surgery.The patient was discharged three weeks after operation with uneventful postoperative period. Four cycles of monthly adjuvant chemotherapy with 5-fluoro uracil (5-FU) (325 mg/mNeuroendocrine carcinomas (NEC) in the duodenum are extremely rare, and are classified as either 'small-cell' or 'non small-cell' types. The small-cell NEC occurring in the duodenum and elsewhere in gastrointestinal tract are similar to the small-cell carcinoma of the lung ,8. Only The natural course of small-cell NECs in the duodenum is still not clear. Most cases reported in the literature show rapid progress of the disease, with radical surgery and/or chemotherapy not altering the clinical course, and thus a poor prognosis. In six of the previous eight cases, patients underwent pancreaticoduodenectomy for removal of the tumor, while the remaining two did not undergo surgery because of multiple liver metastasis or poor general condition. In spite of radical resection with or without adjuvant chemotherapy, most cases showed rapid recurrence and metastasis. Of the eight reported cases, one was unusual as it occurred in a middle-aged female with rapid progress of the disease but effective response to adjuvant chemotherapy using 5FU, tumor necrosis factor, and interferon this patient survived for more than eighteen months . The preThis report identifies a new subtype of small-cell NEC in the duodenum. This subtype appears to have a much better prognosis, and may be amenable to local excision, if the lesion is away from the ampulla of Vater.None declared.NS, MT, MK, KY, KK, HN took part in the operation, performed the literature search and drafted the manuscript for submission. HN supervised the preparation of the manuscript and edited the final version for publication. TS, KS performed pathological investigations and contributed to the pathological content of the manuscript.All authors read and approved the manuscript.


Contrast-induced nephropathy is an important cause of acute renal failure. We assess the efficacy of acetylcysteine for prevention of contrast-induced nephropathy among patients undergoing intravascular angiography.We conducted a systematic review and meta-analysis of randomized controlled trials comparing prophylactic acetylcysteine plus hydration versus hydration alone in patients undergoing intravascular angiography. Studies were identified by searching MEDLINE, EMBASE, and CENTRAL databases. Our main outcome measures were the risk of contrast-induced nephropathy and the difference in serum creatinine between acetylcysteine and control groups at 48 h.higher incidence in six of the latter studies). The pooled odds ratio for contrast-induced nephropathy with acetylcysteine relative to control was 0.54 (95% CI, 0.32–0.91, p = 0.02) and the pooled estimate of difference in 48-h serum creatinine for acetylcysteine relative to control was -7.2 μmol/L (95% CI -19.7 to 5.3, p = 0.26). These pooled values need to be interpreted cautiously because of the heterogeneity across studies, and due to evidence of publication bias. Meta-regression suggested that the heterogeneity might be partially explained by whether the angiography was performed electively or as emergency.Fourteen studies involving 1261 patients were identified and included for analysis, and findings were heterogeneous across studies. Acetylcysteine was associated with a significantly reduced incidence of contrast-induced nephropathy in five studies, and no difference in the other nine (with a trend toward a These findings indicate that published studies of acetylcysteine for prevention of contrast-induced nephropathy yield inconsistent results. The efficacy of acetylcysteine will remain uncertain unless a large well-designed multi-center trial is performed. Contrast-induced nephropathy is a leading cause for acquired acute reductions in kidney function ,2. DespiThe pathophysiology of contrast-induced nephropathy remains incompletely understood. However, current evidence suggests that contrast media induce prolonged vasoconstriction and medullary ischemia coupled with generation of free radicals and oxidative injury to tubular cells -11.Acetylcysteine, a thiol-containing anti-oxidant, has been hypothesized to prevent contrast-induced nephropathy. The potential benefit of acetylcysteine is believed to be mediated by its properties as a scavenger of free-radical species and by increasing the synthesis of nitric oxide, a potent vasodilator, in response to ischemic or other toxic injury in the kidney . Given tWe identified published randomized controlled trials of acetylcysteine for prevention of contrast-induced nephropathy during intravascular angiography using