C. elegans embryos. The two approaches, ChIP-seq and ChIP-chip, yield strongly overlapping results revealing that HLH-1 preferentially binds to promoter regions of genes enriched for E-box sequences (CANNTG), known binding sites for this well-studied class of transcription factors. HLH-1 binding sites were enriched upstream of genes known to be expressed in muscle, consistent with its role as a direct transcriptional regulator. HLH-1 binding was also detected at numerous sites unassociated with muscle gene expression, as has been previously described for its mouse homolog MyoD. These binding sites may reflect several additional functions for HLH-1, including its interactions with one or more co-factors to activate (or repress) gene expression or a role in chromatin organization distinct from direct transcriptional regulation of target genes. Our results also provide a comparison of ChIP methodologies that can overcome limitations commonly encountered in these types of studies while highlighting the complications of assigning in vivo functions to identified target sites.Identifying transcription factor binding sites genome-wide using chromatin immunoprecipitation (ChIP)-based technology is becoming an increasingly important tool in addressing developmental questions. However, technical problems associated with factor abundance and suitable ChIP reagents are common obstacles to these studies in many biological systems. We have used two completely different, widely applicable methods to determine by ChIP the genome-wide binding sites of the master myogenic regulatory transcription factor HLH-1 (CeMyoD) in  Chromatin immunoprecipitation (ChIP) is becoming an increasingly popular means to survey the genome-wide distribution of chromatin-associated proteins and their post-translational modifications. This approach is challenging when applied to many transcription factors because they are often present at low levels within cells and are located discretely, and often infrequently, within chromatin. These problems are amplified in complex tissues if the transcription factor is expressed in only a subset of the cell types within the tissue/organism or is temporally restricted. In addition, many transcription factors lack specific antibodies suitable for ChIP, leaving researchers to opt for indirect methods to interrogate chromatin-associated binding site for the factor of interest.C. elegans mixed stage embryos. One method utilized vast over-production of HLH-1 from a heat shock inducible transgene, immunoprecipitation with an anti-HLH-1 antibody, and hybridization to a genome tiling array (ChIP-chip). The second method employed a Green Fluorescent Protein (GFP)-tagged, low copy number, integrated hlh-1 translational fusion transgene, immunoprecipitation with an anti-GFP antibody, and next-generation sequencing (ChIP-seq). The advantage of over expression is that it can overcome limitations in transcription factor abundance and antibody affinity. The obvious disadvantage of over expression is the potential for ectopic binding to result in misleading profiles of the genome wide distribution. This latter concern may also be applicable to certain epitope-tagged transgenes, although likely less problematic when expression is directed by sequences representing the natural genomic context for the gene of interest that are integrated as one or a few copies. In fact, early results from GFP-tagged coding regions using bacterial recombineering in fosmid clones look very promising in terms of recapitulating endogenous patterns of transcription factor expression and binding To address these issues, we have compared two completely different ChIP methods to determine the genome-wide distribution of HLH-1 (sometimes referred to as CeMyoD) binding sites in The results from each experimental approach were remarkably similar and suggested that there are a large number of HLH-1 binding sites genome-wide, primarily located in promoter regions of candidate target genes. Analysis of these HLH-1-positive ChIP genomic fragments demonstrated that E-box (CANNTG) sequences, known binding sites for HLH-1 and related helix-loop-helix factors, are the predominantly over-represented motif. We have validated our HLH-1 candidate binding sites by ChIP-qPCR, comparisons to independently derived ChIP data, and by reporter gene expression. This analysis demonstrates that both over expression and epitope-tagging of transcription factors can provide a valid profile of DNA binding sites throughout the genome. Moreover, our results highlight the difficulty in ascribing biological functions to ChIP-defined binding sites, a problem common to all current approaches.C. elegans and it functions together with other transcription factors to specify bodywall muscle fate and direct differentiation C. elegans), requiring additional experimental evidence to directly link HLH-1 to downstream target gene activation. Chromatin immunoprecipitation (ChIP), coupled with either whole genome tiling arrays (ChIP-chip) or high throughput sequencing (ChIP-seq), offers the potential to make this link and to provide insight into the molecular mechanisms and logic underlying myogenesis and other differentiation programs.Striated muscle cell fate specification and differentiation in animals is orchestrated at the transcriptional level by Myogenic Regulatory Factors (MRFs) hlh-1 enhancer fragments known to be involved in positive auto-regulation were unsuccessful hlh-1 reporter transgenes. The inability to ChIP endogenous levels of HLH-1 reflects a problem likely to be common for transcription factor studies in many model systems, leading us to explore alternative approaches to profile HLH-1 binding sites on a genomic scale. HLH-1 is an excellent test case for this approach because muscle gene expression has been well documented in C. elegans, providing a predictable set of target genes that should be identified by ChIP if HLH-1 function is direct and restricted to bodywall muscle genes.Polyclonal antibodies have been prepared against full-length HLH-1 fusion proteins in chickens. Previous attempts to use this antibody in chromatin immunoprecipitation experiments in wild type animals to assay binding to cis-acting hlh-1 cDNA from a heat shock promoter C. elegans genome (Nimblegen) with a probe length of 50 bp, centered 36 bp apart. Regions of HLH-1 genomic binding were determined using CARPET We have previously described an integrated transgene expressing a full-length HLH-1 ChIP-chip data from mixed stage embryo populations was collected from three independent, biological replicates. The data was normalized using the Bi-weight and Mean Summarization functions in CARPET hlh-1 translational fusion transgene was generated by recombineering as previously described As part of the modENCODE pipeline, a GFP-tagged C. elegans, we applied increasingly stringent thresholds for peak calls  to further restrict the genes used for our analysis  were near linear at cutoff values between 10−5 and 10−10. This suggested that at stringencies between 10−6 and 10−10, the reduction in peaks and genes identified was reflecting a simple truncation of the number of calls rather than increasing, biologically relevant discrimination. Thus, we chose a conservative cutoff peak p-value at 10−6 for all subsequent analysis of the ChIP-seq data, resulting in 4,016 peaks associated with 2,753 unique candidate target genes (−6 cutoff) was just upstream of the TSS. Data from either method had a distribution that fell off steeply upstream of the TSS, but remained elevated above background downstream of the TSS, extending nearly 6 Kb.Using the CARPET package analysis . At a p-et genes  & S3 wit−6 cutoff) (http://elegans.uky.edu/MA/progs/Compare.html) was used to compare the lists of genes associated with peaks in each experimental approach. Comparison of gene lists revealed that 569 of the 1,032 (55%) candidate genes associated with ChIP-chip were present in the ChIP-seq (10−6 cutoff) list of 2,753 genes (−6 ChIP-seq data failed to identify many ChIP-chip genes (45%), a result that likely reflects not only the more stringent cutoff threshold, but also several technical differences between the methods. For example, the two ChIP methods used different antibodies for immunoprecipitation (chicken anti-HLH versus donkey anti-GFP) that likely cross-react differently with chromatin proteins resulting in very different non-specific background binding sites. However, the HLH-1 binding sites common to these two very different methods suggest that many (55–84%) of the gene calls reflected bona fide regions of HLH-1 localization in embryonic chromatin.We employed the CARPET package  cutoff) .. Softwa53 genes ; the ove53 genes . We notehlh-1, providing a very limited, yet important, internal control set of sites for comparisons to our most recent data. HLH-1 positive auto-regulation of its own expression occurs through several enhancer elements located upstream of the coding region and within the first large intron of hlh-1−6) HLH-1 data to these known sites demonstrates that all four previously described HLH-1 bound enhancers were detected by both genome-wide approaches .Previous ChIP studies have characterized HLH-1 bound enhancer regions regulating proaches , with thhlh-1 promoter previously shown to be unable to preferentially bind HLH-1 http://tock.bcgsc.bc.ca/cgi-bin/sage140), or gut Quantitative PCR (qPCR) was used to confirm HLH-1 bound regions determined by either ChIP-chip or ChIP-seq methods. For the HLH-1 ChIP-chip peak calls, we generated qPCR amplicons to interrogate 20 regions ranked in the top 300 by peak score, 10 peaks ranked in the middle between 301–800, and 10 peaks ranking at the bottom between 801–1,376. As shown in −6) data revealed a number of peaks that were not present in the ChIP-chip data. To determine if these potential sites of HLH-1 binding were valid, we assayed 20 amplicons from ChIP-seq peaks with scores from the top 1,000 as well as 10 amplicons each from peaks scoring in the middle or bottom ranks. As shown in −6) were valid sites of HLH-1 binding and that our ChIP-chip data under-represented these sites.As noted above, the ChIP-seq  were identified after over expression of HLH-1 in our ChIP-chip experiments. Similarly, only 10,543 (3.6%) E-box sites were identified by ChIP-seq (10−6), a number that rises to 24,061 (8.3%) in the unabated, default ChIP-seq data. We concluded from this data that there is a high degree of selectivity for HLH-1 binding that restricts it to only a small fraction of the potential E-box sites present in the genome.While E-boxes are a known consensus basic helix-loop-helix transcription factor binding motif and HLH-1 binds these sequences promiscuously in vitro Gene Ontology (GO) analysis in GENECODIS C. elegans. Examples include 1,064 genes enriched in