Intercellular signaling by cytokines is a vital feature of the innate immune system. In skin, an inflammatory response is mediated by cytokines and an entwined network of cellular communication between T-cells and epidermal keratinocytes. Dysregulated cytokine production, orchestrated by activated T-cells homing to the skin, is believed to be the main cause of psoriasis, a common inflammatory skin disorder. Cytokines are heavily regulated at the transcriptional level, but emerging evidence suggests that regulatory mechanisms that operate after transcription play a key role in balancing the production of cytokines. Herein, we review the nature of cytokine signaling in psoriasis with particular emphasis on regulation by mRNA destabilizing elements and the potential targeting of cytokine-encoding mRNAs by miRNAs. The proposed linkage between mRNA decay mediated by AU-rich elements and miRNA association is described and discussed as a possible general feature of cytokine regulation in skin. Moreover, we describe the latest attempts to therapeutically target cytokines at the RNA level in psoriasis by exploiting the cellular RNA interference machinery. The applicability of cytokine-encoding mRNAs as future clinical drug targets is evaluated, and advances and obstacles related to topical administration of RNA-based drugs targeting the cytokine circuit in psoriasis are described. Cytokines are intercellular signaling proteins that serve as key modulators of the immune system and inflammation. Cells respond to extracellular stress or stimuli by operating intracellular signaling cascades that coordinate cellular gene expression through complex networks of kinase activation, protein phosphorylations, and activation of DNA-binding proteins that translate signals at the cell surface to actions of transcriptional regulation of cellular genes. Cytokines modulate the communication between cells of the immune system and between immune cells and differentiated somatic cells. Upon binding to their cognate receptor on the cell surface, cytokines trigger transcriptional changes and balance cellular activities ranging from growth to differentiation and cell survival. Cytokine-directed transcriptional induction of yet other cytokines may further enhance the innate immune response in an increasingly entangled network of signals.Genome-wide association studies have shown that mutations of genes encoding cytokines, cytokine receptors, or downstream players in the cellular signaling cascades associated with autoimmune disease. Effectors of the different signaling cascades and the transcriptional regulation operated through these pathways have been reviewed at numerous occasions. In this review, we concentrate exclusively on the posttranscriptional mechanisms that act together to balance the expression of cytokines during inflammation. The discovery of RNA interference and the regulatory actions of small RNAs have unveiled a new world of posttranscriptional regulation and yet new layers of complexity in cellular signaling pathways that are in play during inflammation. Small non-coding RNA species, produced from intronic and intergenic regions across the mammalian genome, are key players in post-transcriptional regulatory pathways of gene expression. MicroRNAs (miRNAs) interact with mRNAs and trigger translational suppression or mRNA degradation through recruitment of cellular proteins. Short-lived RNA transcripts, such as several cytokine-encoding mRNAs, contain RNA destabilizing elements and regulatory miRNA binding motifs that may in concert contribute to stringent regulation of cytokine production. Dysregulated cytokine production is a hallmark of tissues affected by chronic inflammatory disease, and miRNAs are likely to play important, but hitherto vaguely characterized, roles in aberrant cytokine regulation and disease development and progression. With emphasis on skin inflammation and psoriasis vulgaris in particular (referred to as psoriasis in the remainder of the review), we focus here on the regulation of cytokines at the RNA level in relation to development of inflammatory disease. We provide an overview of the network of cytokine signaling in psoriasis and its regulation through induced RNA destabilization and miRNA association and the potential linkage between mRNA decay and targeting by miRNA. The applicability of cytokine-encoding mRNAs as future therapeutic targets is evaluated, and obstacles and advances related to topical administration of RNA-based drugs targeting the cytokine circuit in psoriasis are described.Psoriasis is an idiopathic chronic skin disorder which has been estimated to affect about 2% of the population world-wide -3. The dThe epidermal hyperplasia is associated with underexpression of keratinocyte differentiation markers, which causes incomplete differentiation of keratinocytes (parakeratosis). Histopathologically this shows as retention of nuclei of cells in the stratum corneum (the outer stratified cell layer) and an overall thickening of the epidermis (acanthosis). The transit time of keratinocytes from the basal cell layer of the epidermis to the stratum corneum is reduced from 28 days to about 4-7 days in psoriatic lesions.Many advances have been made in recent years in unraveling the molecular mechanisms of psoriasis, but many questions still remain unanswered. It is still not known if the primary nature of the condition is epithelial or immunologic and the autoimmune cause of the inflammation is still to be uncovered . Moreoveh cells). Naïve CD4+ T-cells that are activated through interaction with antigen-presenting cells (APCs) - primarily dendritic cells and macrophages - play a dominant role in both initiation and persistence of the disease. Depending on the exposure to different cytokines, naïve T-cells may develop into one of the at least five different CD4+ T-cell lineages that have been identified to date. Th1 cells play a key role in clearance of intracellular pathogens, whereas Th2 cells trigger and organize a defense response against extracellular pathogens. The Th17 and Th22 lineages, the latter which was just recently discovered [h1 and Th2 lineages in inducing an inflammatory response, whereas Treg cells serve to limit the immune response by production of immunosuppressive cytokines [Psoriasis is the result of an intensifying cross-talk between skin-infiltrating immunocytes and epidermal keratinocytes. An overview of the cell types and primary cytokines involved in this intercellular communication is provided in Figure scovered , assist ytokines .h1/Th2 model by which naïve Th cells differentiate into either Th1 or Th2 cells producing the defining cytokines interferon gamma (IFNγ) and interleukin (IL)-4, respectively, psoriasis was classified as a Th1 disease with a marked type 1 cytokine profile [h1 to Th2 cytokine profile was found to ameliorate the disease [h cells, Th17 cells [h1/Th17 disease. Th17 cells are named according to their production of IL-17, which in turn induces the production of other proinflammatory cytokines, such as TNFα, IL-6, IL-8 and IL-22 , IL-4 (Th2), IL17 (Th17), and IL-22 (Th22), respectively, subsets of Th cells expressing two or even three of these cytokines can be identified [h cells may not necessarily represent distinct lineages but possibly Th cells at different levels of differentiation [h-directed orchestration of the innate immune response in psoriatic skin.According to the classical T profile . This mo disease . However17 cells , which s17 cells , a new por IL-17 , has cauic cells , home prentified . It has ntiation , adding Sabat and co-workers proposed a model for the pathogenesis of psoriasis consisting of three phases . The senCurrent therapies for psoriasis include topical agents, phototherapy and systemic therapies. Topical therapies are mainly used for mild psoriasis and in addition to the widely used corticosteroids, keratolytic ointments, topical retinoids and vitamin D analogues are also used . Photothh differentiation , have been linked to psoriasis [More recently, drugs simultaneously targeting IL-12 and IL-23 have been developed. Both IL-12 and IL-23 are up-regulated in psoriatic lesional skin . As heteh1 cells . Therebyoduction . IL-23, inocytes . This susoriasis  and manysoriasis , Etanercsoriasis  and Alefsoriasis  all redusoriasis .In spite of the recent success of biological cytokine-targeting drugs, there is still a continued need for psoriasis treatments that are safe, effective and convenient. Patients may respond differently to biological agents, and relapses may occur in some patients. Furthermore, there is an increased occurrence of opportunistic infections associated with the general immunosuppressive action of these highly potent cytokine inhibitors. Hence, more evidence is needed concerning the long-term safety of such biological agents. However, the encouraging clinical experience with anti-cytokine treatment has prompted the search for and development of inhibitors of other psoriasis-related cytokines. As a recent example of this, findings in a xenotransplantation mouse model suggest that blockage of IL-20 signaling by systemic administration of anti-IL20 antibodies leads to resolution of psoriasis .The regulation of cytokine production is the very foundation of the innate immune response during stress, infection, and injury. Simultaneous transcriptional regulation by several sets of genes affected by cellular signaling pathways is conceived by a network of transcription factors, co-regulators, and epigenetic moderators. Signaling routes and transcriptional control have been covered in numerous reviews  which are adenosine- and uridine-rich sequences in the 3' UTR of not only multiple cytokines, but also of numerous other genes - as much as 5-8% of human genes . ComputaThe instability of ARE-containing mRNAs is regulated by ARE-binding proteins (AUBPs) that belong to different protein families and among which some promote destabilization whereas others stabilize mRNAs. These proteins modulate different aspects of mRNA metabolism including deadenylation, exosome binding, or decapping and do not seem to execute the RNA degradation themselves. Instead AUBPs may facilitate mRNA recognition by ribonucleases, including 3'-5' exoribonucleases in the multicomponent exosome complex  or 5'-3'Tristetraprolin (TTP), a member of the TIS11 protein family, is a prominent AUBP that induces mRNA destabilization and has been found to associate with a panel of RNA degradation enzymes. TTP binds the ARE motif (with preference for an RNA segment with the sequence UUAUUUAUU) by means of two tandemly repeated zinc finger domains  and was Other AUBPs compete with TTP and BRF-1 for binding to ARE sequences. T-cell intracellular antigen-1 (TIA-1) acts to destabilize TNFα mRNA, whereas Hu protein R (HuR) increases the half-life of ARE-containing mRNAs . Other AThe mitogen activated protein kinase (MAPK) signaling cascades are major pathways linking extracellular signals to the intracellular machinery to regulate a plethora of cellular processes such as embryogenesis and cell death, stress, differentiation, and proliferation . The casThe p38 MAPK pathway has long been known to be implicated in the regulation of the biosynthesis of some cytokines . There aDownstream targets of p38α are multiple and include cytosolic proteins, transcription factors, and several kinases that elongate the cascade , but oneSince then, numerous studies have been made in elucidating the mechanisms of MK2 involvement in cytokine regulation upon stress stimulation ,70,71. pIn contrast, there is much evidence showing that MK2 is involved in post-transcriptional regulation of cytokines including TNFα. The MK2 involvement in ARE-mediated post-transcriptional regulation occurs via TTP, which is, under normal cellular conditions, unphosphorylated and promote rapid ARE-mediated mRNA decay through recruitment of the exosome Figure , left. TOnly a limited number of studies have made a direct link between the p38 MAPK pathway and psoriasis pathogenesis. One of the first studies addressing this aspect reported that up-regulated integrins in the suprabasal epidermal layer of psoriatic skin activate various MAPK pathways which in turn lead to unscheduled production of cytokines and growth factors . The linLater it was shown that also the levels of activated MK2 were increased in psoriatic lesional skin compared to non-lesional skin as measured by the degree of phosphorylated MK2 . Interesin vivo inhibitory potency and inefficient oral bio-availability, but most studies were terminated due to toxicities and significant off-target effects reflecting the multiplicity of pathways and feedback loops in the p38α cascade. Some trials are still ongoing and have completed phase I and II, but there is still quite a lot of skepticism about the clinical value of p38 inhibitors.The implication of p38α in cytokine regulation has, for more than a decade, been of great interest in regards to development of anti-inflammatory therapeutics. In addition to psoriasis, p38α has been shown to be involved in several other inflammatory condition such as rheumatoid arthritis, Crohn's disease, and asthma . Severalin vitro and in vivo preclinical efficacy in inhibiting MK2 involvement in cytokine regulation [The problems regarding p38 inhibition might be circumvented by the use of MK2 inhibitors that can dissect the p38 downstream pathways and interfere only with the inflammatory part without affecting additional cellular functions. Small molecule inhibitors and a peptide inhibitor of MK2 have already been developed showing gulation -89. ThisAn extensive and important group of non-coding RNAs (ncRNAs) appeared with the discovery of miRNAs. More than 700 different miRNAs have been identified in humans and the pathway by which miRNAs exert sequence-specific gene silencing has been elucidated, though numerous questions still remain unanswered. The miRNA pathway was found to converge with the RNA interference (RNAi) pathway  by whichmiRNAs are transcribed mainly from non-coding regions by RNA polymerase II as long primary poly- or monocistronic transcripts called pri-miRNA that are 5' capped and polyadenylated. They can be up to several kilobases long and can contain several distinct miRNAs in short imperfect hairpins. In animals, these transcripts are processed in the nucleus by the endoribonuclease Drosha and the dsRNA-binding protein DGCR8 (a.k.a. Pasha) ,92. DrosThe double-stranded miRNA is associated with a class of effector proteins called Argonaute (AGO) proteins . Humans In vertebrates, miRNAs mainly target the 3' UTRs of the mRNAs, and the biological basis for this seems to be that active translation of the miRNA target site impairs miRNA inhibitory function . The mRNh-cell differentiation with a preference towards Th1 induction and a consequent polarized cytokine production [Computational predictions suggest that a single miRNA can target more than 200 mRNAs and, vice versa, that a single mRNA can be under the regulatory control of multiple miRNAs . The suboduction . By compoduction . Interesoduction . As descIf not direct regulators of cytokine processing, emerging evidence suggest that miRNAs play a role in regulating the network of cytokine signaling. A wealth of studies link miR-155 to numerous pathways in the immune system, strongly implying that miR-155 is a central regulator of immune function. miR-155-deficient dendritic cells showed impaired ability to activate T-cells . miR-155In vivo inhibition of miR-155 by intravenously injected antagomirs, based on locked nucleic acid (LNA), in LPS-treated mice showed a consequent down-regulation of granulocyte-macrophage colony stimulating factor (GM-CSF) mRNA in splenocytes through a yet unknown mechanism [echanism . GM-CSF echanism . ConventLike miR-155, miR-146 (miR-146a and miR-146b) has also been found to be up-regulated in dendritic cells in response to LPS stimulation and it has been shown to target two regulators of TNFα signaling, namely IL-1 receptor associated kinase (IRAK1) and TNF receptor-associated factor 6 (TRAF6) . Thus miCareful miRNA profiling of miRNAs in lesional psoriatic skin has been carried out by Sonkoly and co-workers . InteresThe best described miRNA with connection to psoriasis pathogenesis is miR-203. miR-203 is a skin-specific miRNA expressed predominantly by keratinocytes . miR-203However, another study questioned the link between miR-203 and SOCS-3. The authors showed that exogenously expressed miR-203 in murine keratinocytes did not lead to repression of SOCS-3 protein . On the Numerous miRNA target sites in cytokine mRNAs are predicted by computational analyses. A small subset of such target sites - predicted with stringent search criteria by two separate algorithms - are listed in Figure miR-369-3p was found by Vasudevan and co-workers to mediate translational activation of TNFα during cell cycle arrest . The TNFEven though the complete mechanism is not fully understood, the finding that two other miRNAs, let-7 and an artificial miRNA, also mediated translational activation during cell cycle arrest suggests that translational regulation by miRNAs oscillates between repression and activation as a function of the cell cycle. More studies will have to be conducted to confirm this, but the described relation between miR-369-3p and TNFα lends support to the notion that miR-369-3p might be involved in aberrant TNFα production and possibly other ARE-containing cytokines. Another study links miR-155 to up-regulated levels of a TNFα 3'UTR-containing reporter . The autLike miR-369-3p, miR-16 also bears a partial sequence match with the TNFα ARE - an 8-bp non-seed match Figure . SeveralOur knowledge of small RNA-directed gene regulation is constantly growing, and it seems safe to assume that so far we have only seen the very tip of the iceberg with respect to the many potential roles played by small RNA regulators, adding to the enormous complexity of the innate immune system. Inevitably, our understanding of such regulation gives rise to new ideas of exploiting nature's own regulatory pathways in drug development and treatment of inflammatory disease. As we have seen, cytokines are prominent targets for treatment of inflammatory diseases, like rheumatoid arthritis and psoriasis, by systemically administered inhibitors. Do RNA-based drugs represent new means of regulating cytokine signaling during skin inflammation?In recent years, a plethora of therapeutic possibilities involving the use of double-stranded RNA (dsRNA) as a drug has appeared , and sevTransfection of synthetic siRNAs has the advantage that the siRNAs do not need to reach the cell nucleus, but it also suffers from the disadvantage that repeated administration is necessary to obtain a persistent effect because the siRNAs are degraded and diluted during cell growth. Sustained production of siRNAs is obtained by integratable or episomally stable vector systems that encode short hairpin RNAs (shRNAs) ,136. LikThe applicability of locally administered cytokine-directed RNAi therapeutics in skin inflammation seems to depend on at least two key issues. Firstly, cytokine mRNA targets need to be identified and validated in appropriate animal models. Although prime candidates, like TNFα-, IL23-, and IL20-encoding mRNAs, are easily suggested as targets for RNA-based intervention based on their suitability as targets for systemic protein inhibitors, formal evidence for their potential as therapeutic targets during inflammation is required. Secondly, the delivery of RNAi therapeutics to skin remains an extraordinary challenge to any type of cutaneous RNA-directed therapy. A recent review by Geusens provides an excellent view over the possibilities and hopes of RNAi therapy in skin . Here, wTNFα is produced by skin antigen-presenting cells and by keratinocytes during psoriasis progression and stands out as one of several central cytokines in the pathogenesis of the disease. Recent work by Johansen and co-workers somewhat surprisingly showed that increased levels of TNFα protein in lesional psoriatic skin was not caused by an increase in the levels of TNFα-encoding mRNA but rather by increased translational activity triggered by higher levels of activated MK2 . This suTo test this therapeutic approach, we identified one shRNA variant, among a panel of DNA-encoded TNFα-targeting shRNAs, that consistently induced down-regulated expression of TNFα in cultured cells. We and others have shown that transgenic genetic cargo is efficiently delivered to cells of both the epidermis and dermis by intradermally injected lentiviral vectors -141. VSVDespite its efficiency, intradermal administration of lentiviral vectors is currently more an experimental tool than a therapeutic modality in relation to inflammatory skin diseases. Safety considerations, production costs, and the fact that intradermal injection can be applied to only small areas of skin are among the weighty reasons that such treatment is not currently relevant for psoriasis patients. Ironically, the potency of injected lentiviral vectors illustrates some of the major challenges of cutaneous RNAi therapy, namely the delivery of RNA effectors and the ability of these to cross the major barriers of the skin.As an easily accessible organ, skin stands out an attractive target for RNAi-based therapeutics. However, the skin possesses strong barrier properties that impede easy penetration of topically administered drugs  andin vitro synthesized siRNAs [The tightly packed stratum corneum and narrow intercellular compartments of the skin are the main challenges for drug delivery to skin. Evidently, intradermally injected lentiviral vectors are capable of overcoming both barriers, as cells of both dermis and epidermis are efficiently transduced after a single injection . Similard siRNAs ,147 by kd siRNAs ,148. Sucd siRNAs . The safd siRNAs . IntradeDespite the promises of intradermally injected siRNA therapeutics or shRNA-expressing viral vectors, a topical RNAi therapy remains the most attractive modality for treatment of inflammatory skin disease. However, a topically applied RNA drug is confronted by the skin barrier and may for some applications depend on penetration-enhancing technologies. Topical administration of RNAi therapeutics is generally considered safe and holds some promise at present as a clinically applicable treatment. Routes of skin permeation go through hair follicles and sweat gland pores or through the narrow intercellular space of the stratum corneum and the remainder of the epidermis. The transport of RNA drugs by these routes may be enhanced by microporation techniques that share the common objective of increasing the permeability of the stratum corneum . Such teet al. recently reported efficient topical delivery of siRNAs in mouse skin using the GeneCream formulation for delivery of siRNAs targeting osteopontin mRNA [h1 cytokine with a possible function in the pathogenesis of inflammatory diseases. In situ hybridization of siRNAs delivered with the GeneCream formulation showed detectable levels of siRNA in both epidermis and dermis shortly after topical drug administration. In addition, osteopontin-directed siRNAs reduced the levels of osteopontin mRNA in the skin and suppressed the disease symptoms in a mouse model of rheumatoid arthritis [Physical methods may be accompanied by chemical approaches including the use of chemicals that disrupt the structure of the stratum corneum or lipid-based systems that are able to penetrate, although often with low efficiency, the outer layers of the skin . GeneCretin mRNA . Osteoporthritis . In anotrthritis . CD86 isAnother layer of complexity has been added to the circuit of cytokine signaling with the discovery of ARE-mediated mRNA decay and translational control by miRNAs interacting with cytokine-encoding mRNAs. Computational analyses prophesy numerous potential mRNA-miRNA interactions, but only few have so far been confirmed by experimentation, and we are only starting to appreciate the RNA regulatory pathways that are in play during inflammation and inflammatory disease. We do not yet have a feeling with the proportions of the involvement of miRNAs in ARE-mediated decay of cytokine mRNAs, and the roles of miRNAs mediated by noncanonical binding to cytokine-encoding mRNAs have been impossible to foresee by computer-based predictions. Microarray data have clearly shown that certain miRNAs are up- or down-regulated in psoriasis skin . These fOur understanding of the molecular mechanisms driving an escalated immune response in skin may give rise to alternative therapeutic strategies for treatment of psoriasis. Current therapies for psoriasis include topical agents, phototherapy and systemic therapies including non-biological drugs, like ciclosporin, or biological therapeutics, like recombinant cytokine receptors or antibodies. Does local targeting of cytokine-encoding mRNAs represent a future possible treatment? We have shown that cytokine mRNAs can indeed be targeted in psoriatic skin and that such artificial regulation of gene expression may induce amelioration of the disease in an animal model. This effect was achieved by intradermal injection of viral vectors encoding TNFα-directed shRNAs and demonstrated that knockdown of a single cytokine gene - despite any potential redundancy - may improve the phenotype. One may speculate if such local treatment - as opposed to a systemic approach - will persistently reduce the influx of T cells in the skin. Indeed, skin-homing immune cells will not be exposed to the drug before entering layers of the skin. In any case, the establishment of such drug regimes awaits the development of effective methods for topical delivery of synthetic siRNAs. We have seen the first examples of such formulations and are eager to learn more about the delivery and applicability of cream-emulsified cytokine-directed siRNAs in human skin and for treatment of psoriasis in the xenograft transplantation model. This seems a very promising approach although it may at present suffer from the high costs related to production of siRNAs.If the future tells us that miRNAs are major players in inflammatory disorders, a new generation of RNA drugs could include small antisense RNAs that antagonize miRNA function. Such antagomirs can be synthetically made  or encodThe authors declare that they have no competing interests.The manuscript was prepared by ROB and JGM. Both authors read and approved the final manuscript.


Induction of an inflammatory response is thought to have a significant role in the complications that follow cardiopulmonary bypass (CPB). The statin drugs are increasingly being recognized as having potent anti-inflammatory effects and hence have potential to influence an important mechanism of injury in CPB, although there is no current confirmation that this is indeed the case. Our objective was to systematically review if pre-operative prophylactic statin therapy, compared with placebo or standard of care, can decrease the inflammatory response in people undergoing heart surgery with CPB.We performed a systematic and comprehensive literature search for all randomized controlled trials (RCTs) of open heart surgery with CPB in adults or children who received prophylactic statin treatment prior to CPB, with reported outcomes which included markers of inflammation. Two authors independently identified eligible studies, extracted data, and assessed study quality using standardized instruments. Weighted mean difference (WMD) was the primary summary statistic with data pooled using a random effects model. Descriptive analysis was used when data could not be pooled.Eight RCTs were included in the review, with the number of trials for each inflammatory outcome being even more limited. Pooled data demonstrated benefit with the use of statin to attenuate the post-CPB increase in interleukins 6 and 8 , peak high sensitivity C-reactive protein (hsCRP), and tumor necrosis factor-alpha (TNF-α) post-CPB ] -23.5 pg/ml [-36.6 to -10.5]; -23.4 pg/ml [-35.8 to -11.0]; -15.3 mg/L [CI -26.9 to -3.7]; -2.10 pg/ml [-3.83 to -0.37] respectively). Very limited RCT evidence suggests that prophylactic statin therapy may also decrease adhesion molecules following CPB including neutrophil CD11b and soluble P (sP)-selectin.Although the RCT evidence may suggest a reduction in post-CPB inflammation by statin therapy, the evidence is not definitive due to significant limitations. Several of the trials were not methodologically rigorous and statin intervention was highly variable in this small number of studies. This systematic review demonstrates that there is a significant gap that exists in the current literature in regards to the potential anti-inflammatory effect of statin therapy prior to CPB. The use of cardiopulmonary bypass (CPB) is necessary for many cardiac surgical procedures. However, it is clear that CPB can have deleterious effects, including initiation of cardiopulmonary dysfunction, renal dysfunction, and neurological injury in the acute peri-operative period. Economic costs and the human costs are greater when cardiac surgical patients develop complications following CPB ,2. A numIt is well documented that CPB is associated with a systemic inflammatory response, which involves the synthesis of various cytokines and inflammatory mediators . This inPrevious reviews and meta-analyses that have looked at the clinical impact of pre-operative statin treatment on major adverse events after cardiovascular surgery have suggested a beneficial impact. For example, a systematic review by Liakopoulos and colleagues , which iUnderstanding the biological mechanism by which statins may change the clinical course following cardiac surgery with CPB is important as we move forward in the development of large high-quality RCTs to further address the clinical efficacy of this intervention. It has not been demonstrated by systematic review that statins can modify the biological processes that are related to organ injury post-CPB. It is therefore prudent to systematically review this existing literature. The primary objective of this review was to answer the following question: in people undergoing heart surgery with CPB, does pre-operative prophylactic statin therapy, compared with placebo or standard of care, decrease the inflammatory response after CPB?This systematic review was conducted and reported in accordance with available guidelines .Criteria for types of studies were as follows: randomized clinical trials, with or without placebo. Studies were eligible if they included male or female patients of any age undergoing CPB for elective open-heart surgery . Studies of patients undergoing heart transplant were excluded. Patients already on statins were excluded. Criterion for type of intervention was the pre-operative use of any statin drug administered as prophylaxis pre-CPB, with or without other interventions, compared with a non-statin containing control regimen (either standard of care or placebo). Co-interventions were allowed as long as all arms of the randomized allocation received the same co-interventions. The primary evaluation criterion for outcomes measures was the measurement of inflammatory markers post-operatively , TNF-α and its receptors, high sensitivity C-reactive protein (hsCRP), adhesion molecules , selectins), complement split products ).We conducted a comprehensive search to identify all relevant studies of statin use. Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE (1950 to March 2009), EMBASE (1988 to March 2009), and International Pharmaceutical Abstracts (1970 to March 2009) were searched using relevant search terms relating to heart surgery procedures with CPB and statins [see Additional data file For knowledge of ongoing or unpublished trials, we contacted investigators of all identified trials, a number of groups identified through the United States National Library of Medicine's Directory of Health Organizations and the Cochrane Collaborative Review Groups' Specialized Register, and pharmaceutical companies. Studies in all languages were included.Two authors (CM and either MZ or PG) independently assessed titles and abstracts identified from the electronic database searches, applying predetermined eligibility criteria. If two reviewers were certain that a reference was not relevant it was excluded. Disagreements between authors were solved by consensus. Full text of articles that were judged to be potentially eligible by title and abstract review as above were retrieved and two reviewers (CM and MZ) assessed them for eligibility using the inclusion and exclusion criteria. If both were certain that a study was unsuitable it was excluded and reasons for exclusion noted. Disagreements between authors were solved by consensus. The reviewers were not blinded for authors' names or journal names. The numbers of references retrieved from the searches was recorded. Those eligible or ineligible were documented and a quality of reporting of meta-analysis (QUOROM) statement prepared  Figure .Quality of the studies was assessed by two reviewers (CM and either MZ or PG) without blinding to the journal or authorship, using the established standards of the Cochrane Collaboration (Cochrane collaboration Risk of Bias Assessment Tool) . The quaData was extracted independently by two reviewers (CM and MZ) and was collected on a data extraction form, which had been pre-tested to ensure appropriate data collection. This form included basic identifying information, information on trial quality, and information on trial participants, interventions, and outcomes related to post-operative inflammatory markers. Data regarding the clinical endpoints of mortality and morbidity was also extracted.I2 statistic [Data were analyzed using Review Manager (RevMan) 5 for analysis. For continuous variables (e.g. levels of inflammatory markers) weighted mean difference (WMD) with 95% confidence interval (CI) was used when combining data. Data was pooled where appropriate using random effects models and statistical heterogeneity was quantified using the tatistic .Of the 2322 titles and abstracts found, 17 studies were identified as potentially suitable and the full text retrieved . The two individual studies judged to be low risk of bias and with significantly larger number of participants in each (n = 200) show discrepant results. Patti and colleagues [P = 0.75). In Mannacio and colleagues [P < 0.001). Neither of these studies specified at which time point peak hsCRP was measured. The duration of pre-operative statin therapy was the same in both studies (1 week), although they differed in terms of the statin used  in samples from the placebo group (145% increase in expression) compared with the statin group (102% increase in expression) after 24 hours of culturing.The 2007 study by Chello and colleagues  measuredP = 0.34)One of the studies, judged as high risk of bias, reported the effect of statin on the soluble form of the endothelial ICAM-1 . The levP < 0.001)). This study was judged as unclear risk of bias.Two studies describe measuring P-selectin levels following CPB ,33, FlorStatins are beginning to be used in a number of clinical situations where inflammation is considered a major pathophysiological mechanism including in people undergoing CPB. Pooled data in this systematic review demonstrates that statin therapy before CPB is associated with a reduction in circulating markers of inflammation, specifically IL-6, IL-8, hsCRP, and TNF-α. For IL-6 and IL-8, one additional trial, which was not pooled due to limitations of the original data, illustrates a 75% reduction in IL-6 with statin given pre-CPB and a 20% reduction in IL-8 with statin given pre-CPB. These reductions in inflammatory markers were not reported as significant in the original article; however, the sample size was very small. For TNF-α, un-pooled data which represents one additional study suggests that both treatment and control groups had similar levels of TNF-α throughout the study, although they were all below the detection limit for their assay. In addition, the detection limit of the assay used was not adequate, given the mean for TNF-α concentrations in other studies included in the review.For IL-8 and TNF-α, pooled analysis was limited to only two studies, both of which came from the same investigative group ,30; no hProphylactic statin therapy may decrease adhesion molecules following CPB including neutrophil CD11b and soluble P (sP)-selectin, although the evidence for this is weak. Neutrophil CD11b surface adhesion molecule expression is a marker of neutrophil stimulation. Although statin does not appear to change expression immediately following CPB, other data supports an effect of statin on neutrophils when assessed after a period of time more appropriate for protein transcription to occur.Seven of the eight studies identified for inclusion in this systematic review measured clinical outcomes ,28-33. FOverall this review suggests that several markers of the inflammatory response post-CPB may be attenuated by pre-operative statin therapy. The findings of this review and the strength of the conclusions are limited by several factors related to the current state of evidence; the total number of studies is limited, the trial size is generally small, and the methodological quality of trials is generally not high. Although key investigators in this field who were contacted did not reveal any unpublished data, an additional limitation to this systematic review is that we did not statistically evaluate publication bias; the number of identified trials was small and the utility of such tests to assess the possibility of bias in this situation is extremely limited.A meta-analysis of similar, well-conducted, RCTs is considered one of the highest levels of evidence but the primary trials all have to be conducted with high methodological rigor for the meta-analysis to be definitive. This is not the case for the evidence summarized in this review. In addition, controversy also arises around the interpretation of summarized results when the results of discordant studies are pooled in meta-analysis; this systematic review identified studies that are diverse particularly in regards to the type and dose of statin used and the duration of pre-operative therapy. There is currently no data available regarding type, dose, or duration of statin therapy in regards to modification of either post-operative clinical outcomes or the inflammatory response. The current body of evidence reviewed does not provide adequate data to examine directly treatment regimen to make any final conclusions on this topic. Lastly, small meta-analyses like those published here need to be regarded with caution even in the presence of statistically significant results.Given that the existing RCTs relating to the current topic have important scientific and methodological limitations, including smaller sized samples, the importance of this systematic review lies in its identification of the gaps existing in the literature. In addition, the exploratory nature of the meta-analyses undertaken in this review provides a plausible estimate of effect that can be tested in subsequent studies.Although the RCT evidence may suggest a reduction in post-CPB inflammation by statin therapy, the evidence is not definitive due to significant limitations. Several of the trials were not methodologically rigorous and statin intervention was highly variable in this small number of studies. This systematic review demonstrates that there is a significant gap that exists in the current literature in regards to the potential anti-inflammatory effect of statin therapy prior to CPB. Further well-designed RCTs would help fill this gap and guide a rational development and use of interventions aimed at improving clinical outcomes post-CPB. There are two ongoing RCTs with large target sample sizes , which will hopefully contribute some further evidence to this topic.• Pre-operative statin may decrease inflammatory markers following CPB.• Meta-analysis demonstrates benefit with the use of statin to reduce the post-operative level of IL-6, IL-8, CRP, and TNF-α.• Given that the trial number is very small and some of the studies methodologically weak, the results need to be regarded with caution even in the presence of statistically significant results.• Further well-designed and adequately powered trials are needed.CABG: coronary artery bypass graft; CENTRAL: The Cochrane Central Register of Controlled Trials; CI: confidence interval; CPB: cardiopulmonary bypass; hsCRP: high sensitivity C-reactive protein; ICAM-1: intercellular adhesion molecule-1; IL: interleukin; NNT: number needed to treat; RCT: randomized controlled trials; RD: risk difference; RR: risk ratio; sP-selectin: soluble P-selectin; TNF-α: tumor necrosis factor-alpha; WMD: weighted mean difference.The authors declare that they have no competing interests.CM conceived of the study, designed the protocol, carried out the search, participated in trial selection, assessed methodological quality, extracted the data, carried out the data analysis and drafted the manuscript. MZ participated in trial selection, assessed methodological quality, extracted the data, and helped draft the manuscript. PG participated in trial selection and assessment of methodological quality. All authors read and approved the final manuscript.A word file listing the details of the search strategy used.Click here for fileA word file containing a series of tables providing the characteristics of excluded studies, studies awaiting assessment, and ongoing studies.Click here for file


Previous research has shown an association between preoperative depressive symptoms and a poorer surgery outcome in lumbar spinal stenosis (LSS). It is not known whether depressive symptoms throughout the recovery period are relevant to the outcome of surgery in LSS. In this prospective clinical study the predictive value of preoperative and postoperative depressive symptoms with respect to the surgery outcome is reported.96 patients (mean age 62 years) with symptomatic lumbar spinal stenosis underwent decompressive surgery. They completed the same set of questionnaires preoperatively and 3 months, 6 months, 1 year and 2 years postoperatively. Depressive symptoms were assessed with the 21-item Beck Depression Inventory. Physical functioning and pain were assessed with the Oswestry Disability Index, the Stucki Questionnaire, self-reported walking ability and VAS rating. Logistic regression analyses were used to examine the predictive value of preoperative and postoperative depressive symptoms regarding the surgery outcome. A "good" outcome was defined in two ways: first, by gaining a 30% improvement in relation to the preoperative disability and pain, and second, by having a score at or below the median value for disability and pain on 2-year follow-up.Having elevated depressive symptoms particularly on 3-month follow-up was predictive of a poorer surgery outcome regarding pain and disability: when the outcome was defined as less than 30% improvement from the baseline, the OR's  were 2.94 (1.06-8.12), <0.05 for Oswestry and 3.33 (1.13-9.79), <0.05 for VAS. In median split approach the OR was 4.11 (1.27-13.32), <0.05 for Oswestry. Predictive associations also emerged between having depressive symptoms on 6-month and 1-year follow-ups and a poorer outcome regarding disability. The predictive value of elevated depressive symptoms particularly with respect to 2-yeard disability was evident whether the outcome was defined as a 30% improvement compared to the preoperative status or as belonging to the better scoring half of the study population on 2-year follow-up.Preoperative and postoperative depressive symptoms may indicate those patients at greater risk of a poorer postoperative functional ability. For these patients, further clinical evaluation should be carried out, especially during postoperative stages. Lumbar spinal stenosis (LSS) is a disabling and painful disease that most typically affects middle-aged and older adults ,2. With The factors that influence the outcome remain controversial. Several research groups have tried to identify predictors of the surgical outcome of LSS ,11. The Interestingly, recent research has shown that depressive symptoms have direct biological effects on wound healing and pain among surgical patients through neuroendocrine-immune alterations -16. It hPrevious research has suggested an association between depressive symptoms and a poorer surgery outcome ,13,19, aAs previous research has mainly focused on the development of pre-screening outcome tools , the temThe selection of the sample has been described in more detail by Sinikallio et al. ,23. BrieThe exclusion criteria were: emergency or urgent spinal surgery precluding recruitment and protocol investigations; cognitive impairment prohibiting completion of the questionnaires or other failures in co-operation; and the presence of metallic particles in the body preventing the MRI investigation. A previous spine operation or co-existing disc herniation were not exclusion criteria, but the main diagnosis of the study patients had to be LSS. The surgeons sent the information on patients eligible for operation to the Department of Physical and Rehabilitation Medicine, which organized the study.The patients received an account of the study during their outpatient visit to the Department of Physical and Rehabilitation Medicine and provided informed consent. The study design was approved by the Ethics Committee of the University of Kuopio and Kuopio University Hospital.1) Self-reported walking capacity was recorded. The patients gave an estimate of their walking capacity in metres by answering the question: How long a distance can you walk continuously without pausing, on even ground? 2) Overall back and leg pain intensity was assessed with the visual analogue scale (VAS: range 0-100 mm) [3) Subjective disability was measured by the validated Finnish version of the Oswestry Disability Index (0-100%), where 0% represents no disability and 100% extreme debilitating disability [4) The questionnaire devised by Stucki [Preoperatively, questions concerning socio-demographic background, lifestyle and health were included in the study questionnaire. Data collection took place with the same set of questionnaires before surgery and 3 months, 6 months, 1 year and 2 years postoperatively. The questionnaire included the following items. -100 mm) . The ovesability -27. 4) Ty Stucki  assessed5) Depression was assessed with the Finnish version of the 21-item Beck Depression Inventory (BDI), with scores ranging from 0 to 63 [ 0 to 63 -32. The  0 to 63 -31.a priori defined points: 1) the well-known risk factors for depression were included, as well as 2) the clinically relevant factors regarding disability and pain. Regression analyses were performed using the data for the final 96 subjects who completed the two-year follow-up. A "good" outcome was defined in two ways: first, by gaining a 30% improvement compared to the preoperative disability and pain [All statistical analyses were performed using SPSS/PC . The Student's t-test was used to compare the difference in mean scores between the groups in drop-out analysis. Logistic regression analyses were used to investigate the predictors for the surgical outcome, including preoperative and postoperative depressive symptoms. The rationale for predictor selection was based on two  change"), and secFirst, univariate logistic binomial regression analysis was performed to investigate the unadjusted odds ratios of predictors at different follow-ups with respect to the surgery outcome, defined in the two different ways. Each of the predictors was entered into the model separately. We used four separate models to specifically examine the preoperative and recovery phase depression variables . The following factors were included as the basic covariates in the multivariate logistic regression analyses, regardless of the model: age (years), male (no/yes) and single (no/yes) as categorical variables. In addition, the Stucki symptom severity score, Stucki disability score and having elevated depressive symptoms (BDI ≥ 10: no/yes) were included in the models, according to the follow-up phase. The same set of predictors was used in all the logistic regression analyses.Secondly, in the minimal important change approach, logistic regression analysis  was used to examine the depression score at different follow-up points as a predictor with respect to a 30% decrease from the individual baseline disability (Oswestry score) and pain (VAS score). In practice, a "theoretical" 30% improvement was calculated as 30% decrease from the patient's baseline VAS score and Oswestry score. This calculation was done for each patient separately. Then each patient's actual 2-year score on VAS and Oswestry was compared to the "theoretical" outcome. In case the patient reached lesser than 30% decrease he was classified as having "poor" outcome regarding pain and disability.Thirdly, in the median split approach, corresponding analyses with similar predictor models were performed.The study subjects were 102 patients with both clinically and radiologically defined lumbar spinal stenosis (LSS) selected for surgical treatment. Beck Depression Inventory (BDI) data were missing at baseline for two of the study patients (n = 100) and at the 3-month follow-up for 3 of the 102 baseline patients (n = 99). At the final 2-year follow-up, four patients had died, one patient had dropped out of the study and one patient had missing BDI data (n = 96).In drop-out analysis regarding the main demographic  and clinical  preoperative variables, there were no significant differences between the groups .The main diagnosis of all the study patients was LSS. Thirteen patients in our sample also had radiological disc herniation (DH) in addition to LSS. In the surgery, 19 patients were treated with spondylodesis with or without instrumentation. Preoperatively, the mean BDI score was 8.8 among patients treated with spondylodesis and 10.6 among patients without spondylodesis (p = ns.)The mean age of the study patients preoperatively was 62 SD 11.1) years. Altogether, 41% of the patients were male and 66% were married or living with a partner. Two study patients reported having used antidepressant medication at the preoperative stage, 3 patients at the 1-year postoperative stage and 7 patients at the 2-year postoperative stage. The clinical characteristics of the study patients in different follow-up phases are presented in Table 1.1 yearsFirst, in univariate regression models, all the predictors were entered into the model separately Table . In thesSecondly, in multivariate regression analyses Table , when deThirdly, in multivariate regression models Table  with resThe main finding of the present study was that in LSS patients undergoing decompression surgery, elevated depressive symptoms were a strong predictor of the surgery outcome in multivariate regression analyses. This was evident whether the outcome was defined according to the minimal important change from baseline or according to scoring over the median for pain and disability on 2-year follow-up. Importantly, these associations were particularly evident on 3-month and 6-month follow-up, but not so on 12-month follow-up.In univariate regression analyses, when analyzing the predictive factors separately, the predictive value of 3-month and 6-month depressive symptoms regarding the outcome for disability and pain also became evident. Interestingly, in these analyses, elevated depressive symptoms on one-year follow-up also showed strong associations with greater and less improved disability as outcome. In univariate regression analyses it was also seen that out of the preoperative predictors, a higher age, more LSS-related symptoms and disability as well depressive symptoms were predictive of greater disability on 2-year follow-up.To our best knowledge, this is the first study to examine the predictive role of elevated depressive symptoms assessed in LSS patients in several follow-up phases. From a clinical point of view, these results highlight the particular importance of the early recovery period; postoperative depressive symptoms may indicate those patients at greater risk of a poorer surgery outcome. In our study sample, only a fraction of the patients reported having used antidepressant medication during the follow-up period. As our study was a prospective observational design among a clinical sample of surgically treated LSS patients, psychiatric assessment or treatment was not included in the study protocol. However, when looking at the reported frequencies of antidepressant use, one would be safe to assume that depression among this patient population may well have remained largely undetected.Based on the original and relatively low cut-off point suggested by Beck and Beamesderfer  for deteThe most important remaining question concerns the mechanism explaining our results. Regarding the association between postoperative depression and a poor surgery outcome, one explanation may be the altered motivational state and executive-type cognitive impairments associated with depression, including a low motivation for physical exercise and activity. However, factors other than those of a psychological nature may also affect our findings. Persistent elevation of low-grade inflammatory activity - a physiological state closely associated with the pathophysiology of depression - may have direct effects on the process of physical rehabilitation among LSS patients, including effects on wound-healing and pain ,15. SimiInterestingly, there is growing evidence that antidepressive treatment may reduce the production of pro-inflammatory cytokines ,35. AlthNonetheless, one must be cautious not to label patients as "lost cases" or exclude them from surgical treatment due to their depressive symptoms. Instead, the current findings point to the need for appropriate assessment and treatment practises regarding concurrent depressive symptoms of LSS patients. In particular, our results suggest that research investigating the treatment of depression in patients undergoing surgery for stenosis merits a high priority.Although definite causal conclusions cannot be drawn from our observational study setting, it may be concluded that elevated depressive symptoms are associated with an increased risk for a poor surgery outcome among LSS patients, whether the outcome was defined according to the minimal important change from baseline or according to scoring over the median in pain and disability on 2-year follow-up.This association was evident particularly for patients with elevated depressive symptoms on 3-month and 6-month follow-up. Various pre-screening measures for an optimal outcome have been extensively studied in LSS research , but ourThe authors declare that they have no competing interests.SS performed the statistical analysis and drafted the manuscript.TA, OA, SML, HK, and HV participated in acquisition of data and helped to draft the manuscript.TA, OA, HK and HV also participated in the design of the study.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/11/152/prepub


Manduca sexta, for odor-modulated search behavior to an odor source. When a moth's antennae receive intermittent odor stimulation, the projection neurons (PNs) in the primary olfactory centers , which are analogous to the olfactory bulbs of vertebrates, generate discrete bursts of action potentials separated by periods of inhibition, suggesting that the PNs may use the binary burst/non-burst neural patterns to resolve and enhance the intermittency of the stimulus encountered in the odor plume.An animal navigating to an unseen odor source must accurately resolve the spatiotemporal distribution of that stimulus in order to express appropriate upwind flight behavior. Intermittency of natural odor plumes, caused by air turbulence, is critically important for many insects, including the hawkmoth, We tested this hypothesis first by establishing that bicuculline methiodide reliably and reversibly disrupted the ability of PNs to produce bursting response patterns. Behavioral studies, in turn, demonstrated that after injecting this drug into the antennal lobe at the effective concentration used in the physiological experiments animals could no longer efficiently locate the odor source, even though they had detected the odor signal.Our results establish a direct link between the bursting response pattern of PNs and the odor-tracking behavior of the moth, demonstrating the behavioral significance of resolving the dynamics of a natural odor stimulus in antennal lobe circuits. An animal's nervous system must encode environmental stimuli that are important for the individual's survival and reproduction. According to a generally accepted coding theory, neural-discharge patterns, not the action potential itself, carry information about specific stimulus features . SearchiAlthough specific coding questions differ for different sensory systems, the conceptual issues are similar. For the olfactory system, an important task is to resolve the spatiotemporal dynamics of olfactory stimuli. In nature, odor molecules released from a source form an odor plume with a dynamic, intermittent structure due to turbulent movement of the fluid . AnimalsA-receptor antagonist, bicuculline methiodide , reliably and reversibly disrupted the ability of MGC-PNs to encode intermittent pheromone pulses. While having no significant effect on the sensitivity of MGC-PNs in detecting pheromone, bicuculline injected into the MGC of both ALs caused the moth to navigate ineffectively in a turbulent (or intermittent) odor plume.Extensive previous work on the sex-pheromonal communication system of moths makes it a useful model for studying olfactory processing of stimulus intermittency . When a n = 25), bath application of bicuculline apparently changed the spontaneous activity pattern from randomly bursting to tonic firing, during which the inter-spike interval (ISI) was about 140 ms  and the coefficient of variation (CV) of the ISI was significantly lower  than that during the pre-drug period   or toroid I (T-PNs) . These PM. sexta, E10,E12,Z14-hexadecatrienal [E10,Z12-hexadecadienal), the second key component [2  or high (200 or 500 μM) concentrations showed such extended spiking responses, resulting in a significantly elevated firing rate during the I2 period . At a lower concentration (25 μM), the I2 period did not differ significantly from the control . Interestingly, the peak firing rate during the response decreased with increased drug dosage; however, it was not statistically significant when compared with the saline control . Another example is shown in Figure 2 period clearly followed the excitatory phase until the spontaneous activity resumed. To quantify the PN's ability to follow the repeated odor pulses, the odor-driven bursting responses were assessed with auto-correlation analysis, which revealed periodic peaks separated by 1-s intervals  and the averaged correlation between the central peak and the specified peak . The higher the PFI, the better the PN resolved pulses. During bicuculline application, the silent I2 period was filled with spikes, which resulted in a much-deteriorated periodicity in the autocorrelogram  in affecting the mean PFIs. The interaction between these two factors was also significant (p < 0.01), suggesting the extent of deterioration in tracking odor pulses was pulsing-rate dependent. Similar results were obtained from the single-component data. Together, these results indicate that: first, PN's pulse-following capability was significantly impaired by the actions of bicuculline; and second, although PNs generally improved their accuracy in tracking odor pulses that were delivered at a lower rate, the improvement was compromised under the influence of bicuculline. For example, under saline control, the PNs on average increased their pulse-tracking capability 7.4 times when the stimulus repetition rate dropped from 10 s-1 to 0.2 s-1, but the improvement was only 2.3 times under bicuculline application  and T-PNs (n = 6) in the way they resolved odor pulses . The magnitude of reduction on each pulsing rate, however, was much higher in C-PNs, suggesting the C-PNs followed the odor pulses with higher contrast under control conditions. Nonetheless, application of bicuculline significantly impaired the pulse-following capability of both types of PNs.One potential consequence of the bicuculline-caused prolonged excitation was to decrease the contrast between the excitatory phase and the Im Figure , center.m Figure , right, n Figure . We alsos Figure . Bicucul-1 ; intermediate ; and high  – as analyzed by repeated-measures two-way ANOVA [p > 0.05. These results were in sharp contrast with those of pulse-tracking experiments, where the reduction of PFI values from the saline control due to the bicuculline treatment was statistically significant across a large range of odor-pulsing rates . The response magnitude was defined as the mean instantaneous firing rate within the response window . In general, when the stimulus concentration was increased in decadal steps (0.1 to 100 ng/ml), the PNs' response magnitude also increased, regardless of whether a single pheromone component  or the binary blend  was used as stimulus. Moreover, the slope of the dose-response curve under bicuculline treatment was similar to that under the saline control, indicating that bicuculline did not alter PN's gain control mechanisms. Furthermore, the difference in response magnitude between the bicuculline treatment and the saline control was not statistically significant across the four odor concentration steps for all three bicuculline dosages – low . Only 40% of the bicuculline-injected moths, however, hovered in front of the pheromone source, whereas nearly 80% of the unoperated and saline-injected moths did so, a difference that was statistically significant . Similarly, a significantly smaller fraction of the bicuculline-injected animals contacted the odor source  or displayed abdomen curling , which is a typical attribute of mating behavior  were dynamically scaled such that the estimated frequency of filaments within the odor plume was within the range of odor-pulsing frequencies where the bicuculline-induced reduction of PFIs was significant . Among all these three groups only 20–30% of the animals contacted the solvent source. None of these moths showed the stereotypical close hovering and abdomen curling . The results showed that none of the behavioral measurements in the bicuculline group was significantly different from those of the other two control groups , which alternated more frequently between upwind surge and cross-wind casting. Similarly, the transit probability surface plots [If the behavioral defects resulting from bicuculline injection were due to a disruption of the pulse-following capability of PNs, as shown in the physiological experiments Figure , one wouce plots  demonstrn = 8) and saline-injected moths (n = 3), 100% of the bicuculline-injected moths (n = 8) progressed upwind and hovered in front of the odor source, which was a white paper 'flower' loaded with a mixture of known, behaviorally effective floral volatiles that mimic the odor of an important floral food resource for M. sexta in southern Arizona [G test: p > 0.05). Moreover, the percentage of moths in the bicuculline treatment and unoperated groups that extended their proboscis into the paper flower was not significantly different . As a positive control, a few bicuculline-injected moths were flown to a pheromone source. They exhibited frequent alternation of upwind progression and cross-wind casting, confirming the disruptive effects of bicuculline on pheromone-plume tracking . Moreover, the behavioral disruption was caused by effects of bicuculline within the MGC because the same drug treatment did not disrupt the orientation of moths to floral odors.Searching for a particular pattern of neural activity responsible for a defined behavior is challenging because of the difficulty of establishing a causal link. In this study we confronted this problem by successfully disrupting MGC-PNs' ability to generate discrete bursts of action potentials and to follow repeated odor pulses that mimic the intermittency of natural odor plumes. Such a bursting response pattern was also observed in a previous study in which the moth was exposed to a pheromone plume and the electroantennogram (EAG) and firing activity of MGC-PNs were simultaneously recorded . The disA receptors in PNs [-1 , and the estimated filament-encounter frequency was about 4 pulses s-1 . Because of the boundary-layer effect around the moth antennae, which prolongs the pheromone concentration decay time [Bicuculline methiodide effectively and reversibly disrupted the ability of PNs to encode intermittent odor pulses Figure , consists in PNs ,30. This1 Figure , implyincay time , the ORCcay time ,33, makeIn our experiments, the flight-track analysis showed that although the unoperated and saline-injected animals spent most of the time heading directly toward the odor source, the bicuculline-injected moths were unsuccessful at steering a zero-degree track angle relative to the odor source despite being capable of making upwind progress Figure . As a coDrosophila melanogaster differ noticeably from findings with moths in showing upwind surge even with a homogeneous odor cloud [According to a model proposed by Baker  based onor cloud . Our resThe bursting response generated by PNs upon contact with each odor filament is a critical component of the olfactory code responsible for upwind surges. In a natural odor plume, the arrival of odor packets at appropriate frequencies produces a series of fused upwind surges, which often appear as approximately straight flight tracks toward an odor source Figure . TransfoTo determine the relationship between MGC-PNs' pulse-following ability and the pheromone-modulated orientation behavior of male moths, it is important to ask if the treatment with bicuculline also caused other changes, such as an altered firing rate, that might contribute to the moth's inability to track the odor plume in the wind tunnel. Experimental results showed, however, that the bicuculline treatment did not significantly change the response magnitude over a large range of pheromone concentrations . Moreover, bicuculline treatment had no detectable effect on ORC activities whereas it did affect simultaneously recorded PNs . This was probably due to some differences in ion conductances between ORCs and PNs. Thus, we conclude that the ability of PNs to respond to olfactory stimuli and encode odor concentrations  are not affected by bicuculline treatment. Instead, the temporal response pattern is the feature that is significantly modified by the treatment.Although bicuculline may not affect only the neurons associated with MGC, non-MGC neurons are unlikely to contribute to pheromone-mediated behaviors as pheromonal stimuli do not cross-excite non-MGC glomeruli ,37. MoreOn the basis of our findings, we conclude that the temporal pattern of MGC-PN responses (spiking bursts entrained to odor pulses), and not their magnitude (frequency of spiking), is significantly disrupted by injection of bicuculline into the MGC. The inability of moths to navigate successfully to and locate the pheromone source therefore most likely results from the loss of PNs' ability to track the individual filaments in an odor plume, rather than impaired detection and/or concentration coding of the pheromonal signal, and thus reveals a format of neural representation necessary for natural odor-seeking behavior.Manduca sexta (L.) (Lepidoptera: Sphingidae) were reared in the laboratory on an artificial diet under a long-day photoperiod, and adult male moths, 4 days post-emergence, were prepared for experiments as described previously [2, 3 mM KCl, 10 mM TES buffer pH 6.9, and 25 mM sucrose.eviously ,39. For To allow long-term recording from single neurons, which is needed for the pharmacological experiments in this study, we used a juxtacellular recording and dye-deposition technique modified from . In shorE10,Z12-hexadecadiennal  [E11,Z13-pentadecadiennal  [Olfactory stimuli were delivered to the preparation by injecting odor-laden air puffs onto a constant air flow (1 liter per minute) that was directed at the middle of the antenna ipsilateral to the AL from which recordings were made. Trains of five air puffs (50 or 100 ms) with various inter-pulse intervals  were generated by means of a solenoid-activated valve controlled by an electronic stimulator (WPI). These air puffs were directed through a glass syringe containing a piece of filter paper, bearing various amounts of a single pheromone component  or a blend of the same quantities of the two key pheromone components. The stimulus compounds used were: eromone) ,40; E11,eromone) ; and a mM. sexta, uniglomerular MGC-PNs have been shown repeatedly to give predictable responses to the pheromone components according to the MGC glomerulus in which their dendrites arborize [1) preceding a depolarization phase that is then followed by a period of delayed after-hyperpolarization (I2). This characteristic pattern results from synaptic inputs from GABAergic, inhibitory local interneurons (LNs) as well as intrinsic properties of the PNs [1 is difficult to detect; however, the silent I2 period is clearly visible. Finally, the spontaneous activity of MGC-PNs typically is more randomly bursting, whereas that of LNs is more tonic.MGC-PNs were characterized using three physiological criteria: randomly bursting spontaneous firing pattern; response specificity to pheromone components; and multiphasic pattern of responses. In arborize ,39,41,422+ in inducing the bicuculline effects, we replaced the CaCl2 in the physiological saline solution with MgCl2 and then equalized the osmolarity with sucrose. Spontaneous activity and odor-evoked responses were first recorded under the normal physiological saline solution and then repeated under the Ca2+-free saline solution, bicuculline diluted in the Ca2+-free saline solution, bicuculline diluted in normal saline solution, and finally the normal saline wash. This series of treatments was designed to perform on a single MGC-PN.Bicuculline methiodide  was diluted in physiological saline solution to different concentrations  and then bath-applied to moth preparations. A drip system comprising multiple 60-cc syringes converging on a central Teflon tube was used to facilitate quick switching from normal physiological saline solution to bicuculline solution and back. To minimize the disturbance to the physiological recordings, close attention was paid to the level of solution between syringes when switching from one syringe to another, ensuring an approximately constant rate of flow. The time when bicuculline took effect was determined by the change of spontaneous activity from randomly bursting to tonic. This time was about 10 minutes for concentrations of bicuculline >100 μM. To determine the role of extracellular CaTo determine whether bicuculline-induced changes in MGC-PNs originate locally in the AL or from the periphery, sensillum tip recordings were performed simultaneously with the juxtacellular recordings from MGC-PNs. The antenna ipsilateral to the AL in which juxtacellular recordings were performed was gently twisted so that the long sensilla pointed upward. The tips of pheromone-sensitive type-I trichoid sensilla  were carSpike traces were digitized at 25 kHz sampling rate using Datapack 2k2 software , and the time stamp of each spike was extracted offline with the event-extraction function within the software package. The spike train data (columns of time stamps) were imported into a custom-written Matlab  script, which first transformed the data column into a rate histogram at 5-ms bin width, and then calculated the autocorrelograms using the internal correlation function of Matlab. A simple PFI, which is based on the autocorrelograms, was calculated to reflect a PN's pulse-following ability. The peaks flanking the central peak on either side in the autocorrelograms are directly locked by odor pulses. Therefore,ipi is the inter-pulse-interval , Cipi is the correlation value at the specified ipi derived from correlograms, C is a vector of correlation values between the central peak  and the peak at ipi, and n represents the number of time bins between these two peaks. This ratio index essentially reflects the contrast of spike density between the bursting response driven by odor pulses and the period between two adjacent bursts. The better a neuron resolves odor pulses, the higher the PFI value is.where To determine the width of the response window, the spike train data were exported into Neuroexplorer  for plotting the peristimulus time histograms, which allowed approximate estimation of response duration. Then the average of instantaneous spiking frequency  within the response window was calculated using a custom-written Matlab script for each odor-evoked spike burst, and finally these averages from all five trials (pulses) were averaged again to obtain the grand average. The prolonged excitatory responses caused by the bicuculline application were cut off at the 250 ms window, in which most of the odor-evoked responses under physiological saline condition fell. The measurement of response magnitude, defined as the grand average of instantaneous spiking frequency, is robust to the variations in actual response durations. We compared the dose-response curves calculated using a 250 ms window with that using a 500 ms window and did not observe significant differences. All statistical comparisons were performed using the Statistics Toolbox of Matlab or a third-party program downloaded from the Matlab website .A 4-day-old moth was restrained in a plastic tube 60-90 minutes prior to scotophase and kept at room temperature in the light awaiting surgery and injection. Moths were de-scaled entirely from the nape of the neck to the labial palps. A rectangular window was cut in the head capsule, horizontally above the nape of the neck, extending the length between the antennae and short of the labial palps. The window was removed and pushed forward, keeping the connective tissues and muscles attached. The MGC regions in both ALs were located by gently pushing muscle fibers and connective tissues aside with fine forceps and then were injected with 500 μM bicuculline or physiological saline solution. Injection was accomplished via Quartz pipettes  pulled with a Model P-2000 laser puller (Sutter Instruments) using the same program for pulling intracellular electrodes. Pipettes were filled with the solution to be injected and connected with an output line of a dual-channel Picopritzer . The pipettes were then clipped at the tip with fine forceps to allow solution passage. Pipettes were manually inserted into the MGC in each AL and 10 drops (mean diameter ± SD: 76 ± 9.2 μm) were administered quickly in succession with a step pedal that controls the Picospritzer. After injection, the cuticle window was repositioned and sealed with myristic acid (Sigma), and the moth was removed from the plastic tube and placed in an individual cage to recuperate under the same conditions in which it had eclosed. Post-surgery moths were taken 20-30 minutes into scotophase for flying in the wind tunnel. To see whether moths could recover from bicuculline injection, these animals were kept in the dark for at least 2 h before testing them in the wind tunnel.A Plexiglas wind tunnel (L × W × H = 4 × 1.5 × 1.5 m) was used to create a highly controlled wind-flow environment for examining upwind flight behavior in response to pheromone plumes. The wind-tunnel conditions were physicochemically scaled to match the odor emission rate equivalent to that of one female moth and the filament frequencies used in physiological experiments . Longitudinal (u) wind speeds in experiments were 20 cm/s. At the beginning of scotophase, naive, adult male moths were placed individually 3.5 m downwind from the odor source. Each moth was allowed to fly freely inside the wind tunnel for 5 minutes, during which its behavior was recorded. Two types of behavioral data were acquired during experiments: video acquisition and subsequent motion analysis of moth flight behavior for each treatment group ; and scoring of moth behaviors. Scored behaviors were: wing fanning , upwind flight (moth comes within 0.75 m of odor source), close hover (moth hovered within 10 cm of odor source), source contact, and abdomen curl .n = 10, 6, and 9 moths for the unoperated, saline-injected, and bicuculline-injected treatment groups, respectively.Experimental treatment groups included three drug treatments and two odor stimuli, thereby creating a 3 × 2 experimental series. Treatments were tested in a randomized-block design where each block contained positive and negative controls for stimuli and drug treatments. Drug treatments were unoperated moths (to control for surgery effects), saline-injected moths (to control for injection effects), and bicuculline-injected moths. Moths were flown individually to either of two odor stimuli: the two-component pheromone blend or the cyclohexane (negative) control. Four microliters of 500 ng/μl of the pheromone blend , or 4 μl of cyclohexane, were pipetted onto a filter paper placed in the upwind portion of the wind tunnel. This pheromone concentration closely mimicked pheromone emission rates of calling females . For the pheromone odor stimuli, 15, 10, and 12 moths were used for the unoperated, saline-injected, and bicuculline-injected treatment groups, respectively. For the cyclohexane (control), n = 8, 8, and 3, respectively) were flown to floral odors.Two more series of control experiments were conducted to determine the duration of the drug effect and the approximate diffusion range. To determine if moths recovered their ability to track the odor plumes after injection, a similar injection protocol, drug-treatment group , and pheromone stimulus were used, but this experimental series differed from the previous one in one important aspect; moths were flown 2-3 h post-injection. For these experiments, 8, 7, and 9 moths were used for the un-operated, saline-injected, drug-injected treatment groups, respectively. A last experimental series examined whether bicuculline injected into the MGC diffused into neighboring regions of the olfactory system devoted to food odors and impaired behavior to those odors. Three treatment groups  when testing overall treatment effects and when comparing pairs of proportions. An α-level of significance of 0.05 was used. The digitized flight-track analyses  were analyzed using one-way analysis of variance (ANOVA) because data met the assumptions of this test.The scored categorical variables 'wing fanning', 'upwind flight', 'close hover', 'source contact', and 'abdomen curl' were analyzed by means of a log-likelihood test -10,12-hexadecadienal  and -10,12,14-hexadecatrienal (EEZ).Additional data file Additional experimental procedures and additional figures and tablesClick here for file


Celecoxib (Celebrex®) was developed as a selective cyclooxygenase-2 (COX-2) inhibitor for the treatment of chronic pain. However, it now appears that this compound harbours additional pharmacologic activities that are entirely independent of its COX-2-inhibitory activity. This review presents the recently emerged direct non-COX-2 targets of celecoxib and their proposed role in mediating this drug's antitumour effects. Although celecoxib (Celebrex) was developed as a selective cyclooxygenase-2 (COX-2) inhibitor, additional pharmacologic activities have emerged outside of its analgesic activity. For instance, its potency to inhibit COX-2 in the nanomolar range has been eclipsed by its ability to inhibit various isoforms of carbonic anhydrase (CA) at even lower concentrations (see below). Moreover, when added to cells in culture at moderate micromolar concentrations, celecoxib was shown to affect several additional cellular components with roles in cellular proliferation and survival . Therefore, these non-COX-2 targets of celecoxib should be taken into account when interpreting experimental results obtained from the use of celecoxib.When employed in the micromolar range in cell culture, the molecular mechanism by which celecoxib exerts its myriad of pharmacologic activities has not been fully elucidated. But among the various targets, there are at least two cellular components that seem to be controlled directly by celecoxib and thus appear to represent receptors that are engaged at moderate micromolar concentrations. One of these is 3-phosphoinositide-dependent protein kinase-1 (PDK1) and the other is sarcoplasmic/endoplasmic reticulum (ER) calcium ATPase (SERCA). When studied The above findings indicate that the ‘selective’ inhibitor celecoxib has lost its selectivity over time, simply due to the discovery of additional drug targets. This poses the obvious dilemma that observed drug effects cannot be easily ascribed solely to the inhibition of COX-2, but must be supported by careful controls in order to establish the relevant mechanism of drug action. In other words, even though inhibition of COX-2 may take place, this observation by itself does not establish this particular pharmacologic activity as the underlying mechanism by which celecoxib exerts its antitumour properties. For instance, if certain phenotypic consequences observed with celecoxib were attributed to the inhibition of COX-2 activity alone, one would expect that other COX-2 inhibitors, such as rofecoxib (Vioxx®), valdecoxib (Bextra®) or those traditional non-steroidal anti-inflammatory drugs (NSAIDs) that inhibit both COX-1 and COX-2, should also demonstrate these types of biological activities. This is clearly the case in many inflammatory conditions, where the activities seen with NSAIDs can be attributed to COX-2 inhibition. In addition, chemoprevention of colon cancer is also an established pharmacologic activity where inhibition of COX-2 correlates with the suppression of tumour development .in vitro but also in various xenograft animal tumour models in vivo , are expressed in a wide variety of malignancies and implicated in tumour growth , which causes a prominent, yet transient, shutdown of general protein synthesis  . In fact in vivo , furtherin vivo, arguing against earlier concerns that such effects might be artefacts of the high drug concentrations used in cell culture systems in vitro. Additionally, celecoxib is able to inhibit the tumour-associated CAs IX and XII at nanomolar concentrations that are below those required for inhibition of its original target, COX-2.Celecoxib is unique among the coxibs and traditional NSAIDs, because this particular drug displays the greatest potency to induce apoptotic cell death. This activity does not correlate with the inhibition of COX-2, but is congruent with its unique ability to inhibit the non-COX-2 targets PDK1 and SERCA at moderate micromolar concentrations. The inhibition of SERCA constitutes a very rapid drug effect, as increased concentrations of cytosolic calcium levels can be measured within seconds after the addition of celecoxib to intact cells. Significantly, inhibition of these non-COX-2 targets by celecoxib also seems to occur in animal tumour models Although it shall remain undisputed that the inhibition of COX-2, as exerted by celecoxib, has clinically relevant antineoplastic applications, it is also apparent that the celecoxib molecule harbours additional activities that may exert antitumour functions independent of the COX-2 inhibitory activity. It is therefore important to consider the entirety of these multifaceted effects when interpreting data obtained from the experimental use of celecoxib.