both electronic and manual search strategies. We supplemented this by scanning the reference lists of all identified articles, reviewing selected conference proceedings, and by contacting experts in the field. All languages and types of publications were considered eligible. The comprehensive literature search was initially performed in April 2003 and updated in June 2004 to identify any potential new studies that may have appeared.MEDLINE (1966 through April, 2003), EMBASE (1980 through April, 2003) and CENTRAL  databases were searched via OVID using an approach recommended for systematic reviews of randomized trials . PubMed Two individuals (SMB and WAG) independently evaluated identified articles for eligibility on the basis of four inclusion criteria: 1) study design , 2) target population (patients undergoing intravascular angiography), 3) intervention  and 4) outcome .Two reviewers (SMB and WAG) independently extracted data from all primary studies fulfilling eligibility criteria. Any discrepancies in extracted data were resolved by consensus. Data extracted included identifying information, focus of the study, details of study protocol and demographic data. The primary outcome measures were the incidence of contrast-induced nephropathy and change in serum creatinine. The secondary outcome measure was requirement for renal replacement therapy. Authors of the studies were contacted for additional information when applicable.et al [Two reviewers (SMB and WAG) independently assessed methodological quality of individual studies. Any disagreements were resolved by consensus. Items used to assess study quality were methods of randomization, any blinding, use of a placebo, reporting of losses to follow-up or missing outcome assessments, and evidence of important baseline differences between the groups -18. An oet al .The presence of heterogeneity can compromise the interpretation and validity of meta-analyses and can result from significant differences in methodology, study populations, interventions, outcomes, or chance . A priorData from all of the selected randomized controlled trials were combined to estimate the pooled odds ratio (OR) with 95% confidence intervals (CIs) using a random-effects model as described by Der Simonian and Laird ,21. The A total of 66 unique citations were identified by our initial search strategy Figure . After tAll the randomized controlled trials were published in the years 2002 through 2004. Tables The definition of contrast-induced nephropathy was variable across studies. Four studies defined contrast-induced nephropathy as a > 44.2 μmol/L increase in serum creatinine from baseline ,29,32,37The reported incidence of contrast-induced nephropathy was variable across studies. Table Table Meta-regression was performed to assess a number of clinical and study quality factors that may have led to heterogeneity across studies. Interestingly, these analyses suggest that the heterogeneity may be partially explained by whether the angiography procedures were performed electively or as emergency, because studies where all enrolled patients were undergoing elective procedures had significantly lower odds ratios than did studies where emergency cases were included (coefficient for "elective-only" studies, -0.6, 95% CI, -1.24 to 0.03, p = 0.06).Other meta-regression analyses demonstrated that the heterogeneity could not be accounted for by differences in patient age (coefficient -0.04, 95% CI, -0.2 to 0.1, p = 0.6), baseline serum creatinine (coefficient -0.001, 95% CI, -0.01 to 0.01, p = 0.9), volume of contrast media (- 0.006, 95% CI, -0.02 to 0.07, p = 0.4) or diabetes mellitus (coefficient -0.01, 95% CI, -0.03 to 0.02, p = 0.6). Likewise, heterogeneity was not accounted for by differences in study quality including use of blinding (coefficient -0.6, 95% CI, -1.7 to 0.5, p = 0.3), concealment of randomization (coefficient -0.8, 95% CI, -3.8 to 2.1, p = 0.6), use of placebo (coefficient -0.6, 95% CI, -1.7 to 0.5, p = 0.30, consecutive patient enrollment (coefficient 0.5, 95% CI, -1.5 to 2.4, p = 0.6) or overall Jadad score (coefficient 0.05, 95% CI, -0.4 to 0.5, p = 0.8).There was some evidence to suggest possible publication bias according to Begg's test (p = 0.03, with continuity correction) and a trend with Egger's test (coefficient -3.03, 95% CI, -6.71 to 0.65, p = 0.09). Figure Our meta-analysis of 14 peer-reviewed studies of patients undergoing intravascular angiography may lead some to conclude that the administration of acetylcysteine causes a reduced incidence of contrast-induced nephropathy. However, such a conclusion may be premature based on data published to date because our