bodywall muscles of L1 larvae using polyA binding protein tagging hlh-1 transgene used for ChIP-chip in this study hlh-1 over expression 6 hours after induction resulted in a list of 927 genes. Finally, a compilation of community data at WormAtlas (http://www.wormatlas.org) was used to generate a list of 480 genes expressed in bodywall muscle that was further culled of genes also expressed in the hypodermis to yield a more tissue restricted list of 297 bodywall muscle genes.If HLH-1 is involved in the direct activation of genes expressed in bodywall muscle, there should be a correlation between the HLH-1 target genes identified by ChIP and the bodywall muscle transcriptome. Several groups, using a variety of techniques and approaches, have profiled bodywall muscle gene expression in http://www.wormbase.org as well as lists of genes previously described as enriched in the adult germline We compared our HLH-1 ChIP-chip and ChIP-seq candidate target gene lists with each of these previously identified bodywall muscle gene lists, normalizing to the number of genes in each list. As controls for these comparisons, we used three randomly generated sets of 2,000 genes each using an alphabetical sort of all genes from WormBase (WS195) data Previous studies have suggested that the distribution of bodywall muscle genes throughout the genome is non-random, with evidence for clustering and under-representation of muscle genes on certain linkage groups, including the X chromosome myo-2::gfp minimal promoter. We, and others, have used the myo-2::gfp minimal promoter (Fire vector L3136) in the past as a robust assay for bodywall-specific transcriptional enhancer activity for test sequences ye-1, etr-1, let-60, unc-60, and ric-3) were positive for bodywall muscle enhancer activity when introduced as stable extrachromosomal transgenes in an otherwise wild type background  and two that were positive for expression in wild type animals . All seven reporter constructs were injected, along with a rescuing unc-119 plasmid, into the hsp::hlh-1, unc-119 stain and non-Unc animals selected for the assay. None of the seven reporters showed a strong or reproducible response to heat shock induced HLH-1 when assayed at multiple times after induction in embryos, larvae, or adults . As an alternative, we used an epitope-tagged (GFP) HLH-1 transgene that was ChIPed with anti-GFP antibodies and the immunoprecipitated DNA analyzed by next generation sequencing (ChIP-seq). Using these alternative approaches for the transcription factor HLH-1 in C. elegans. Both ChIP datasets demonstrate that HLH-1 can be detected just upstream of the TSS of thousands of potential target genes, that an E-box (CANNTG) is the dominant over-represented motif in these bound sequence intervals, and the genes associated with binding sites are enriched in those known to be expressed in bodywall muscle. The widespread localization of HLH-1 predominantly to promoter regions is consistent with the expectation that HLH-1 acts directly to regulate target gene expression. HLH-1 activity is likely mediated through direct binding to E-box sequences (CANNTG) in the promoter of target genes. The symmetry of the top returned E-box motifs is suggestive of homodimer binding, consistent with the fact that C. elegans bodywall myogenesis occurs in the absence of the E protein binding partner for the MRFs typically found in other biological systems This study provides valuable insights into HLH-1 biology in C. elegans in the ChIP-seq data using default threshold values. Equally important was the lack of evidence for widespread, ectopic binding of HLH-1 after over expression in the ChIP-chip data. We found that only a small fraction (∼3.5%) of all possible E-boxes genome wide were identified by either our ChIP-chip or ChIP-seq (10−6) approaches, arguing for a high degree of selectivity for HLH-1 binding. A comparison of the HLH-1 ChIP data to that recently published for another transcription factor, PHA-4, also suggests highly selective binding (1). Although the peak overlaps between HLH-1 and PHA-4 embryonic datasets was slightly above random expectations, the associated gene overlap was at, or far below, random expectations for all pair wise comparisons. HLH-1 and PHA-4 target genes would not be expected to overlap in embryos as these two transcription factors are expressed in mutually exclusive tissues at this stage of development. Further studies are required to determine if the binding site specificity we observed is an intrinsic property of HLH-1 or reflects additional constraints, including required cooperativity with other factors or the limited accessibility to potential binding sites in the context of general or tissue-specific chromatin organization.There were also several surprising aspects of our results from this study. We did not anticipate the large number of HLH-1 bound intervals and associated genes, up to half of all protein coding genes in −6) and the 297 WormAtlas bodywall muscle expression gene list. The inability to identify all bodywall muscle genes by mixed stage embryo ChIP technology is not completely unexpected because some bodywall muscle genes may be activated late in embryogenesis or post-embryonically. However, the discordance between the ChIP data and bodywall muscle transcriptome profiles also highlights the inherent flaws in experimental manipulation, current technologies employed for gene expression and ChIP studies, and bioinformatic treatment of the resulting data. At the level of gene expression, each of the several attempts to define the bodywall muscle transcriptome in C. elegans has generated a gene list, yet there is limited consensus among these. These differences arise due to many factors, including methodological efficiencies, life stage variability, and the use of various experimental platforms to assay expression. At the ChIP level, our results similarly highlight the variability associated with experimental and platform differences. For example, our ability to validate ChIP-seq peaks not detected by ChIP-chip, using the ChIP-chip reagents and methods demonstrates that our ChIP-chip data is under representing the HLH-1 binding sites. One likely explanation of this under representation is the limited dynamic range of chip hybridizations that makes the discrimination of signal and background more difficult compared to the more quantitative next generation sequencing approach. In addition, our ChIP-chip peak calls required consensus between three biological replicates, a stringency that may have eliminated true positives. The identification and validation of ChIP-chip HLH-1 binding sites that were not detected by ChIP-seq are more difficult to explain. Clearly, no single approach captures a clear and complete view of transcriptional systems suggesting a combination of approaches is likely needed to more fully understand the relationship of transcription factor DNA binding and gene expression.We were also surprised to find that neither of the ChIP methods identified a majority of the genes defined previously by various groups cataloging the bodywall muscle transcriptome. The best observed overlap (45%) was between the ChIP-seq ; the heat shock promoter driven hlh-1 strains KM472 and KM267 unc-119 (ed3) for testing enhancer reporter genes in the presence of over expressed HLH-1.The following hlh-1 coding region in the context of a fosmid clone was introduced into animals by biolistic transformation using the unc-119 selection marker, as previously described rol-6 plasmid pRF4. At least two stable transgenic lines were generated for each construct and expression in both adult and embryonic bodywall muscle lineages was determined. The 20 fragments corresponding to the selected peak sequences  according to the protocols provided by Nimblegen. Immunoprecipitated DNA with chicken anti-HLH-1  or pre-immune chicken serum (control) samples was prepared in biological triplicates. Nimblegen C. elegans tiling array contain 385,000 oligonucleotides covering the C. elegans genome at a resolution of 50 bp with 36 bp gaps between probes. ChIP-chip hybridization and scanning were done by Nimblegen. ChIP-seq assays were conducted using the modENCODE pipeline as previously described The ChIP assay was conducted as described previously Quantile normalization, peak detection, gene annotation, peak distribution was done using the CARPET tiling analysis software package http://frodo.wi.mit.edu/primer3/) Primers were designed for HLH-1 ChIP enriched genomic DNA regions using Primer3  relative to gene coding regions on the plus and minus coding strands below (green boxes). ChIP-seq intervals using both default and e−6 thresholds are shown for comparison to the ChIP-chip intervals. Note the similarity in patterns among ChIP datasets with the ChIP-chip intervals constituting an increasingly smaller subset as the ChIP-seq data threshold is reduced.Genome browser view comparison of ChIP data. An Integrated Genome Browser (IGB) view of the ChIP data for a 1,000 kb region of LGII of (TIF)Click here for additional data file.Figure S2etr-1 drives bodywall muscle expression. The 2,150 bp interval identified by HLH-1 ChIP-chip and ChIP-seq methods was fused to a reporter gene used to assay bodywall muscle activity and introduced into a wild-type background by transgenesis. This genomic fragment was sufficient to drive bodywall muscle expression of the reporter in embryos, larvae, and adults.The HLH-1 bound interval upstream of (TIF)Click here for additional data file.Figure S3hlh-1 cDNA transgene (paired lanes 1–7) were assayed by Western blot probed with chicken anti-HLH-1 antibody. Lysates were prepared from embryos of gravid adults three hours after mock (−) or heat shock treatment (+) and equal amounts of total protein was loaded in each lane. All transgenic strains show strong induction of HLH-1 in response to heat shock. M is molecular size marker. Samples 1 & 2 are the heat shock hlh-1 strains, KM267 & KM472, respectively, which have been previously characterized . Samples 3–7 are embryo extracts from strains harboring both the heat shock hlh-1 transgene and reporter genes of amplicons associated with the following genes: sample 3: aqp-2, sample 4: etr-1, sample 5: ric-3, sample 6: ceh-20, sample 7: emb-9. Arrows indicate full-length HLH-1.Heat shock induction of HLH-1. Total embryonic lysates from wild type animals (Cntrl) or strains harboring a heat shock inducible (TIF)Click here for additional data file.Table S1ChiP-chip peak call data.(XLS)Click here for additional data file.Table S2Gene list calls for ChiP-chip, ChIP-seq, and the overlap of each method.(XLS)Click here for additional data file.Table S3−6) peak call data.ChiP-seq (e(XLS)Click here for additional data file.Table S4Primer sequence information.(XLS)Click here for additional data file.


C-type natriuretic peptide (CNP) is a paracrine molecule which is mainly synthesized in the vasculature. High levels have been reported in sepsis, and CNP has been proposed as a biomarker predicting sepsis in traumatized patients. We aimed at evaluating the diagnostic and prognostic value of N-terminal pro-CNP (NT-proCNP) for predicting sepsis, disease severity and mortality in critically ill medical patients.273 critically ill patients  and 43 healthy controls were consecutively included in a prospective clinical single-center non-interventional study at the Medical Intensive Care Unit, RWTH-University Aachen, Germany. Patients' outcome was followed for about 1 year. NT-proCNP serum concentrations were determined upon ICU admission, as well as in the mornings of day 3 and day 7 after admission. Intensive care treatment measures as well as routine and experimental laboratory parameters were recorded and analyzed.NT-proCNP serum concentrations upon admission to the ICU were elevated in critically ill patients as compared with healthy controls. Patients with sepsis had significantly higher NT-proCNP levels than non-sepsis patients. NT-proCNP was strongly associated with inflammatory parameters , biomarkers of organ dysfunction and clinical composite scores . NT-proCNP levels at admission and day 3 were found to be a strong predictive marker for ICU- and overall survival. Moreover, a decline of serum NT-proCNP after admission to the ICU was associated with reduced mortality. The predictive power of serum NT-proCNP was similar to 'conventional' prognostic tools such as clinical scores.NT-proCNP is significantly elevated in critically ill patients, with highest levels in sepsis. Inflammation as well as organ function are strongly associated with NT-proCNP serum concentrations. Low initial NT-proCNP levels and a decline during initial treatment indicate a favourable ICU- and long-term outcome. The natriuretic peptide family consists of three distinctive members: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). These peptides exert multiple potent diuretic, natriuretic and vasorelaxant functions, thereby directly influencing body-fluid homeostasis and blood pressure control ,2. As ANCNP is synthesized as a precursor proCNP protein, and conversion of proCNP to the biologically active hormone CNP is processed by the intracellular endoprotease furin . Amino-tWe therefore conducted a large study with critically ill patients in a medical ICU, performing longitudinal measurements of NT-proCNP serum concentrations during the first week of ICU treatment, to address whether NT-proCNP is activated in critical illness, whether NT-proCNP has diagnostic value for sepsis and/or multiorgan failure, and whether NT-proCNP can serve as a prognostic predictor for ICU and long-term survival.The study protocol was conducted in accordance with the ethical standards laid down in the Declaration of Helsinki and approved by the local ethics committee . We investigated 273 patients  who were admitted consecutively to the General Internal Medicine ICU at the RWTH-University Hospital Aachen, Germany  from the local blood transfusion institute at the University Hospital Aachen. At our blood transfusion institute, all volunteers that donate blood agreed (after informed consent) to contribute to ongoing biomarker studies. All control subjects had normal values for blood counts, C-reactive protein, and liver enzymes, and tested negative for hepatitis B and C and HIV.In addition, ICU patients were divided according to their Acute Physiology and Chronic Health Evaluation (APACHE) II score into patients with "moderate disease severity"  and "high disease severity" (APACHE-II > 10) .Among the 273 critically ill patients enrolled in this study, 197 patients conformed to the criteria of bacterial sepsis . Intra-assay coefficient of variation was 5.3 to 8.3%, and inter-assay coefficient of variation was 7 to 9%. IL-6, IL-10, TNF-α , and procalcitonin  were measured by commercial chemiluminescence assays, following manufacturers' instructions.U-test and multiple comparisons between more than two groups have been conducted by Kruskal-Wallis analysis of variance and Mann-Whitney-U-test for post hoc analysis. Box plot graphics illustrate comparisons between subgroups and they display a statistical summary of the median, quartiles, range, and extreme values. The whiskers extend from the minimum to the maximum value excluding outside and far out values, which are displayed as separate points. An outside value (indicated by an open circle) was defined as a value that is smaller than the lower quartile minus 1.5-times interquartile range, or larger than the upper quartile plus 1.5-times the interquartile range. A far out value (indicated by an asterisk) was defined as a value that is smaller than the lower quartile minus three times interquartile range, or larger than the upper quartile plus three times the interquartile range [P < 0.05 were considered statistically significant [Data are given as median and range due to the skewed distribution of most of the parameters. Differences between two groups were assessed by Mann-Whitney-le range . All valnificant . The pronificant . After sP < 0.001; Figure We first tested whether NT-proCNP is activated in critically ill patients. Upon admission to the medical ICU, critically ill patients displayed significantly higher NT-proCNP serum concentrations as compared with healthy controls  and SAPS2 Table . These fBased on the clear associations between NT-proCNP, inflammatory markers, organ dysfunction and prognostic clinical scores, we hypothesized that NT-proCNP measurements could predict mortality in critically ill medical patients. We determined NT-proCNP serum concentrations at ICU admission, and at days three and seven of ICU treatment. Its prognostic impact on ICU and overall survival among all critically ill patients and the subgroups of sepsis and non-sepsis patients was assessed over a long-term follow-up period .Patients that died during the subsequent ICU treatment showed significantly higher NT-proCNP levels at admission and on day three Figure . On day P = 0.001, day three P = 0.001, day seven not significant; Cox regression analyses). In this respect, NT-proCNP levels showed comparable prognostic accuracy like established multifactorial scores such as SOFA or the SOFA score change during the first three days of ICU treatment . Kaplan-Meier curves showed, using a cut-off value for serum NT-proCNP of 8 pmol/L, significantly improved ICU survival for critically ill patients with low NT-proCNP levels at admission and on day three  were included, NT-proCNP did not reach independent prognostic significance (detailed data not shown).P = 0.002) and day three (P = 0.013) predicted long-term mortality in critically ill patients. We also observed a trend to high levels predicting mortality for NT-proCNP measured at day seven. Using Kaplan-Meier curves, with cut-off values for serum NT-proCNP of 8 pmol/L, we demonstrated significantly improved overall survival for critically ill patients with low NT-proCNP . Larger studies should evaluate whether adjustment of NT-proCNP to individual renal and hepatic functions might further improve its diagnostic power.NT-proCNP was significantly elevated in critically ill patients with highest levels in sepsis. Inflammation as well as organ function strongly impact NT-proCNP concentrations. Low NT-proCNP levels and a decline during initial treatment were associated with favorable ICU and long-term outcomes. The predictive power of serum NT-proCNP was similar to 'conventional' prognostic tools such as clinical scores. Yet, further studies are needed to elucidate the underlying pathomechanisms of NT-proCNP in patients with critical illness.• NT-proCNP, a paracrine molecule released mainly from vasculature, has been implicated in inflammatory and metabolic pathways and has been recently proposed as a novel biomarker for predicting sepsis in traumatized patients.• In critically ill medical patients, NT-proCNP serum concentrations upon ICU admission are elevated as compared with healthy controls and are higher in sepsis patients than in non-sepsis patients.• In the initial course of ICU treatment, NT-proCNP serum concentrations significantly decline between admission and day three.• NT-proCNP levels at admission to the ICU are correlated to biomarkers of inflammation, organ dysfunction, and disease severity.• NT-proCNP is a strong outcome predictor, and a decline of NT-proCNP serum concentrations after ICU admission is associated with a reduced mortality.ANP: atrial natriuretic peptide; APACHE: Acute Physiology and Chronic Health Evaluation; AUC: area under the curve; BNP: brain natriuretic peptide; CNP: C-type natriuretic peptide; CRP: C-reactive protein; IL: interleukin; NO: nitric oxide; NT-proCNP: amino-terminal pro-C-type natriuretic peptide; PCT: procalcitonin; ROC: receiver operating characteristic; SAPS: simplified acute physiology score; SOFA: sequential organ failure assessment; TNF-α: tumor necrosis factor α.The authors declare that they have no competing interests.AK, FT, and CT designed the study, analyzed data and wrote the manuscript. SV performed measurements. ES, HZ, HD, and AH collected data and assisted in patient recruitment.


Calcitonin gene-related peptide (CGRP) plays an important role in migraine pathogenesis. Transient receptor potential vanilloid-1 (TRPV1) is a cation channel which is expressed in sensory ganglia and can be activated by a number of pain inducing stimuli. The relationship between CGRP and TRPV-1 is still unclear.to investigate the functional role of CGRP on pain sensitization via the mediation of TRPV1 expression in rat trigeminal ganglion (TG).CGRP (600 ng/kg) was intravenously injected to the Wistar rats (250-300 g). Rat trigeminal ganglia and brainstem were removed 45 and 60 minutes after CGRP or saline injection. The levels of CGRP, TRPV-1, phosphorylated PKC (p-PKC) and cyclic AMP responsive element-binding protein (CREB) in TG were determined by western blotting and by immunochemical and immunofluorescence staining. The c-fos level in trigeminal nucleus caudalis (TNC) was also determined.Following CGRP injection, there was a significant increase in the amount of TRPV1, CGRP, p-PKC and CREB were observed in rat TG. In addition, an increase in c-fos levels was also determined in TNC of CGRP-treated rats indicating trigeminal nociception.These results indicate the role of TRPV-1 in CGRP-evoked trigeminal sensitization. This may explain the process of peripheral sensitization developed during the attack of migraine.


Overcoming cellular senescence is strictly required for virus-driven tumors, including those associated with Epstein-Barr virus (EBV). This critical step is successfully accomplished by EBV through TERT expression and telomerase activation in infected cells. We herein review the complex interplay between EBV and TERT/telomerase in EBV-driven tumorigenesis. Evidence accumulated so far clearly indicates that elucidation of this issue may offer promising opportunities for the design of innovative treatment modalities for EBV-associated malignancies. Indeed, several therapeutic strategies for telomerase inhibition have been developed and are being investigated in clinical trials. In this respect, our recent finding that TERT inhibition sensitizes EBV+ lymphoma cells to antivirals through activation of EBV lytic replication is particularly promising and provides a rationale for the activation of clinical studies aimed at assessing the effects of combination therapies with TERT inhibitors and antivirals for the treatment of EBV-associated malignancies. Infection of B lymphocytes is usually non-productive or latent, whereas intermittent reactivation and virus replication at epithelial surfaces allow the spreading of EBV to new hosts. Despite its widespread diffusion and apparent harmlessness, EBV is causally linked to the development of both lymphoid and epithelial malignancies, including Burkitt lymphoma (BL), Hodgkin lymphoma, post-transplant lymphoproliferations, AIDS-associated lymphomas, nasopharyngeal and gastric carcinoma [More than 90% of the world’s population is infected by Epstein-Barr virus (EBV), a ubiquitous gammaherpesvirus that may silently persist in memory B lymphocytes. Primary infection is usually asymptomatic and when it is delayed until adolescence or adulthood a benign lymphoproliferative disease, known as infectious mononucleosis, may occur. B lymphocytes are the main target of EBV infection arcinoma . EBV infarcinoma ,2. A secarcinoma . EBV mayin vitro. Available evidence indicates that EBNA-2 and LMP-1 are absolutely required for EBV-mediated B-cell transformation, whereas a crucial role is played by EBNA-1, EBNA-3, -5, and -6 [Studies carried out with recombinant EBV strains lacking individual latent genes allowed the identification of the genes that are essential for B-cell immortalization , and -6 ,5. LMP-1, and -6 . LMP-1 h, and -6 .in vitro of B lymphocytes, is considered a useful model to investigate the relationships between virus and host in the EBV-driven lymphomagenesis. Indeed, these immortalized cells are similar to those regularly produced in vivo but negatively controlled by EBV-specific CD8+ cytotoxic T cells [in vivo are telomerase-positive confirms the relevance of telomerase expression in the process of tumorigenesis.LCLs obtained by EBV infection  T cells . Studies T cells ,11. The reviewed in[Genetic instability is a hallmark of cancer and tumor cells should circumvent replicative senescence and acquire the ability to sustain unlimited proliferation. Telomere/telomerase interplay is an important mechanism involved in the genomic stability and cellular replicative potential and its dysfunction has emerged as playing a key role in carcinogenesis . Telomerviewed in).TERT gene is likely the key determinant in the regulation of telomerase activity; TERT transcriptional activity is specifically up-regulated in cancer cells, but it is silent in most normal cells. More than 20 transcription factor-binding sites acting as activators or repressors have been identified within the TERT promoter. Cooperation of MYC and SP1 is required for full activation of the TERT promoter, while TP53, through its interaction with SP1, down-regulates TERT. TERT is also directly activated by nuclear factor (NF)-kB, hypoxia-inducible factor-1, and the ETS/MYC complex. The histone methyltransferase SMYD3 also directly contributes to inducible and constitutive TERT expression in normal and malignant human cells. TERT expression is suppressed by the oncosuppressor genes WT127 and MEN1, and through the MAD/MYC and the TGF-β/SMAD pathways. Cell cycle inhibitors p16INK4a and p27KIP1 have also been shown to down-regulate TERT expression in cancer cells . Regula complex . Telomer complex . Notably complex , promote complex -24. High complex ,26.Although it is well recognized that the establishment of latent EBV infection and TERT activation are both required for EBV-driven cell transformation, temporal and possible causal relationships between these two events remain to be clarified. Early passages EBV-infected B lymphocytes greatly differed in their timing of TERT expression and telomerase activation; EBV-driven B-cell activation may fail in the induction of telomerase activity and telomerase-negative EBV-infected B cells may have a prolonged life-span compared to normal B lymphocytes ,20. Althvia NF-kB and MAPK/ERK1/2 pathways [MYC is in germ-line configuration, TERT is likely to be activated by LMP-1, as it occurs in LCLs [In primary B lymphocytes, activation of TERT occurs concomitantly with the induction of latent EBV proteins and down-regulation of EBV lytic gene expression . We havepathways . LMP-1 ipathways . In epitpathways . In B cepathways . This is in LCLs .in vitro using the LCL model is that TERT expression plays a relevant role in inhibiting the virus lytic cycle, thereby favouring the induction and maintenance of EBV latency in primary B lymphocytes, a prerequisite for EBV-driven transformation. Indeed, high level of endogenous TERT expression or ectopic TERT expression on telomerase-negative EBV-infected cells prevents the induction of viral lytic cycle. By contrast, TERT silencing by specific siRNA or short-hairpin (sh)RNA induced the expression of BZLF1, EA-D, and gp350 EBV lytic proteins and triggered a complete lytic cycle. This occurs in both EBV-immortalized LCL and fully transformed EBV-positive BL cell lines thus supporting the notion that TERT is a critical regulator of the balance between viral latent and lytic cycles [Notably, in addition to maintaining telomere length and allowing EBV-infected cells to overcome senescence and apoptosis, TERT may promote EBV-driven lymphomagenesis through extra-telomeric functions. One interesting result achieved c cycles ,31. MoreThe fine mechanism(s) by which TERT prevents the expression of lytic proteins is still an interesting open question. It has been demonstrated that the treatment of primary EBV-positive BL with zidovudine (AZT), a thymidine analog, induced EBV lytic cycle and cell death through the NF-κB pathway ,33. GiveThe high expression of TERT in tumor cells and the requirement of a sustained telomerase activity for their unlimited proliferation capability make telomerase a particularly attractive target for cancer therapy. Moreover, the fact that rapidly proliferating cancer cells have shorter telomeres compared to normal somatic cells and stem cells further corroborates the enhanced specificity for tumor cells of the cytotoxic effects exerted by drugs targeting telomerase . Severalex vivo with a chimeric mRNA encoding the entire TERT sequence and a portion of the lysosomal-associated membrane protein, which redirects the TERT protein to the lysosome degradation pathway favoring its degradation into immunogenic peptides [A second main therapeutic approach involves immunotherapy strategies targeting telomerase . Considepeptides . SeveralThe observation that EBV-driven tumors generally express telomerase at high levels provides the rationale supporting the use of the aforementioned pharmacological and immunological approaches targeting telomerase/TERT also for the treatment of these malignancies. Nevertheless, exploitation of telomerase inhibition in this setting may offer additional advantages of potential clinical relevance. In fact, our observation that inhibition of TERT by shRNA triggers virus replication in both EBV-immortalized and fully transformed B cells  providesIn EBV-associated tumors, telomerase can be also targeted indirectly by strategies that inhibit the expression of LMP-1, the major viral oncoprotein endowed with pleiotropic effects also including the ability to up-regulate TERT at the transcriptional level ,29. In tAvailable evidence indicates that TERT expression and telomerase activation play a critical role in EBV-driven tumorigenesis. Several therapeutic strategies for telomerase inhibition have been developed and are being investigated in clinical trials, although data concerning the response rates obtained in patients with EBV-associated lymphomas are limited. Most therapeutics have shown to be more effective when used in combination with standard therapies. In this respect, our finding that TERT inhibition sensitizes EBV+ lymphoma cells to antivirals through activation of EBV lytic replication is particularly promising and provides a rationale for conducting further research to assess the effects of combination therapies with TERT inhibitors and antivirals for the treatment of EBV-associated malignancies.AZT: Zidovudine; ATL: Alternative lengthening of telomerases; BL: Burkitt lymphoma; EBV: EBV nuclear antigens; EBNAs: Epstein-Barr virus; LCL: Lymphoblastoid cell lines; MAPK: Mitogen-activated protein kinase; NK: Natural killer; NF: Nuclear factor; JNK: c-Jun N-terminal kinase; PI3K: Phosphatidylinositol 3-kinase; sh: Short-hairpin; TERT: Telomerase reverse transcriptase.The authors declare that they have no competing interests.All authors performed the literature research, composed the article and approved the final version to be submitted. All authors read and approved the final manuscript.


The medullary respiratory network generates respiratory rhythm via sequential phase switching, which in turn is controlled by multiple feedbacks including those from the pons and nucleus tractus solitarii; the latter mediates pulmonary afferent feedback to the medullary circuits. It is hypothesized that both pontine and pulmonary feedback pathways operate via activation of medullary respiratory neurons that are critically involved in phase switching. Moreover, the pontine and pulmonary control loops interact, so that pulmonary afferents control the gain of pontine influence of the respiratory pattern.IT) and expiration (ET).We used an established computational model of the respiratory network  and exteI Tand ET, (b) the suppression of pontine-medullary interactions led to the prolongation of I Tat relatively constant, but variable ET, and (c) these perturbations applied together resulted in "apneusis", characterized by a significantly prolonged IT. The results of modeling were compared with, and provided a reasonable explanation for, multiple experimental data. The model was able to reproduce the experimentally demonstrated changes in I Tand E Tand phrenic pattern following vagotomy and/or pontine suppression by NMDA receptor blockers MK801and AP-5. According to the model these changes reflect the characteristic changes in the balance between the pontine and pulmonary feedback mechanisms involved in control of breathing during various cardio-respiratory disorders and diseases.In our model: (a) the simulated vagotomy resulted in increases of both AP-5 - amino-5-phosphonovaleric acid, NMDA receptor antagonist; BötC - Bötzinger Complex; E - Expiratory or Expiration; I - Inspiratory or Inspiration; KF - Kölliker-Fuse nucleus; MK801 - dizocilpine maleate, NMDA receptor antagonist; NTS - Nucleus Tractus Solitarii; PBN - ParaBrachial Nucleus; PSRs - Pulmonary Stretch Receptors; VRC - Ventral Respiratory Column; VRG - Ventral Respiratory Group.


The pathogenic mechanisms of toluene diisocyanate (TDI)-induced asthma are not understood clearly.In this review, we summarized recent data to further understand the pathogenic mechanisms through recent approach of molecular genetic studies, and specific antibody responses to cellular mechanisms.Genetic polymorphism studies suggested the involvement of oxidant/antioxidant-related genes and neurogenic inflammatory response genes. A high-resolution technique of HLA allele suggested some HLA genetic markers presenting the phenotype of TDI-induced asthma. The new volatile type of TDI-human serum albumin conjugate could improve specific immunoglobulin E and immunoglobulin G bindings in enzyme-linked immunosorbent assay studies for identifying TDI-induced asthma patients. The role of bronchial epithelial cells and cytokeratins derived from epithelial cells could be involved in autoantibody productions, suggesting that an autoimmune mechanism may be involved in airway inflammation. Neutrophil activation and oxidant/antioxidant-related mechanisms were also suggested.The pathogenic mechanism of TDI-induced asthma is complicated as various humoral and cellular mechanisms are combined and involved differently on an individual basis. Therefore, additional efforts for further elucidation should follow. Although more than 300 causative agents of asthma have been reported, isocyanate, especially toluene diisocyanate (TDI), is the most prevalent cause of occupational asthma worldwide. Although incidences are varied depending on the forms and types of isocyanate, it is generally reported that 5% of TDI-exposed workers could develop TDI-induced asthma . The annA European study of 142 patients with TDI-induced asthma and 50 asymptomatic exposed controls demonstrated that HLA class I alleles were not significantly associated with TDI-induced asthma . HoweverIL4RA, CD14, and IL13. Based on studies showing the involvement of neurogenic inflammation in TDI-induced asthma, we used a single base extension to screen for 2 single-nucleotide polymorphism of neurokinin 2 receptor (NK2R) gene, 7853C > T and 11424G > A, in 70 patients with TDI-induced occupational asthma, 59 asymptomatic exposed controls, and 93 unexposed healthy controls [The genes for glutathione S-transferase  and N-accontrols . No signSeveral investigators have detected IgE antibodies specific for TDI-human serum albumin (HSA) conjugate in the sera of workers showing a positive bronchial challenge response to TDI, with a reported prevalence of 0% to 50% of workers ,4,7. MaeThus, it is certain that IgE-mediated responses contribute to the development of asthmatic symptoms in TDI-induced asthmatic patients; however, additional studies are needed to develop a more effective TDI-HSA conjugate and to investigate the role of non-IgE-mediated mechanisms.A few studies have suggested that specific IgG antibodies against hexamethylene diisocyanate (HDI)-HSA and methylene diphenyl diisocyanate (MDI)-HSA conjugates play a pathogenic role in occupational asthma based on an association between IgG antibodies and the results of bronchoprovocation tests (BPTs),  whereas h2 cells occur in the bronchial mucosae of patients with TDI-induced asthma, as in patients with allergic asthma. Other immune responses to diisocyanate-related asthma include lymphoproliferative responses and cytokine/chemokine production with exposure to isocyanate [+ T lymphocytes in the airway mucosa of patients with TDI-induced asthma, and a specific T-cell line was successfully derived from T cells in the airway mucosa of a patient with TDI-induced asthma [h2 lymphocytes and other inflammatory cells, such as eosinophils, mast cells, and activated neutrophils, may contribute to the development of TDI-induced bronchoconstriction. A role of CD8+ T lymphocytes was suggested.Inflammatory reactions involving eosinophils, mast cells, and T lymphocytes, especially those bearing IL-2 receptors, ,23 and iocyanate ,25. Immuocyanate . A signiocyanate . A few sd asthma . In addid asthma . These dMounting evidence suggests that diisocyanates are able to bind airway epithelial cell proteins, resulting in airway inflammation with cytokine and chemokine production and cellular recruitment . The toxRecent microarray analyses of epithelioid cells demonstrated increased expression of cytokeratin (CK) 19 in cultured bronchial epithelial cells after TDI exposure. The prevalence of serum IgGs against CK-19 was significantly higher in TDI-induced asthma patients than in asymptomatic exposed controls or normal controls,  and the The bronchoalveolar lavage fluid of isocyanate-induced asthma patients, especially those with latent asthmatic responses, showed neutrophilia . The numA remarkable increase in exhaled nitric oxide was detected after exposure to isocyanate in TDI-induced asthma patients . ReducedRecent studies demonstrate that the pathogenic mechanism of TDI-induced asthma is complicated as various humoral and cellular mechanisms are combined and involved differently on an individual basis. More efforts should be devoted to increase the sensitivity of specific antibody detection techniques with a better TDI-HSA conjugate, and to study the cellular and molecular genetic mechanisms.NotesSupported by a grant from the Korean Health 21 R&D Project, Ministry of Health and Welfare, R.O.K. (A 050571).


Frankia form a nitrogen-fixing symbiosis with 8 different families of actinorhizal plants. We report a 5.57-Mbp draft genome sequence for Frankia sp. strain CcI6, a salt-tolerant nitrogen-fixing actinobacterium isolated from root nodules of Casurina cunninghamiana grown in Egyptian soils.Members of the actinomycete genus  Frankia are well known for their facultative lifestyles as free-living soil dwellers and as plant symbionts of wood angiosperms termed actinorhizal plants  using Illumina technology  platform developed by the Joint Genome Institute, Walnut Creek, CA (The high-quality draft genome of reek, CA , and resAYTZ00000000. The version described in this paper is version AYTZ01000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no.


Previous studies identifying predictors for transmural infarction in patients with ST-elevation myocardial infarction (STEMI) reperfused by primary percutaneous intervention (PCI), especially analyzing the time-dependency of transmural infarction, achieved inconsistent results and are limited due to small and highly-selected study samples. Furthermore it remains unclear whether transmural infarction assessed within the acute phase of STEMI is associated with adverse clinical outcome.STEMI patients reperfused by primary PCI (n=322) within 720 min after symptom-onset underwent contrast-enhanced magnetic resonance imaging (MRI) at a median of 3 days after the index event . Patients were subcategorized into tertiles according to time-to-reperfusion: lower tertile (< 175 min), middle tertile (175-320 min) and upper tertile (> 320 min). Infarct transmurality was assessed by a score with late-enhancement grading as <25%, 25-50%, 51-75% and >75% transmurality analyzing all 17 left ventricular segments. Transmural infarction was assumed if the hyperenhancement extended 75% of wall thickness in at least one segment. Clinical follow-up was performed after a median of 20 months . The primary endpoint was defined as a composite of death and congestive heart failure.Overall, transmural infarction occurred in 50.6% (n=157) of all patients. The infarct transmurality score progressed significantly with increasing ischemic time .Using multivariable logistic regression analysis including parameters such as post-PCI TIMI-flow, ST-segment resolution and maximum creatine kinase levels, time-to-reperfusion was identified as the only independent predictor for transmural infarction .Furthermore, in Cox regression analysis neither the presence of transmural infarction nor the transmurality score were associated with the occurrence of the primary composite endpoint .In STEMI patients reperfused by primary PCI time-to-reperfusion is the only independent predictor for transmural infarction. However, infarct transmurality is not associated with the occurrence of death and congestive heart failure.


Female genital tract mucosae are bathed in acidic secretions, which, in the case of HIV-1 infected women, likely contain virus coated with antibody. Thus, uninfected male sexual partners are often exposed to an acidic milieu containing virus in the form of immune complexes. We investigated the impact of low pH and antibody on transcytosis, a potentially critical mechanism by which HIV-1 passes through genital tract epithelia to infect susceptible target cells.HIV-1 was incubated with Env-specific monoclonal and polyclonal antibodies at pH 6.0 or pH 7.4 and exposed to the apical surface of tight junction-forming human endometrial carcinoma (HEC-1) cells in transwell plates. The quantity and infectivity of transcytosed virus was measured by RT-PCR and infection of TZMbl cells, respectively.We found that the combination of acidic pH and Env-specific antibody augmented transcytosis as much as 30-fold compared with Env-specific antibody at neutral pH or compared with non-specific antibody or no antibody at neutral or acidic pH. The pH and antibody dependence of enhanced transcytosis was blocked by antibody specific for the Fc neonatal receptor (FcRn) or by treatment with bafilomycin A1 (which inhibits acidification of endosomes). Non-neutralizing antibodies resulted in a lower quantity of total transcytosed virus, measured by RT-PCR, than did neutralizing antibodies. However, the ratio of total to infectious virus was much higher for neutralizing antibodies, indicating that neutralizing antibodies efficiently allow transcytosis while blocking infectivity of the transcytosed virus; the non-neutralizing antibodies facilitate transcytosis  without blocking infectivity.These results demonstrate that acidity and Env-specific antibody greatly enhance transcytosis of virus across mucosal epithelial cells via FcRn. Since male penile and urethral tissues express FcRn, our results suggest a novel mechanism by which antibody, and in particular, non-neutralizing antibody, might facilitate female-to-male transmission following sexual exposure.


In recent years MRI-guided interventional electrophysiology (EP) has rapidly emerged as a promising alternative to conventional X-ray guided ablation. We present a novel MRI-compatible electrophysiologic ablation and recording system, performing realistic biatrial simulated procedures.To date, preclinical studies evaluating MRI-guided ablation have used non-irrigated catheters limited to test lesions in the right heart. Modern complex EP procedures require externally irrigated catheters to produce adequate lesion depth. We present the first feasibility study of an externally-irrigated MRI-compatible ablation catheter, and integrated EP pacing and recording system. We demonstrate the feasibility of pulmonary vein (PV) and cavotricuspid isthmus (CTI) ablation, the targets of ablation in atrial fibrillation and typical atrial flutter respectively.An externally irrigated MRI-compatible ablation and diagnostic electrophysiology catheters and integrated EP recording system  were tested in n=11 sheep, in a 1.5T MRI scanner. The diagnostic catheters were placed in the coronary sinus, and right ventricular apex. Power controlled  lesions were formed at the pulmonary veins and cavotricuspid isthmus with the ablation catheter. Real-time intracardiac electrograms were recorded during MR imaging. Steady state free precession (SSFP) non-breath-hold images were repeatedly acquired to guide the catheter to the target region using passive catheter tracking. Markers in the catheter were used to ensure adequate visualization of the catheter. Lesion visualization was performed using both non-contrast (T2-weighted turbo spin echo pulse sequence) and gadolinium-DTPA enhanced T1-weighted imaging (inversion-recovery gradient echo pulse sequence).The catheters were able to be visualised and navigated throughout the heart. An average of 8±2.5 lesions were formed near the ostia of the pulmonary veins, and 6.5±1.3 lesions were formed at the cavotricuspid isthmus. The electrophysiological endpoint of bidirectional block was achieved in all cases. Mean procedure duration was 150±55 minutes. Histologic examination demonstrated transmurality of lesion formation at both the pulmonary vein ostia, and cavotricuspid isthmus. Lesion visualisation with both T2W imaging and contrast-enhanced imaging correlated with sites of injury at autopsy (see image). Non-contrast agent-enhanced techniques were suitable for repetitive lesion visualization during interventions, thus allowing for intra-procedural monitoring of ablation success.These data represent the most realistic simulated interventional MRI-guided electrophysiology procedures in the published literature, demonstrating the use of multiple catheters, an integrated EP pacing and recording system, and the use of an externally-irrigated ablation catheter. For the first time, the feasibility of CMR guided pulmonary vein ablation is shown, opening the possibility of MR-guided complex electrophysiology procedures, including pulmonary vein isolation for atrial fibrillation.Dr Ganesan is funded by a Cardiovascular Lipid award.


Among paraneoplastic neurologic disorders (PND), opsoclonus-myoclonus syndrome, so-called “dancing eye syndrome,” is a rare disorder combining multivectorial eye movements, involuntary multifocal myoclonus, and cerebellar ataxia. Although several paraneoplastic antibodies against postsynaptic or cell-surface antigens have been reported, usually most patients are serum antibody negative. We report a 65-year-old patient with opsoclonus-myoclonus syndrome revealing a small-cell lung carcinoma. If serologic antineuronal anti-body screening was negative, autoantibodies against glutamic acid decarboxylase (anti-GAD) were positive. Despite the specific anticancer treatment and high dose corticosteroids, the patient developed a severe and progressive encephalopathy and died 10 days later. Opsoclonus-myoclonus syndrome (OMS) is characterized by rapid, involuntary multivectorial eye movements without intersaccadic intervals. It is associated with involuntary multifocal myoclonus mainly affecting the trunk, limbs, and head. Cerebellar ataxia and uncoordinated voluntary movements may also be seen. Pathophysiologically OMS appears to be an immune-mediated disease where positive titres directed at anti-neuronal antibodies in the serum and cerebrospinal fluid (CSF) are occasionally detected. OMS has been linked to infections and metabolic disorders and has been documented as a paraneoplastic syndrome.The paraneoplastic opsoclonus-myoclonus syndrome, also called “dancing eyes syndrome,” is a rare entity among the paraneoplastic neurologic disorders (PND) . OMS is In this paper, we report the case of a male patient with pathologically proven small-cell lung cancer who presented with symptoms compatible with OMS. This was the first clinical manifestation of an underlying malignancy. He demonstrated a rapid deterioration in his clinical state leading to death despite the introduction of chemotherapy active against the small-cell lung cancer.A 65-year-old man presented to the emergency department of our institution with a history of left hemithoracic pain, weight loss, gait ataxia, and tremor. He had a 120-pack-year smoking history and known ischemic cardiomyopathy with previous coronary stenting.On physical examination he had a reduced performance status  and neurological assessment revealed tremor, gait ataxia, and mild involuntary limb jerking which resulted in the patient remaining bedbound despite any evidence of cognitive impairment and sensory or motor deficit. The initial chest X-ray demonstrated an enlarged left hilum. Thoracic imaging by computed tomography (CT) revealed a left hilar mass measuring 26 mm with associated left hilar and mediastinal lymphadenopathy (>1 cm short axis) . EndobroFurther workup for a potential cerebellar paraneoplastic disorder revealed a normal electroencephalography, normal cell counts, protein levels, viral markers, and negative cytology in the cerebrospinal fluid (CSF) analysis. Magnetic resonance imaging (MRI) of the brain did not reveal ischemic changes, metastatic lesions, or abnormal enhancement with gadolinium . Brain 1As the index of suspicion for PND remained high, we performed serologic anti-neuronal antibody screening. Anti-Hu, Ant-Ri, anti-Yo, anti-CV2, anti-Ma1, anti-Ma2, anti-amphiphysin, and anti-VGKC were all negative. A serum glutamic acid decarboxylase antibody radio immunoassay (GAD65 Ab Assay—AntiGAD Antibodies blood test) was positive (1.1 U/mL) .Chemotherapy was commenced using the combination of carboplatinum and etoposide with intravenous corticosteroids. Cisplatinum administration was contraindicated because of the recent history of ischemic cardiomyopathy and a measured ventricular ejection fraction at 40%. Despite the initiation of specific treatment of the underlying malignancy, dramatic neurologic deterioration occurred and the patient developed multivectorial saccadic eye movements with associated massive myoclonic head, trunk, and limb movements. This deterioration resulted in a comatose state necessitating endotracheal intubation, sedation, and transfer to the intensive care unit.The patient died 10 days later due to severe and progressive encephalopathy despite the specific anticancer treatment and high dose corticosteroids.Paraneoplastic OMS has been associated with SCLC and breast and ovarian malignancies in adults and antineuronal antibodies have been occasionally detected , 7. AmonOMS is an involuntary multidirectional eye movement syndrome  accompanied by myoclonic jerks . Cerebellar ataxia (POMA syndrome), tremor, and encephalopathy are common. As with the majority of PND, OMS precedes the diagnosis of cancer (in up to two-thirds of cases), but it can also be detected during the follow-up period suggesting recurrent disease . The patIn our case the patient initially presented with cerebellar symptoms. Investigations were consistent with a cerebellar paraneoplastic disorder supported by a histologically proven diagnosis of SCLC, brain 18-FDG-PET findings, and positive anti-GAD antibodies , 13. OurThe rapid progression towards OMS with multidirectional eye movements and jerking body movements and ultimately coma strongly suggests a paraneoplastic phenomenon, even if standard serological screening for anti-neuronal antibodies associated with OMS remained negative. It is therefore not surprising that current recommendations are to repeat the screening tests every six months if malignancy is not initially discovered . HoweverIn summary we believe that new approaches to detect anti-neuronal antibodies will allow faster detection of the underlying malignancy of PND leading to an earlier treatment of the disease with better outcomes for the patients.


Development of treatments for acute and chronic pain conditions remains a challenge, with an unmet need for improved sensitivity and reproducibility in measuring pain in patients. Here we used pulsed-continuous arterial spin-labelling [pCASL], a relatively novel perfusion magnetic-resonance imaging technique, in conjunction with a commonly-used post-surgical model, to measure changes in regional cerebral blood flow [rCBF] associated with the experience of being in ongoing pain. We demonstrate repeatable, reproducible assessment of ongoing pain that is independent of patient self-report. In a cross-over trial design, 16 participants requiring bilateral removal of lower-jaw third molars underwent pain-free pre-surgical pCASL scans. Following extraction of either left or right tooth, repeat scans were acquired during post-operative ongoing pain. When pain-free following surgical recovery, the pre/post-surgical scanning procedure was repeated for the remaining tooth. Voxelwise statistical comparison of pre and post-surgical scans was performed to reveal rCBF changes representing ongoing pain. In addition, rCBF values in predefined pain and control brain regions were obtained. rCBF increases (5–10%) representing post-surgical ongoing pain were identified bilaterally in a network including primary and secondary somatosensory, insula and cingulate cortices, thalamus, amygdala, hippocampus, midbrain and brainstem , but not in a control region in visual cortex. rCBF changes were reproducible, with no rCBF differences identified across scans within-session or between post-surgical pain sessions. This is the first report of the cerebral representation of ongoing post-surgical pain without the need for exogenous tracers. Regions of rCBF increases are plausibly associated with pain and the technique is reproducible, providing an attractive proposition for testing interventions for on-going pain that do not rely solely on patient self-report. Our findings have the potential to improve our understanding of the cerebral representation of persistent painful conditions, leading to improved identification of specific patient sub-types and implementation of mechanism-based treatments. As many as 80% of individuals experience moderate to severe post-operative painModern neuroimaging techniques, such as Positron Emission Tomography (PET) and functional Magnetic Resonance Imaging (fMRI), show great promise in the development of novel measurement techniques, allowing non-invasive investigation of the cerebral mechanisms underpinning the pain experience. Many imaging studies to date, however, have relied on ‘experimental pain’ models using healthy volunteers to derive brain responses to acute, repeated, short-duration nociceptive stimuli , have limited the impact of their findings. Similarly, reports using Blood Oxygenation Level Dependent [BOLD] fMRI, for exampleCompared to studies examining responses to evoked pain, there are relatively few neuroimaging reports describing the cerebral representation of ongoing pain; fewer still describe clinical ongoing pain. There are several reports using PET, for example Here we assess the validity of pulsed-continuous ASL [pCASL] a priori. Further, we provide novel insights into the central representation of post-surgical trigeminal pain in humans. Our findings are discussed in terms of their potential impact on development of novel interventions for treatment of acute and chronic pain conditions and how the pCASL technique might be utilised in translational research.In this study we applied pCASL to the challenge of representing the cerebral basis of ongoing pain. We imposed three constraints, namely that the ongoing pain experience was induced by genuine tissue damage, could not be modulated by the experimenter within a single session, and that assessments of ongoing pain could be repeated to fulfil the requirements of a cross-over trial design. We demonstrate quantitative, reproducible rCBF increases that represent the experience of being in ongoing pain following TME including those in a network of brain regions specified This study was approved by Kings College Hospital Research Ethics Committee (REC Reference 07/H0808/115).16 right-handed, healthy male volunteers aged 20–41, (mean = 26.4 years) provided informed consent to participate in the study. Females were excluded due to potential variability in the phase of the menstrual cycle affecting reproducibility of the response to post-surgical painParticipants were scanned on five separate occasions (S1–S5); screening/familiarisation (S1), pre-surgical (S2) and post-surgical sessions (S3) for the first extraction and pre-surgical (S4) and post-surgical (S5) sessions for the second extraction. An interval of at least two weeks separated S3 and S4, following complete recovery from the first surgery. Order of left and right tooth extraction was balanced and pseudo-randomised across the group. At each session, pulse rate and blood pressure were recorded, an alcohol and drug-screen performed and a psychometric assessment completed. Analgesic medication (1000 mg paracetamol & 400 mg ibuprofen) was provided to participants immediately following scanning during S3 & S5.At S1, standardised screening questionnaires were administered to assess presence of any pain and baseline psychometric information (see Baseline Psychometry). A short MR examination was performed for familiarisation with the imaging environment and participants received training on using a computerised, joystick-operated visual analogue scale (VAS). MR examinations during sessions S2–S6 were identical, each comprised of six separate consecutive pCASL scans, each lasting six minutes. Participants were instructed to lie still with their eyes open. Prior to and following acquisition of each rCBF map, participants subjectively rated pain intensity and alertness using a computerised VAS.Baseline psychometric screening assessments were performed for all participants prior to scanning at S1. Screening for depression was performed using the Center for Epidemiological Studies Depression Scale [CES-D]Unilateral TME was performed under local anaesthesia  using a standardized technique. Surgical difficulty was rated on a 1-5 scaleImaging was performed on a 3 Tesla Signa HDx whole-body MR imaging system  fitted with an 8-channel, phased-array receive-only head coil. High-resolution T1- and T2-weighted MR structural sequences were acquired for radiological assessment and image registration. Resting-state rCBF measurements were made using pCASLhttp://www.fmrib.ox.ac.uk/fsl]Preprocessing and analysis were performed using FSL v4.1.0 , primary [S1], and secondary [SII] somatosensory cortices, insula [INS], thalamus [THAL], amygdala [AMY] and hippocampus [HIP] in each cerebral hemisphere. Finally, an ROI was created for V5/MT, an a priori-defined, comparably-sized control ROI involved in visual motion perception and eye movementsAnatomical ROIs in MNI-template space were derived from Harvard-Oxford Cortical/Subcortical and Juelich-Histological Atlases. Based on Two ROI datasets were created. In both datasets, the mean of the 20% voxels with greatest CBF values was computedAll ROI analyses were performed using GenStat v11.1.Temporal variation within-session rCBF values extracted from set one were plotted to examine temporal variation in rCBF value within a single session. For each ROI in each hemisphere, rCBF estimates from each pCASL scan were fitted to a mixed effect model, with Participant, Participant-by-Session, Participant-by-Session-by-Time and Participant-by-Session-by-Hemisphere as random effects, Session-Pair (Pair 1/Pair 2) as fixed effect, and a 3-way factorial of Surgery (Pre-surgery/Left/Right tooth Post-surgery) Hemiphere (Left/Right) and Timepoint (1–6). P-values were Bonferroni corrected for multiple comparisons.For each ROI in each hemisphere, rCBF values for each subject in each session were fitted to a mixed effect model. Participant and Participant-by-Session were fitted as random effects, and Surgery (Pre-surgery/Left/Right-tooth post-surgery), Session-pair  and Hemisphere (Left/Right) were fitted as fixed effects. Significance thresholds were imposed after Bonferroni correction.w) for each ROIFor each ROI, an ANCOVA model was fitted to rCBF values obtained from each hemisphere in set two. Subject was fitted as a fixed effect and VAS estimate of pain [VAS] fitted as a covariate. The model was used to calculate intra-subject correlation co-efficients (ρThere were no differences relating to site of surgery (left versus right). Perceived surgical difficulty and time taken from local anaesthesia to first CBF map did not differ between left and right surgeries . = 432.99, p<0.001), but there were no differences following extraction of left, compared to right, third molars (p = 0.97). There was no session order effect (p = 0.55).Mean alertness ratings did not differ between pre- and post-surgical MRIs , . There wa priori would demonstrate CBF changes during post-surgical pain; for brevity, only clusters with highest Z-scores per anatomical region have been reported. We did not observe any post-surgical decreases in CBF in these regions or elsewhere. In particular, bilateral increases in rCBF during post-surgical pain were identified in postcentral gyrus, specifically the somatotopic region of S1 relating to the face/jawA distributed network of brain regions demonstrated significant increases in rCBF relating to pain following extraction of left and right third molars, compared to pain-free pre-surgical periods in the same subjects. a priori. In the frontal lobe, clusters were identified in superior, middle, medial and orbital-frontal cortices, in precentral gyrus and superior and inferior parietal lobules bilaterally. In the temporal lobe, bilateral regions of increased CBF were identified in superior, middle inferior temporal and fusiform gyri, and in the lingual gyrus and precuneus in the occipital lobe. In the basal ganglia, clusters were identified in caudate and lentiform nuclei bilaterally. In the brainstem, increased post-surgical CBF was identified bilaterally adjacent to the lateral mid-pons, approximating to the trigeminal ganglion/roots  was identified within a single session. There were no other significant second or third order interactions of Time with Hemisphere, or Surgery, indicating that within-session temporal variation across pCASL scans did not differ between cerebral hemispheres, either in pre-surgical or post-surgical scanning sessions following either left or right TME. In the light of these findings, assessment of between session variation in rCBF was studied using ROIs derived from set two, the average of all 6 cASL maps acquired within a single session.The anatomical location of each ROI, post-surgical CBF change and associated time courses is illustrated in Mixed effect models were computed for all pain and control ROIs. Main effects and interactions for ROIs are summarised in w) were computed for each ROI in each hemisphere to assess the relationship between post-surgical pain rCBF and patients' self-reported pain VAS scores. Significant linear relationships were identified in AMY, HIP, S1, SII, THAL, INS, PCC & ACC ROIs, (Within-subject correlation co-efficients (ρCC ROIs,  but not a priori hypothesised regions inherent in the central processing of pain, but not in control brain regions hypothesised to be unchanged by pain. rCBF assessments were stable within a single session and there were no between-session differences in post-surgical rCBF following extraction of left, compared to right, teeth, indicating a viable test-retest paradigm. Post-surgical CBF changes correlated with VAS estimates of self-reported pain, but only in brain regions known to underpin the processing of pain and not in a control brain region. Quantitative changes in rCBF that represent ongoing pain have potential as markers of treatment efficacy for acute and persistent painful conditions.Using pCASL, we have demonstrated reproducible, rCBF-derived markers of ongoing, clinically-relevant pain. Increases in rCBF were established following surgery, compared to pain-free pre-surgical periods, in an un-biased voxel-wise analysis and in Our findings of rCBF increases during pain following TME provide valuable new insights into the representation of ongoing post-surgical trigeminal pain. Independently of site of removal, the pain resulting from tooth extraction is represented by a largely bilateral pattern of rCBF changes throughout the brain. No hemispheric differences in rCBF changes related to extraction were found. These findings differ from earlier pain studies using PET imaging, which have largely reported rCBF changes contralateral to the painful body-site, for example, contralateral increases in rCBF in PFC, insula cortex, and lentiform nucleus were reported following a composite third molar extraction and thermal heat pain challengeOur finding of bilateral post-surgical rCBF increases in S1 is supported by primate electrophysiological studies of S1 neurones with bilateral receptive fieldsDemonstration of local increases in CBF in Vp during post-surgical, ongoing trigeminal pain echo recent reports of changes in brain activation in Vp in preclinical studiesTaken together, our findings have potential to impact positively upon the role of neuroimaging in assessing novel treatments for painOur finding of reproducible rCBF data, within and between sessions, makes ‘cross-over’ assessments of pain treatments tenable. A critical next step to develop ASL as a methodology for assessing modulation of ongoing pain will be to demonstrate pain-related CBF changes that are attenuated by an analgesic of known efficacy. Successful demonstration of analgesic-modulated CBF changes should provide the evidence necessary to refine decision-making techniques for assessing efficacy of novel interventions. We envisage several potential uses for the pCASL methodologya priori ROIs based on previous knowledge of the specific pain condition should be preferred. While we believe ASL has utility in analgesic trials, the method should be equally applicable to assessing changes in ongoing pain in other, non-pharmaceutical scenarios; for example, pain modulation following cognitive behavioural therapyImproved knowledge of acute ongoing pain should impact upon understanding the central representation of chronic pain; bridging this gap might facilitate developing new medications for intractable pain conditions that are often resistant to currently approved analgesicsIn summary, using perfusion MRI, in concert with the TME model, we have described a network of rCBF increases representing ongoing post-surgical pain. Post-surgical CBF changes are reproducible within- and between sessions. Our findings represent the beginning of a novel approach to measure ongoing pain as an alternative to self-report. The approach is stable and provides robust, repeatable results in a relatively small group of participants, compared to conventional studies solely using self-reported pain as endpoints


In economically developed countries, AIDS-related lymphoma (ARL) accounts for a large proportion of malignances in HIV-infected individuals. Since the introduction of highly active anti-retroviral therapy (HAART) in 1996, epidemiology and prognosis of ARL have changed. While there is a slight increase in the incidence of Hodgkin's lymphoma in HIV-infected individuals, use of HAART has contributed to a decline in the incidence of non-Hodgkin's lymphoma (NHL) and also a decrease in the overall incidence of ARL. Strategies that employ HAART, improved supportive care, and the use of Rituximab with multi-agent chemotherapy have contributed to improved rates of complete remission and survival of patients with ARL that rival those seen in stage and histology matched HIV negative NHL patients. Most recent clinical trials demonstrate better outcomes with the use of rituximab in ARL. Tumor histogenesis  is associated with lymphoma-specific outcomes in the setting of AIDS-related diffuse-large B cell lymphoma. High-dose chemotherapy (HDCT) and autologous stem cell rescue (ASCT) can be effective for a subset of patients with relapsed ARL. HIV sero-status alone should not preclude consideration of ASCT in the setting of ARL relapse. Clinical trials investigating the role of allogeneic hematopoietic stem cell transplant in ARL are currently underway. Non-Hodgkin's lymphoma (NHL) has been associated with human immunodeficiency virus (HIV) infections since the beginning of the acquired immune deficiency syndrome (AIDS) epidemic. The initial case definition of AIDS by the US Centers for Disease Control and Prevention (CDC) in 1982 included AIDS-defining malignancies such as Kaposi's sarcoma and primary central nervous system lymphoma (PCNSL), with subsequent inclusion of peripheral intermediate and high-grade B-cell NHL ..13].HIV creates a milieu of combined immune suppression and chronic antigenic stimulation in lymph nodes . This en-MYC oncogene, inactivation of p53 tumor-suppressor gene, somatic mutations in Bcl-6, and overexpression of EBV oncoprotein 4 . These chromosome breaks and/or molecular lesions likely have significant downstream effects which lead to impaired lymphocyte differentiation and cell-cycle control [The cytokine profile of HIV-infected individuals at risk for ARL demonstrates increased markers of B-cell activation such as interleukin- (IL-) 6, IL-10, and soluble CD-30 compared to HIV-positive controls who do not develop ARL . Althoug control , 17. Rec control , 19. Use control , 21. HAA control . μL, age older than 35, history of injection drug use, poor performance status, elevated serum lactate dehydrogenase (LDH), advanced stage of disease, and prior AIDS diagnosis [Early in the AIDS epidemic the clinical course of ARL was dominated by advanced stage disease, concomitant and life-threatening opportunistic infections (OIs), and poor response to treatment. Efforts to treat patients with ARL using aggressive and complex chemotherapy regimens led to unacceptable toxicity and early death while low-dose chemotherapy regimens yielded modest benefit—only 10% of patients survived for 2 years . Variousiagnosis . ExpectaμL had a predicted 1-year survival of 82% compared to 15% in those with an unfavorable IPI and CD4+ count <100 cells/μL [μL), and the IPI [Control of HIV viral replication through HAART has emerged as a major positive prognostic factor for patients with ARL with several studies showing dramatic improvements in OS among those who received HAART –30. The cells/μL . Furthercells/μL . A time- the IPI . More reARLs are aggressive lymphomas and roughly 80% of patients present with advanced stage disease. Half of these patients will have gastrointestinal involvement and 30% will have bone marrow involvement . In addiSeveral prospective studies have reported using regimens such as cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP); methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, and dexamethasone (m-BACOD); infusional cyclophosphamide, doxorubicin, and etoposide , 23, 36.The introduction of HAART has led to dramatic improvement s in morbidity and mortality for PLWA. Such individuals now have an OS that is near or comparable to that of HIV seronegative individuals , 37. The use of HAART has facilitated the use of standard-dose and even dose-intensive chemotherapy regimens with reasonable safety to patients with ARL. With use of HAART, leading to improvements in HIV viral suppression and patients' immune status, more recent studies have demonstrated that patients with ARL can be treated with standard-dose lymphoma protocols without experiencing undue toxicity , 38, 39.μL, prior OI and performance status ≥3. Standard-risk group patients had a CR of 79%, similar to that achieved in non-HIV infected patients with similar histologically aggressive lymphomas, while the CR was 29% in the high-risk group [μL. A total of 218 good-risk patients (HIV score 0) received ACVBP  or CHOP; 177 intermediate-risk patients (HIV score 1) received CHOP or low-dose CHOP (Ld-CHOP); 90 poor-risk patients (HIV score 2-3) received Ld-CHOP or VS (vincristine and steroid). The 5-year OS in the good-risk group was 51% for ACVBP versus 47% for CHOP; in the intermediate-risk group, 28% for CHOP versus 24% for Ld-CHOP; in the poor-risk group, 11% for Ld-CHOP versus 3% for vs. The only significant factors for OS were HAART therapy, HIV score, and the IPI score, but not chemotherapy regimen, suggesting that in ARL patients, HIV score, IPI score, and HAART affect survival but not the dose intensity of the CHOP-based chemotherapy.The United States National Cancer Institute (NCI)-sponsored AIDS Malignancy Clinical Trials Consortium (AMC) compared dose-reduced or full-dose cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) with concomitant HAART with complete remission (CR) of 30% and 48% in the reduced dose and full-dose groups, respectively . The Gersk group . A more Infusional therapy may overcome some drug resistance and high tumor proliferation in aggressive lymphomas. A study of infusional dose-adjusted EPOCH  in 39 newly diagnosed ARL reported a CR in 74% of patients, and at a median followup of 53 months, disease-free survival (DFS), and OS of 92% and 60%, respectively [P = 0.147), there were also substantially more deaths due to treatment-related infections in the rituximab arm  with the majority of those deaths occurring in patients with CD4+ counts <50 cells/μL at study entry. A number of recent clinical trials have further sought to combine anti-CD20 chimeric monoclonal antibody (rituximab) with ARL-based chemotherapy based on its superior efficacy when added to treatment of lymphoma in non-HIV infected patients. In a pooled analysis of three ARL prospective phase II studies evaluating rituximab in combination with infusional CDE, the CR was 70% and the estimated 2-year FFS and OS rates were 59% and 64%, respectively . An incrIn contrast to AMC 010, a phase II study from France did not show an increase in the risk of life-threatening infections while treating newly diagnosed ARL patients with R-CHOP [μL had a high infectious death rate in the concurrent arm. The investigators concluded that concurrent rituximab and infusional EPOCH is an effective regimen for ARL that merits further evaluation, but that caution is needed for those patients with severe immunodeficiency. Rituximab has also been evaluated in conjunction with infusional EPOCH. In a randomized phase II AMC trial 034, rituximab was given either concurrently just before each infusional EPOCH chemotherapy cycle or sequentially (weekly for 6 weeks) after completion of all chemotherapy (4–6 cycles) in ARL . CR was To summarize, rituximab has been shown to improve response rates and overall survival for patients with aggressive NHL who were HIV negative and treated with CHOP, without an increased risk of infectious complications. Rituximab, when given sequentially with chemotherapy, is associated with improved control of ARL; however, in a single clinical trial, the use of rituximab accounted for a higher risk of severe infection than has been seen for patients with NHL who were HIV negative. Several more recent ARL studies combining chemotherapy with rituximab have not, however, led to an increased risk of death due to infectious complications. μL. Mature results from an ECOG study which randomizes non-HIV-infected patients with DLBCL to R-EPOCH or RCHOP will likely influence the way ARL is treated [Most oncologists favor administration of rituximab with chemotherapy, although caution should be exercised for using rituximab in severely immunosuppressed patients with CD4+ counts of less than 50 cells/ treated . R-CHOP Several ongoing clinical trials are evaluating novel chemotherapeutic combinations with biologic agents in this setting of ARL as listed in The appropriate timing of institution of HAART in relationship to multiagent chemotherapy has been evaluated in several clinical trials but with no clear answers. In a small study involving ARL patients, antiretroviral control was a significant factor in the ability to attain CR . In a moChemotherapy without concomitant antiretroviral therapy also has been studied in the HAART era. Reasons for HAART omission include concerns of drug interactions with chemotherapy and poor patient adherence because of nausea or vomiting. This could lead to heightened toxicity and the emergence of multidrug-resistant HIV quasispecies. In the NCI EPOCH study, HIV viral load increased and CD4+ cell counts decreased while patients received chemotherapy, but both parameters improved rapidly following the reintroduction of HAART at the completion of chemotherapy . In the Prospective studies of ARL generally have combined the high-grade DLBCL and Burkitt lymphoma/Burkitt-like lymphoma histologies, with little difference in outcome between the subtypes. In contrast, two retrospective reviews demonstrated substantially inferior outcomes for AIDS-associated Burkitts lymphoma in the HAART era in comparison with HIV-associated DLBCL when CHOP-like treatments were used . Median 9/L was 11 days . The median time to achievement of an unsupported platelet count of at least 20 × 109/L was 13 days . One patient died on day +33 after ASCT as a result of hepatic and renal occlusive disease and multiorgan failure. No other fatal regimen-related toxicity occurred and 10 (53%) of 19 evaluable patients were in complete remission at day +100. Optimal chemotherapy for patients with relapsed/refractory ARL has not been defined and such patients are usually treated with regimens that are used for non-HIV-infected patients with relapsed/refractory aggressive lymphoma, such as ifosfamide, cisplatin and etopside (ICE), or etoposide, solumedrol, high-dose cytosine arabinoside, platinum (ESHAP) , 48. The2, and etoposide 60 mg/kg). The median time to engraftment was 10 days . Treatment-related toxicities between the groups were comparable and 2-year overall survival at 75% was the same for both groups. Recently, investigators from the City of Hope compared their experiences treating 29 consecutive patients with relapsed or progressive ARL who underwent ASCT to age, gender, histology, and stage-matched NHL controls who also underwent ASCT . The medP = 0.18), 2-year DFS  and the 2-year point estimates of OS  were comparable to matched HIV-negative NHL controls [European investigators have also reported results involving 53 HIV-positive and 53 HIV-negative matched lymphoma patients who underwent ASCT . Incidencontrols . These scontrols , 56–59. Gene therapy is an emerging technology that holds promise for patients with relapsed ARL. Recently, a stable expression of a lentiviral vector encoding anti-HIV RNAs in blood stem cells was transplanted into four such patients who were undergoing HDT followed by ASCT . They eaAttention to supportive care measures is essential for patients with ARL. Judicious use of hematopoietic stimulants including the newest formulation of pegylated granulocyte-colony stimulating factor (G-CSF) may help ameliorate chemotherapy-induced neutropenia. The US Food and Drug Administration (FDA) has issued a warning to medical providers that erythropoiesis-stimulating agents  are associated with a high rate of thrombosis for patients with cancer . For the To minimize cardiac toxicities, pegylated anthracyclines have been used for the treatment of ARL, and liposomal anthracyclines also may offer some of the pharmacokinetic benefits of infusional doxorubicin but without the need for patients to be connected to cumbersome infusion pumps for several days' duration. Single institution studies suggested that substituting liposomal anthracyclines for doxorubicin in multiagent lymphoma protocols was both safe and effective , 63. AMCjiroveci prophylaxis  regardless of initial CD4+ cell count. Trimethoprim-Sulfamethoxazole is infrequently used because of its potential to exacerbate myelosuppresasion. Infectious complications associated with ARL may be minimized by using prophylactic fluroquinolone antibiotics and azoles during periods of protracted neutropenia. All patients should receive Pneumocystitis With longer life expectancy following chemotherapy, late-treatment-related complications are emerging as a serious concern after treatment for ARL. Chemotherapy-related acute myelogenous leukemia  has been reported in an HIV-positive individual 48 months after therapy with R-EPOCH for ARL . SimilarEpidemiologic reports clearly indicate that the incidence of ARL has decreased since the introduction of HAART among patients with AIDS. Chemo-immunotherapy for the most common subtype of ARL, DLBCL, is improving, with several phase II clinical trials showing good outcomes employing R-CHOP or R-EPOCH. CR rates for those patients with good prognosis features rival those of similarly matched non- HIV infected patients with DLBCL. Better supportive care, employing the use of G-CSF, prophylactic antibiotics, and HAART either during or shortly after completion of chemotherapy may ameliorate the increased toxicity of concurrent immunotherapy, thereby improving survival of patients with ARL. Clinical trials investigating the role of autologous and allogeneic HSCT in relapsed/refractory ARL are currently underway. With effective treatments and longer life expectancy following chemotherapy, late-treatment-related complications are emerging, and how best to mitigate and manage these complications is an emerging challenge.