systematic review reveals considerable heterogeneity of findings across trials. Furthermore, our meta-analysis of post-treatment creatinine values does not reveal any truly meaningful difference in serum creatinine levels at 48 h between the acetylcysteine and control groups. Finally, insufficient data are available to allow inferences to be drawn about the efficacy of acetylcysteine on clinically meaningful endpoints such as dialysis, length of hospitalization or mortality.et al that recently received considerable attention, and that rather firmly concluded that acetylcysteine is beneficial [et al, and more directly address the relevance of this heterogeneity in the overall interpretation of study results. Two other meta-analyses have also recently been published, and similarly concluded that acetylcysteine is beneficial; however, these studies also failed to adequately address the issue of the considerable heterogeneity across studies [This meta-analysis has several features that distinguish it from a similar meta-analysis by Birck neficial . First, neficial . Fourth, studies ,42. Coll studies . Many wi studies ,45 make The presence of heterogeneity and/or publication bias can compromise the interpretation of meta-analyses and result in erroneous and potentially misleading conclusions ,43. A stdefinitely efficacious based on data published to date. Our firm conclusion based on this meta-analysis of published trails is that although the data seem quite promising, the efficacy of acetylcysteine has not been definitively proven.There are many parallels between the intravenous magnesium story and our meta-analysis findings for acetylcysteine. The marked heterogeneity of findings across studies, and the finding of funnel plot asymmetry (indicating possible publication bias), ought to be viewed as strong cautionary points against making firm conclusions about the efficacy of acetylcysteine. And while it is true that acetylcysteine is inexpensive, easy to use and has a favorable side-effect profile, it is probably premature to conclude scientifically that it is To isolate potential sources of heterogeneity we performed a meta-regression analysis exploring several clinical and study quality factors. There was no evidence of association between effect size and baseline serum creatinine, volume of contrast media, or diabetes mellitus, all independently identified risk factors for development of contrast-induced nephropathy ,53. HoweFunnel plot asymmetry is often interpreted to indicate publication bias. However, it is important to consider that this asymmetry may also be due to other sources of bias that deserve further examination. In particular, fundamental disparities in study design, inconsistencies in methodological quality and differences in the definition of primary outcomes may have contributed to funnel plot asymmetry. Our meta-regression analysis explored the potential role of several study quality factors, and none were identified as statistically significant predictors of apparent acetylcysteine efficacy across trials. Nonetheless, it is quite possible that other unmeasured study quality factors may have contributed to biased results and accompanying funnel plot asymmetry.Contrast-induced nephropathy continues to be an active subject matter for clinical investigation ,56. A deAll of the above leads us to conclude that while acetylcysteine appears to be safe and inexpensive, its efficacy for the prevention of contrast-induced nephropathy remains unproven. The results of the trials that we reviewed to date should be viewed as early promising evidence of benefit, and suggest that it is now perhaps reasonable to use acetylcysteine in routine care because of its relative ease of use and safety. However, its true efficacy will remain uncertain unless a definitive well-designed multi-center trial is performed. Such a clinical trial will be most relevant if it addresses a priori clinically meaningful endpoints of renal insufficiency, rather than surrogate endpoints based on changes in creatinine levels alone, and further considers stratification on hypothesized important subgroups that may benefit such as those with a low ejection fraction .The authors declare that they have no competing interests.SMB developed the study protocol, conducted literature search, screened articles for eligibility, extracted data, analyzed data, interpreted results, wrote and revised the manuscript. WAG contributed to protocol development, screened articles for eligibility, extracted data, analyzed data, interpreted results, and provided critique of successive drafts of the manuscript. Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: