The topical role of uric acid and its relation to cardiovascular disease, renal disease, and hypertension is rapidly evolving. Its important role both historically and currently in the clinical clustering phenomenon of the metabolic syndrome (MS), type 2 diabetes mellitus (T2DM), atheroscleropathy, and non-diabetic atherosclerosis is of great importance.Uric acid is a marker of risk and it remains controversial as to its importance as a risk factor (causative role). In this review we will attempt to justify its important role as one of the many risk factors in the development of accelerated atherosclerosis and discuss its importance of being one of the multiple injurious stimuli to the endothelium, the arterial vessel wall, and capillaries. The role of uric acid, oxidative – redox stress, reactive oxygen species, and decreased endothelial nitric oxide and endothelial dysfunction cannot be over emphasized.In the atherosclerotic prooxidative environmental milieu the original antioxidant properties of uric acid paradoxically becomes prooxidant, thus contributing to the oxidation of lipoproteins within atherosclerotic plaques, regardless of their origins in the MS, T2DM, accelerated atherosclerosis (atheroscleropathy), or non-diabetic vulnerable atherosclerotic plaques. In this milieu there exists an antioxidant – prooxidant urate redox shuttle.Elevations of uric acid > 4 mg/dl should be considered a "red flag" in those patients at risk for cardiovascular disease and should alert the clinician to strive to utilize a global risk reduction program in a team effort to reduce the complications of the atherogenic process resulting in the morbid – mortal outcomes of cardiovascular disease. While the topicality of serum uric acid (SUA) being a risk factor is currently controversial ,2, thereet al. recently published article has demonstrated new information regarding this subject. They were able to demonstrate that elevations of SUA levels were independent of variables commonly associated with gout or the metabolic syndrome in association with CVD mortality in middle aged men [LK Niskanen's aged men .et al. sat out to determine the clinical aspects of premature coronary heart disease in 100 male patients 40 years old and younger. Their findings were increased mesomorphic body build, shorter stature, increased anterior posterior chest wall diameter, and increased cholesterol and uric acid (5.13 +/- .11 vs. 4.64 +/-.06) as compared to the normal population [In 1951, Gertler MM and White PD pulation .et al. noted an elevated SUA was also associated with an increased risk of coronary heart disease for men aged 30–59 [A much larger trial (1967) confirmed the initial interest in SUA and CVD with the publication of the early, large (5,127 participants), epidemiologic, seminal Framingham study. This classical paper by Kannel ed 30–59 . In addied 30–59 .et al. have nicely demonstrated that hyperuricemia predicts cardiovascular events in the general population, the hypertensive population, and patients with pre-existing CVD. Furthermore hyperuricemia predicts the development of future hypertension [This controversy regarding SUA being a risk factor or a risk marker is not as important as understanding its overall role in the association with endothelial cell damage, dysfunction, decreased endothelial nitric oxide (eNO) bioavailability, and how SUA interacts with other substrate toxicities and increased reactive oxygen species (ROS) of the A-FLIGHT-U acronym, which result in accelerated atherosclerosis table . Johnsonrtension .There are certain clinical clustering groups with increased cardiovascular risk, which have associated hyperuricemia table . Non-diaet al. suggested that hyperuricemia be added to the cluster of metabolic and hemodynamic abnormalities associated with insulin resistance and/or hyperinsulinemia of Syndrome X [The importance of hyperuricemia and the clustering phenomenon of the metabolic syndrome were first described by Kylin in 1923 when he described the clustering of three clinical syndromes: hypertension, hyperglycemia, and hyperuricemia . In 1988ndrome X .The four major players in the MS are hyperinsulinemia, hypertension, hyperlipidemia, and hyperglycemia. Each member of this deadly quartet has been demonstrated to be an independent risk factor for CHD and capable of working together in a synergistic manner to accelerate both non-diabetic atherosclerosis and the atheroscleropathy associated with MS, PD, and T2DM.In a like manner, hyperuricemia, hyperhomocysteinemia, ROS, and highly sensitive C- reactive protein (hsCRP) each play an important role in expanding the original Syndrome X described by Reaven in the atherosclerotic process. The above quartet does not stand alone but interacts in a synergistic manner resulting in the progression of accelerated atherosclerosis and arterial vessel wall remodeling along with the original players and the A-FLIGHT-U toxicities table . The MS 2-•) [Insulin, proinsulin, and amylin individually and synergistically activate the renin – angiotensin system (RAS) with subsequent increase in Ang II. Ang II is the most potent endogenous inducer of NAD(P)H oxidase, increasing NAD(P)H, which increases vascular – intimal reactive oxygen species (ROS) and superoxide (O2-•) ,20. Ther2-•) .Hypertension is strongly associated with hyperuricemia. SUA levels are elevated in hypertension and are present in 25% of untreated hypertensive subjects, 50% of subjects taking diuretics, and greater than 75% of patients with malignant hypertension . PotentiBecause endothelial dysfunction, local oxidant generation, elevated circulating cytokines, and a proinflammatory state are common in patients with cardiovascular disease and hypertension there is an increased level of oxidative – redox stress within vascular tissues. Oxidative – redox stress results in impaired endothelium-dependent vasodilation with quenching of endothelial nitric oxide (eNO) and allows the endothelium to become a net producer of ROS specifically superoxide as the endothelial nitric oxide synthase (eNOS) enzyme uncouples to produce superoxide instead of eNO. This similar mechanism applies equally well to that associated with type 2 diabetes and congestive heart failure ,19. It iet al. were able to demonstrate that blood pressure levels were predictive for cardiovascular disease incidence synergistically with serum uric acid level [Lin KC id level . Two sepid level ,29. ThisThese above models have provided the first challenging evidence that uric acid may have a pathogenic role in the development of hypertension, vascular disease, and renal disease .Obesity has reached epidemic proportions in the past decade and represents one of the confounding factors associated with the MS and T2DM ,30 figu.Hyperuricemia has been associated with increasing body mass index (BMI) in recent studies and are even apparent in the adolescent youth -33.et al. have recently discussed the role of leptin as possibly being a regulator of SUA concentrations in humans and even suggested that leptin might be one of the possible candidates for the missing link between obesity and hyperuricemia [Leptin levels are elevated and associated with insulin resistance in MS and early T2DM. Bedir A uricemia . Furtheruricemia ,33  – ROS oxidation products) and a reductive stress through pseudohypoxia with the accumulation of NADH and NAD(P)H in the vascular intima ,35,36.This redox stress consumes the natural occurring local antioxidants such as: SOD, GPX, and catalase table . Once thA direct relation between homocysteine levels and SUA levels is known to occur in patients with atherosclerosis. Not only do these two track together (possibly reflecting an underlying elevated tension of redox stress) but also may be synergistic in creating an elevated tension of redox stress, especially in the rupture prone, vulnerable atherosclerotic plaque with depletion of local occurring antioxidants -41 (figuNon-diabetic atherosclerosis and atheroscleropathy (accelerated atherosclerosis associated with MS, prediabetes, and T2DM) are each impacted with the elevation of uric acid ,43.In MS and T2DM there is an observed increased thrombogenecity, hyperactive platelets, increased PAI-1 (resulting in impaired fibrinolysis), and increased fibrinogen in the atherosclerotic milieu associated with the dysfunctional endothelial cell. Additionally, the vulnerable atherosclerotic plaque includes increased tissue factor, which increases the potential for thrombus formation when the plaque ruptures and exposes its contents to the lumen ,42,43.The upper 1/3 of the normal physiologic – homeostatic range (> 4 mg/dl) and abnormal elevations (> 6.5 or 7 mg/dl in men and > 6.0 mg/dl in women) in SUA definitely should be considered as one of the multiple injurious stimuli to the arterial vessel wall and capillary, which may contribute to endothelial dysfunction and arterial – capillary vessel wall remodeling through oxidative – redox stress ,3,19 fi. There aEach of these A-FLIGHT-U toxicities may be viewed as an independent risk marker – factor and is known to have a synergistic effect when acting in concert ,39,42,43The simple concept that SUA in patients with CVD, MS, T2DM, hypertension, and renal disease may reflect a compensatory mechanism to counter oxidative stress is intriguing. However, this does not explain why higher SUA levels in patients with these diseases are generally associated with worse outcomes .Antioxidants may become prooxidants in certain situations -55. TherSUA in the early stages of the atherosclerotic process is known to act as an antioxidant and may be one of the strongest determinates of plasma antioxidative capacity .However, later in the atherosclerotic process when SUA levels are known to be elevated  this previously antioxidant (SUA) paradoxically becomes prooxidant. This antioxidant – prooxidant urate redox shuttle seems to rely heavily on its surrounding environment such as timing (early or late in the disease process), location of the tissue and substrate, acidity , the surrounding oxidant milieu, depletion of other local antioxidants, the supply and duration of oxidant substrate and its oxidant enzyme. In the accelerated atherosclerotic – vulnerable plaque the intima has been shown to be acidic , depleteNitric oxide and vitamin C have each been shown to inhibit the prooxidant actions of uric acid during copper-mediated LDL-C oxidation ,55.We have devised an acronym, to better understand the increase in SUA synthesis within the accelerated atherosclerotic plaque termed: ANAi. A – apoptosis, N – necrosis, A – acidic atherosclerotic plaque, angiogenesis (both induced by excessive redox stress), i – inflammation, intraplaque hemorrhage increasing red blood cells – iron and copper transition metal ions within the plaque.This acronym describes the excess production of purines: (A) adenine and (G) guanine base pairs from RNA and DNA breakdown due to apoptosis and necrosis of vascular cells in the vulnerable – accelerated atherosclerotic plaques; allowing SUA to undergo the antioxidant – prooxidant urate redox shuttle figure .Reactions involving transitional metal ions such as copper and iron are important to the oxidative stress within atherosclerotic plaques. Reactions such as the Fenton and Haber- Weiss reactions and similar reactions with copper lead to an elevated tension of oxidative – redox stress.FENTON REACTION:2+ + H2O2 → Fe3+ + OH• + OH-Fe3+ + H2O2 → Fe2+ + OOH• + H+FeHABER – WEISS REACTION:2O2 + O2- → O2 + OH- + OHH2O2 + OH- → H2O + O2- + H+H3+ - Cu2+ - Cu1+) metal ions can undergo similar reactions with formation of lipid peroxides and ROS. This makes the leakage of iron and copper from ruptured vasa vasorum very important in accelerating oxidative damage to the vulnerable accelerated atherosclerotic plaques, as well as, providing a milieu to induce the SUA antioxidant – prooxidant switch within these plaques [The hydroxyl radicals can then proceed to undergo further reactions with the production of ROS through addition reactions, hydrogen abstraction, electron transfer, and radical interactions. Additionally, copper  and the inflammatory cells (primarily the macrophage and to a lesser extent the lymphocyte).The endothelium is an elegant symphony responsible for the synthesis and secretion of several biologically active molecules. It is responsible for regulation of vascular tone, inflammation, lipid metabolism, vessel growth (angiogenesis – arteriogenesis), arterial vessel wall – capillary sub endothelial matrix remodeling, and modulation of coagulation and fibrinolysis. One particular enzyme system seems to act as the maestro: The endothelial nitric oxide synthase (eNOS) enzyme and its omnipotent product: endothelial nitric oxide (eNO) figure .The endothelial nitric oxide synthase (eNOS) enzyme reaction is of utmost importance to the normal functioning of the endothelial cell and the intimal interstitium. When this enzyme system uncouples the endothelium becomes a net producer of superoxide and ROS instead of the net production of the protective antioxidant properties of eNO table  figure .There are multiple causes for endothelial uncoupling in addition to hyperuricemia and the antioxidant – prooxidant urate redox shuttle: A-FLIGHT -U toxicities, ROS, T2DM, prediabetes, T1DM, insulin resistance, MS, renin angiotensin aldosterone activation, angiotensin II, hypertension, endothelin, dyslipidemia – hyperlipidemia, homocysteine, and asymmetrical dimethyl arginine (ADMA) ,39,43.Xanthine oxidase – oxioreductase (XO) has been shown to localize immunohistochemically within atherosclerotic plaques allowing the endothelial cell to be equipped with the proper machinery to undergo active purine metabolism at the plasma membrane surface, as well as, within the cytoplasm and is therefore capable of overproducing uric acid while at the same time generating excessive and detrimental ROS  figure ,4. To suThe healthy endothelium is a net producer of endothelial nitric oxide (eNO).2-) associated with MS, T2DM, and atheroscleropathy [The activated, dysfunctional endothelium is a net producer of superoxide (Oeropathy .Uric acid and highly sensitive C reactive protein (hsCRP) each now share a respected inclusion as two of the novel risk markers – risk factors associated with the metabolic syndrome. It is not surprising that these two markers of risk track together within the MS. If there is increased apoptosis and necrosis of vascular cells and inflammatory cells in accelerated – vulnerable atherosclerotic plaques as noted in the above section then one would expect to see an increase in the metabolic breakdown products of RNA and DNA with arginine and guanine to its end product of uric acid. SUA elevation may indeed be a sensitive marker for underlying vascular inflammation and remodeling within the arterial vessel wall and capillary interstitium.Is it possible that SUA levels could be as similarly predictive as hsCRP since it is a sensitive marker for underlying inflammation and remodeling within the arterial vessel wall and the myocardium .Should the measurement of SUA be part of the national cholesterol educational program adult treatment panel III and future IV (NCEP ATPIII or the future NCEP ATPIV) clinical guidelines ?et al. conclude that SUA may reflect the severity of systolic dysfunction and the activation of an inflammatory reaction in patients with congestive heart failure [Uric acid is known to induce the nuclear transcription factor (NF-kappaB) and monocyte chemoattractant protein-1 (MCP-1) . Regardi failure . Additio failure .et al. have shown a statistically significant positive correlation between CRP and body mass index (BMI), total cholesterol, triglycerides, LDL-C, fasting glucose, fasting insulin, uric acid, systolic blood pressure, and diastolic blood pressure and a significant negative correlation of CRP with HDL-C in a study of 3692 Japanese men aged 34–69 years of age. They conclude that there are a variety of components of the MS, which are associated with elevated CRP levels in a systemic low-grade inflammatory state [Tamakoshi K ry state .CRP and IL-6 are important confounders in the relationship between SUA and overall mortality in elderly persons, thus when evaluating this association the potential confounding effect of underlying inflammation and other risk factors should be considered .2O2 generation and oxidative stress within the renal architecture with resultant increased remodeling. Hyperuricemia could increase the potential for urate crystal formation and in addition to elevated levels of soluble uric acid could induce inflammatory and remodeling changes within the medullary tubulo-interstitium.Hyperuricemia can be the consequence of increased uric acid production or decreased excretion. Any cause for decreased glomerular filtration, tubular excretion or increased reabsorption would result in an elevated SUA. Increased SUA has been found to predict the development of renal insufficiency in individuals with normal renal function . In T2DMet al. revealed a J-shaped curve association with SUA levels and all-cause mortality in hemodialysis patients [A recent publication by Hsu SP patients . They wepatients .et al. have speculated that the increased risk for the lowest quartile reflects a decreased antioxidant activity, while the increased risk at higher levels reflects the role of uric acid in inducing vascular disease and hypertension through the mechanism of the previously discussed antioxidant prooxidant urate redox shuttle. This would suggest that treatment with xanthine oxidase inhibitors  should strive to bring levels to the 3–4 mg/dl range and not go lower [Johnson RJ go lower .While it is not within the scope of this review to discuss this important topic with an in- depth examination, it is important to discuss some prevailing concepts and provide some clinical nutritional guidelines for hyperuricemia table .Moderation is the key element in any diet approaching hyperuricemia. The nutritional "gold standard" for the treatment of hyperuricemia has been "the low purine diet". This traditional diet has recently come into question as it may limit the intake of high purine vegetables and fruits. Vegetables and fruits are important for the fiber they supply in addition to naturally occurring antioxidants. Recently, of greater importance is controlling obesity through generalized caloric restriction and increased exercise to combat the overnutrition and underexercise of our modern-day society, as well as, controlling the consumption of alcohol .Nutritional support by the nutritionist and the diabetic educator  is of utmost importance when dealing with the metabolic syndrome, T2DM, and the cardiovascular atherosclerotic afflicted patients in order to obtain global risk reduction, because we are what we eat.From a clinical standpoint, hyperuricemia should alert the clinician to an overall increased risk of cardiovascular disease and especially those patients with an increased risk of cardiovascular events. Hyperuricemia should therefore be a "red flag" to the clinician to utilize a team effort in achieving an overall approach to obtain a global risk reduction program through the use of the RAAS acronym table .SUA may or may not be an independent risk factor especially since its linkage to other risk factors is so strong, however there is not much controversy regarding its role as a marker of risk, or that it is clinically significant and relevant.Regarding the MS and epidemiologic evaluations: A multivariate model could well eliminate hyperuricemia as an independent risk factor even if it were contributing to the overall phenotypic risk of the syndrome. Additionally, we must remember that it was Reaven that called for the inclusion of hyperuricemia to Syndrome X we now call MS – insulin resistance syndrome -IRS in 1993 .A quote by Johnson RJ and Tuttle KR is appropriate for the concluding remarks:"The bottom line is that measuring uric acid is a useful test for the clinician, as it carries important prognostic information. An elevation of uric acid is associated with an increased risk for cardiovascular disease and mortality, especially in women" .2-•); xanthine oxidase (XO); type 2 diabetes mellitus (T2DM); angiotensin converting enzyme (ACE); renin-angiotensin-aldosterone system (RAAS); advanced glycosylation endproducts (AGE); superoxide dismutase (SOD); glutathione (GPX); plasminogen activator inhibitor (PAI-1); angiotensin II (AngII); low density lipoprotein cholesterol (LDL-C); asymmetrical dimethyl arginine (ADMA); highly sensitive C reactive protein (hsCRP); national cholesterol educational program adult treatment panel III (NCEP ATPIII); nuclear transcription factor (NF-kappaB); monocyte chemoattractant protein-1 (MCP-1); tumor necrosis factor alpha ; interleukin one beta (IL-1beta); interleukin 6 (IL-6); body mass index (BMI); high density lipoprotein (HDL); hydrogen peroxide (H2O2); free fatty acids (FFA).Serum uric acid (SUA); cardiovascular disease (CVD); coronary heart disease (CHD); endothelial nitric oxide (eNO); endothelial nitric oxide synthase (eNOS); endothelial nitric oxide (eNO); reactive oxygen species (ROS); metabolic syndrome (MS); insulin resistance syndrome (IRS); nicotine adenine dinucleotide phosphate oxidase reduced NAD(P)H; superoxide (OThe authors declare that they have no competing interests.MRH and SCT envisioned, wrote and edited jointly.


Metarhizium anisopliae var. anisopliae, and its leaf-cutting ant host Acromyrmex echinatior as the model system.Parasite heterogeneity and within-host competition are thought to be important factors influencing the dynamics of host-parasite relationships. Yet, while there have been many theoretical investigations of how these factors may act, empirical data is more limited. We investigated the effects of parasite density and heterogeneity on parasite virulence and fitness using four strains of the entomopathogenic fungus, The relationship between parasite density and infection was sigmoidal, with there being an invasion threshold for an infection to occur (an Allee effect). Although spore production was positively density-dependent, parasite fitness decreased with increasing parasite density, indicating within-host scramble competition. The dynamics differed little between the four strains tested. In mixed infections of three strains the infection-growth dynamics were unaffected by parasite heterogeneity.Metarhizium, that produce aggregated transmission stages. Such parasites make useful models for investigating infection dynamics and the impact of parasite competition.The strength of within-host competition makes dispersal the best strategy for the parasite. Parasite heterogeneity may not have effected virulence or the infection dynamics either because the most virulent strain outcompeted the others, or because the interaction involved scramble competition that was impervious to parasite heterogeneity. The dynamics observed may be common for virulent parasites, such as  However, as the transmission stages of parasites tend to be clustered, the majority of host-parasite interactions will rather involve multiple parasite individuals. This is particularly the case for those parasites that exhibit a semelparous life-history, releasing all their transmission propagules in a single event that normally coincides with host death ,2. In thnfection . Thus thnfection ,5.The co-occurrence of multiple parasites within a single host makes within-host competition between the parasites inevitable. Hosts represent limited resources and there is a carrying capacity for the total biomass of parasites that the host resources can support. The within-host growth of the parasites will thus normally be density-dependent, decreasing as the carrying capacity is approached ,7. InterIn spite of the fundamental importance of interactions between different strains of parasites, empirical studies of mixed infections are relatively rare and a number of them have produced results that conflict with the theoretical predictions ,24. MostM. anisopliae var. anisopliae (Metschnikoff) (Deuteromycotina: Hyphomycetes) in the leaf-cutting ant Acromyrmex echinatior Forel (Hymenoptera: Formicidae: Attini). M. anisopliae var. anisopliae is a generalist entomopathogenic fungus that is known to infect leaf-cutting ants [Metarhizium spores have a tendency to remain attached to one another, making even dispersed spores likely to be clustered, and soil has been estimated to contain as many as 1,000 to 50,000 spores g-1 [Metarhizium thus has a semelparous life-history and is an 'obligate killer' [Metarhizium are common, fundamental studies of its infection dynamics are rare.Here we examine the infection dynamics of the parasite ing ants -41, as wores g-1 . Inside ores g-1 ,21,22. AM. anisopliae var. anisopliae strains near leaf-cutting ant nests in Panama [M. anisopliae var. anisopliae adhered to the mass action principle and whether parasite growth and fitness was density-dependent. We then examined whether the infection dynamics were consistent between different strains of parasite, and how the dynamics were affected by intraspecific within-host competition involving multiple parasite genotypes. We used two Panamanian strains that had had the opportunity to coevolve with A. echinatior and one exotic strain that had never previously encountered the host. A number of studies suggest that exotic strains are less competitive during within-host interactions than are native strains that have coevolved with the host [There is a diversity of n Panama , indicatthe host -48. We dthe host . Note ththe host , will noMetarhizium anisopliae var. anisopliae spores applied had a significant effect on ant mortality  . This supports the assumption that sporulation was complete at the time of assessment and also indicates that parasites that took longer to kill their host did not produce more spores. The proportions of cadavers sporulating were significantly less than expected based upon the number of ants estimated (from the control data) to have died from other causes (F1,17 = 8.55, P = 0.0095). This indicates that certain ants killed by Metarhizium failed to sporulate. Both the proportion of ant cadavers sporulating  and the mean number of spores produced per sporulating cadaver (F9,127 = 1.97, P = 0.048) increased significantly with dose , it was found that k increased significantly with dose  nor the number of spores produced , the strain , and also the colony of origin . There were no significant interactions between strain and concentration . Mortality was generally positively correlated with dose, although the significance of pairwise differences between doses did vary somewhat between strains  (Breslow statistic = 8.36, d.f. = 1, P = 0.004) or the mixture of all three isolates (Breslow statistic = 5.00, d.f. = 1, P = 0.025)  and to increase slightly with dose in all treatments  and the strains also did not differ overall (F3,270 = 0.19, P = 0.903). The k-values for all three strains were positively correlated with the number of spores applied (F1,15 = 3612.9, P < 0.0001) and had similar slopes .Although the proportion of ant cadavers sporulating was consistently high (>80%) in strain 02–73 and was positively correlated with dose in the other strains Figure , the intMetarhizium [e.g. [7 spores per ml. In addition, the mortality caused by doses of 1 × 104 spores per ml or less did not differ from that of ants treated with the control solution , originating from a different geographical location and from a different host order (Lepidoptera) that had the lowest virulence, and the strain isolated from a leaf-cutting ant worker (KVL 02–72) that had the highest. Many studies have found Metarhizium strains to vary in virulence [e.g. [When the different strains of ce e.g. ,61]. Dif. DifM. ace [e.g. . As viruce [e.g. ,63. Althce [e.g. ,64,65. Dk-value vs. dose relationship was identical for the mixed and single infections, indicating that the density-dependent growth patterns were unaffected by host heterogeneity.A fundamental assumption of most models of host-parasite relationships is that within-host competition is more intense, and results in heightened virulence, when it involves more than one parasite genotype -10. HoweMetarhizium and other entomopathogenic fungi [There are two possible explanations for the results. The most parsimonious is that the most virulent strain in the mixed infections simply outcompeted the other strains and drove them to extinction within the host. Such superinfection dynamics have previously been suggested for ic fungi ,66,67 anic fungi ,31. It rMetarhizium strains were engaged in scramble competition with dynamics that are impervious to interactions being inter- or intraclone.Importantly, the infection dynamics did not differ between the single and mixed infections. The occurrence of within-host competition, whether it involves the production of antagonistic compounds or is mediated by the host's immune system, might be expected to force parasites to divert some resources from growth and the production of transmission stages to producing or coping with competitive mechanisms. While there is some evidence for this , the proMetarhizium-Acromyrmex system studied here. Even though the probability of a successful infection was increased substantially by parasites occurring in aggregations, the effect of competition between parasite propagules of the same clone makes dispersal the best strategy. It seems likely that this is generally true for semelparous parasites. Further investigations of the impact of within-host competition on parasite fitness are needed and should endeavour to establish where on the superinfection-coinfection continuum the interaction lies by identifying which parasites produce transmission stages. The fact that the production of transmission stages of semelparous parasites, such as Metarhizium, is concentrated into a single bout, and thus that their lifetime fitness can be readily quantified, makes them excellent models for doing this.The importance of within-host competition between parasites is well illustrated by the A. echinatior were collected from Gamboa, Panama, and maintained in the lab under controlled conditions (ca. 24°C, 70% RH) on a diet of bramble leaves (Rubus fruticosus) and rice grains. For the experimental replicates, large workers (head width 2.1 to 2.4 mm) were removed from their colonies and placed individually in plastic pots (diameter: 2.5 cm, height: 4 cm) where they were maintained at 24°C with an ad libitum supply of water and sugar water. A number of isolates of M. anisopliae var. anisopliae were collected from the vicinity of leaf-cutting ant nests at the same location in Gamboa, Panama, and were cultured as monospore isolates on Sabouraud dextrose agar [Colonies of ose agar . Spore  and thus provide a reliable estimate of spore production.Ants were treated with ous work ,40,56, tous work , and plaM. anisopliae var. anisopliae isolate KVL 02–56, which had been isolated from the dump pile of an Atta colombica nest in Gamboa, Panama [8 to 5 × 103 spores ml-1 . Given that soil at the site in Panama has been estimated to contain as many as 1,000 to 50,000 spores g-1 [A. echinatior (Ae47 and Ae109) were treated with each of these doses, or with the control solution, and their survival monitored for twenty days after application. The effect of dose and colony of origin on ant mortality was analysed with a Cox proportional hazard regression model to examine the effect of parasite density (dose) on virulence and infection rate. This incorporates both case mortality and the time of death. Pairwise comparisons of the doses were done using Kaplan-Meir survival analyses and the Breslow statistic. The ant cadavers were left for ten days after the end of the experimental period, in order to allow ample time for all cadavers to sporulate fully. The numbers of cadavers sporulating and the numbers of spores produced by these cadavers were assessed with binary logistic and general linear models respectively. These data were used to calculate k-values to assess whether parasite growth was density dependent, as done previously by Ebert et al. [The experiment involved , Panama . A sporeores g-1 , it seemki = log10 (M0 Di / Mi D0)ki is the k-value at dose i, M is the number of spores produced, D is the number of spores applied, and M0 and D0 represent the number of spores produced and applied respectively at the lowest dose tested. A zero value for k indicates that the number of spores produced at dose i are exactly proportional to the increased dose (so, for example, a doubling of dose results in a doubling of the number of spores produced). The equation gives a positive value for k when the number of spores produced at dose i are less than proportional to the increase in dose. Zero values for k thus indicate that spore production is independent of parasite density, while positive values of k indicate that spore production is negatively density dependent.where M. anisopliae var. anisopliae were compared. These were KVL 02–73 (isolated from soil at the field site from which the ant colonies were collected), KVL 02–72 (isolated from an Atta colombica leaf-cutting ant worker), and the strain Ma275 (isolated from Cydia pomonella (Lepidoptera: Tortricidae) in Germany). The strains therefore represented a range in terms of their potential coevolution with A. echinatior, with the former two being likely to have had some interaction while Ma275 would not previously have encountered A. echinatior. Each strain was diluted tenfold and tested at six doses from 5 × 108 to 5 × 103 spores ml-1. In addition, a mixed spore suspension was made up to examine inter-strain competition. The suspension contained equal numbers of spores of each of the three strains. This was serially diluted and applied at the same doses as the individual isolates. Four ants from each of five colonies of A. echinatior (Ae48, 109, 143, 153 and 154) were treated with each dose of each isolate or with the control solution and their survival monitored for twenty days. The survival of the ants was analysed as in the preceding experiment to examine if strains differed in virulence and if the growth of each strain was density-dependent. Ten days after the end of the experimental period, spore production was assessed by ranking each cadaver for the amount of sporulation as described earlier. These ranks were used to estimate the actual number of spores produced and these values were then used to calculate k-values. A regression analysis was carried out on these data in order to assess if the growth of each strain was density-dependent and whether the density-growth dynamics differed between strains.To establish whether the dynamics recorded in the first experiment were consistent for different strains of the parasite, three isolates of WOHH conceived the study, assisted with the experiments, analysed the results, and drafted and revised the manuscript. KSP, LVU and DP carried out Experiment 1 and KSP also participated in Experiment 2. MP assisted with both experiments. LT assisted with mycological aspects and JJB provided support throughout. All authors contributed to the writing of the manuscript.


Expression microarrays are increasingly used to characterize environmental responses and host-parasite interactions for many different organisms. Probe selection for cDNA microarrays using expressed sequence tags (ESTs) is challenging due to high sequence redundancy and potential cross-hybridization between paralogous genes. In organisms with limited genomic information, like marine organisms, this challenge is even greater due to annotation uncertainty. No general tool is available for cDNA microarray probe selection for these organisms. Therefore, the goal of the design procedure described here is to select a subset of ESTs that will minimize sequence redundancy and characterize potential cross-hybridization while providing functionally representative probes.Litopenaeus setiferus) was used to test this algorithm so that the detailed results could be examined. The functional representative level of the selected probes was quantified using Gene Ontology (GO) annotations.Sequence similarity between ESTs, quantified by the E-value of pair-wise alignment, was used as a surrogate for expected hybridization between corresponding sequences. Using this value as a measure of dissimilarity, sequence redundancy reduction was performed by hierarchical cluster analyses. The choice of how many microarray probes to retain was made based on an index developed for this research: a sequence diversity index (SDI) within a sequence diversity plot (SDP). This index tracked the decreasing within-cluster sequence diversity as the number of clusters increased. For a given stage in the agglomeration procedure, the EST having the highest similarity to all the other sequences within each cluster, the centroid EST, was selected as a microarray probe. A small dataset of ESTs from Atlantic white shrimp (For organisms with limited genomic information, combining hierarchical clustering methods to analyze ESTs can yield an optimal cDNA microarray design. If biomarker discovery is the goal of the microarray experiments, the average linkage method is more effective, while single linkage is more suitable if identification of physiological mechanisms is more of interest. This general design procedure is not limited to designing single-species cDNA microarrays for marine organisms, and it can equally be applied to multiple-species microarrays of any organisms with limited genomic information. Expression microarrays are powerful tools for human disease diagnosis, prognosis and treatment  offeringThe two most widely used expression microarray systems are oligonucleotide and cDNA microarrays. Oligonucleotide microarrays are generated by chemically synthesizing short oligo probes 20–70 bp) onto the slides . In cont0 bp ontoIn the absence of cDNA microarray probe selection algorithms, EST selection for spotting on microarrays has been approached using various informal methods. These methods included spotting ESTs without sequencing information, spotting only sequenced ESTs with annotations, or forcing the selection on gene-oriented clusters . The choIn this study, we propose a probe selection procedure for cDNA microarray that tracks both sequence redundancies and functional representativeness of the selected probes in an integrated sequence diversity plot (SDP). SDP includes a sequence diversity index (SDI) to measure the sequence similarities within EST clusters quantitatively. The issue of how many probes are sufficiently representative for all collected ESTs is approached in a manner similar to the choice of dimensions to retain in principle component analysis (PCA). This approach reflects the fact that there is no definitive right answer to the question ; the numLitopenaeus setiferus) from the Marine Genomics website [A small data set of 1047 ESTs from Atlantic white shrimp :This index is the ratio of within-cluster sequence dissimilarities to the total sequence dissimilarity when m clusters are formed (dmi is the distance (dissimilarity), the E-value from blast result (details see methods), between the ith pair of sequences for a total km pairs of within-cluster comparisons when m clusters are formed. D is defined as , the average distance of the total N pair-wise distances among all n sequences (where  in this data set).where m, is the sum of the number of assembled contigs and singlets (single sequences, which cannot be assembled with any other sequences) when m clusters (m = 1,2...n) are formed. The contiguity index (CI) at a given number of clusters (m) is defined as the inverse of the average number of putative genes per cluster, which equals the number of clusters per gene:The within-cluster sequence contiguity is evaluated using CAP3 , commonlThis index reflects how contiguous the sequence members are within a cluster. Maximum value of CI is 1 when all the members are contiguous (one cluster per gene).m clusters of ESTs are formed is defined as the number of unique GO terms associated with all m probes (m = 1, 2...n) divided by the number of the GO terms associated with all n sequences (n = 971).The unique probe GO index (UPGI) when ProbeGOmj is the number of unique GO terms associated with the probe representing the jth cluster when m clusters are formed and sequenceGOi is the number of GO terms of the ith sequence. This index measures functionally how representative the selected probes are among all functionally unique sequences in the entire EST collection. Three UPGIs are calculated for three GO domains, respectively: molecular function (UPGI-MF), biological process (UPGI-BP), and cellular components (UPGI-CC) ) has shown similar results observed using the probe functional index, UPGI (Eq. (3); Figure ) has sho) has shoESTs were annotated based on Gene Ontology (GO) terms (details see methods). Three types of functionally unassigned sequences were generated through the GO annotation process: the first type was the sequences having no similar sequences found in the GO database. The majority of ESTs 63%) belonged to this category , the functional uniqueness of the selected probes using single linkage method is superior than that of the other two methods while average linkage is the most effective method for dimension reduction . The magnitude of SDI decreases as the number of clusters increases; sequences are more similar within clusters as the number of clusters increases. SDI is analogous to the F-statistics. That is, SDI is the "within" variation divided by the "total" variation while the F-statistics is "within" variation divided by "between" variation. Two ancillary indexes  and a sequence contiguity index (CI)) were designed to evaluate the functional representativeness of the selected probes and identify the numbers of putative genes each probe potentially would cross-hybridize. These indexes aid the probe selection processes by bringing in the functional annotations of ESTs as the main goal of the microarray experiments is generally to interpret the biological significances and interactions of genes of interest. A common goal of microarray experiments is to identify co-regulated genes. This is based on the assumption that if two genes are co-expressed, they are likely to be co-regulated through the same mechanism . It has Different clustering processes mimic different scenarios of cross hybridization between sequences. Sequences from the same transcript will hybridize with each other, and this is reflected in the clusters formed using the single linkage method. In contrast, some of the sequences in the clusters formed by the complete or average linkage methods could be paralogs or alternative splicing variants of the same gene. It might be argued that if a sequence, for example Penaeidin 2, was chosen as a probe from the cluster of sequences containing different subtypes to spot on the microarray, this sequence will likely hybridize to the sequences in the same cluster, for example, Penaeidin 3a and Penaeidin 3c. The contiguity index and probe functional index developed in our study will identify the cross hybridization potential for the users. Potential cross-hybridization has become a more apparent problem for the transcriptomics community. A tool was developed to identify potential cross-hybridized probes lately , howeverdmi) could be easily replaced in the future by any good cross-hybridization predicting parameter(s) developed for long cDNA sequence hybridization. The design procedure we described here will work in the exact same fashion. In a similar manner, although the traditional hierarchical clustering algorithm with three linkage methods was used in our study, any bottom-up clustering algorithm (e.g., K-nearest means clustering) or top-down approach  could be easily performed, and the corresponding SDIs, UPGIs and CIs will be generated in the same way and summarized in the SDP. The performances of these different bottom-up or top-down algorithms (to group or partition the sequences) could be compared using the SDP. In brief, other distance matrix and clustering algorithms other than what we used in this study could be easily applied using our algorithm, and their performances could be evaluated quantitatively using the suite of indexes in SDP.Cross-hybridization potential between long cDNA seqeuences is harder to model than that between short (oligonucleotide) sequences. Although several studies have shown that local sequence percent identity seem to be a reasonable predictor for cross hybridization for cDNA microarray experiments -33, the Litopenaeus vannamei [Annotation of functionally unknown sequences is not a trivial task itself. Gene Ontology has become a standard ontology to annotate unknown sequences. Sequence similarity search against the GO database using BLAST was used in this study. The completeness of the GO database and sensitivity/selectivity of the BLAST procedure would dictate the annotation capability. Several different approaches could potentially improve the annotation in the future. One example found in this study was Penaeidin family, a unique family of antimicrobial peptides with both proline and cysteine-rich domains that were first identified and characterized as peptides in the hemolymph of the Pacific white shrimp, vannamei . No homovannamei  to searcvannamei ,37) coulThe integration of this cDNA probe selection procedure with the database through Marine Genomics web-based interface  is curreThe sequence diversity index (SDI) was developed in this study to select probes using ESTs for designing cDNA microarrays. Two ancillary mathematic indexes (sequence contiguity index [CI] and unique probe GO index [UPGI]) were used to identify potential cross-hybridization between different transcripts  and to quantify biologically how representative the probes were. These three indexes were summarized in a sequence diversity plot (SDP) and were used to assist cDNA microarray probe selections for organisms without any genomic information. This method allows the user-defined number of probes to be selected for the cDNA microarray experiments. Different clustering methods balance the representativeness of the probe functional annotations and minimization of the sequence redundancies. Accordingly, different linkage methods can be used to decide between microarray designs for biomarker discovery or for functional genomics.It is clear that sequence assembly into contigs is not necessary for microarray probe selection although it is informative to identify the relationship among sequence members within clusters based on the CI. The microarray design procedure described here could also be used for multi-species or cross-species microarray design in a scenario where the sequences with high similarity from different species cross hybridize to each other , but notThis method is not limited to the ESTs collected from single or multiple marine organisms. Furthermore, this method can be applied to any organisms without the complete sequenced genomes.L. setiferus) were used in this study. Pre-processing included customized low quality filtering, poly-A tail, vector, adaptor screening, trimming, and low-complexity masking by DUST [Twenty six thousand and six hundred fifty-six (26,656) Expressed Sequence Tags (ESTs) from 14 marine species were generated and stored in a postgresSQL database through a user-friendly interface at the Marine Genomics website . All the by DUST . After pq and n, the statistics of HSP (high-scoring segment pairs) scores are characterized by two parameters, K and lambda. The E-value, the expected number of HSPs with score of at least S, given by the formula E = -λSKqne, was used as the distance measurement (dmi in Eq (1) in results) between ESTs for cluster analysis to determine sequence redundancies. dmi is the distance between the ith pair of sequences for a total km pairs of within-cluster comparisons when m clusters are formed.All against all pair-wise BLASTN  was perfHierarchical cluster analyses with three common linkage methods (single linkage, average linkage, complete linkage) were performed to reduce the redundancies among sequences.Two sequence indexes, the sequence diversity index [SDI; Eq. (1) in results] and the contiguity index [CI; Eq. (2) in results], were used throughout the sequence redundancy reduction. SDI was used to aid the number of probes to select. The within-cluster sequence contiguity (CI) is evaluated using CAP3 assembly software with default parameters . UnweighTo maximize the hybridization probability between the selected probe and the sequences within the cluster, the sequence has the highest similarity to all the other sequences within the cluster is selected. That is, the centroid EST, the sequence has the minimum average distance to all the other sequences within each cluster was spotted on the array.-6 for GO annotation are considered as similar, and they potentially share the same molecular functions, cellular components, or biological processes. The GO terms found associated with the EST sequences, if any, were recorded separately for each of the three domains. If there were multiple GO terms in any single domain (e.g., molecular function), the inverse of the number of GO terms in that domain is used for functional quantification . For example, there are three molecular functional annotations (GO:0005515, GO:0004866, GO:0004867) associated with the sequence 1046, then each of them is considered 1/3 in the GO quantification for this particular sequence. Therefore, the quantification for each GO domain will sum up to the original analyzed sequence numbers at the end when we quantify the percentage of each category (n = 971).Functions of all 971 sequences were annotated using the functional categorizations of similar sequences stored in Gene Ontology (GO) database . GO termThe unique probe GO index [UPGI; Eq. (3) in results] was used to quantify functionally how representative the selected probes were within EST clusters.Two sequence indexes (CI and SDI) and one functional index (UPGI) mentioned above were included in the sequence diversity plot (SDP) Figure . SequencSDP: sequence diversity plot, SDI: sequence diversity index, CI: sequence contiguity index, and UPGI: unique probe GO index


Cigarette smoking takes a staggering toll on human health and attracts considerable public health attention, yet real solutions seem distant. The 2004 Family Smoking Prevention and Tobacco Control Act (US Senate bill S2461) would have given the US Food and Drug Administration limited authority to regulate cigarettes to "protect the public health." However, such legislation is unlikely to substantially reduce smoking or related deaths.The past 500 years of tobacco control efforts demonstrate that nicotine prohibition is a practical impossibility for numerous reasons, state revenue being one of the most ominous. The FDA already has regulatory authority over pharmaceutical grade nicotine products, and requires pharmacists to dispense the most addictive of these only with prescriptions. Meanwhile, every corner store can sell far more addictive and dangerous cigarettes to any adult. The FDA could immediately increase competition between cigarettes and clean nicotine products by approving available nicotine products for over-the-counter sales to adults. Similarly permissive regulation of cigarettes and addictive nicotine products will reduce tobacco use and improve smokers' health, but increase nicotine use in the population. Fortunately, restricted youth access and accurate labeling of nicotine's absolute risks will dissuade many non-smokers from experimenting with it, while accurate depiction of its risks relative to cigarette smoking will encourage many smokers to switch. The FDA could take a series of small steps that might ultimately replace a large proportion of cigarette smoking with equally addictive nicotine products, without risking serious public health setbacks. Vaccine, methadone, and injury prevention policies establish relevant public health precedents.Cigarettes, or an equally addictive alternative, will be a permanent and common product in most societies. Regulations restricting only the safest addictive nicotine products are hard to justify. Addictive nicotine compliments other tobacco control strategies. Modern tobacco control policies are applicable to addictive nicotine. Controlled trials and test market studies are urgently needed to evaluate addictive nicotine as an alternative to smoking. Meanwhile, legislators should preserve the Food and Drug Administration's option to permit non-prescription sales of addictive nicotine. Cigarette smoking is a source of worldwide misery ,2 and reThe recently defeated  2004 FamA complimentary strategy called "harm reduction" proposes that smokers could improve their health by frequently substituting less hazardous tobacco products, such as 'smokeless cigarettes,' chewing tobacco, and newer smokeless tobacco products for cigarettes -14. 'SmoSome tobacco control advocates suggest a third strategy: national policies encouraging competition between safer nicotine products and cigarettes ,23-34. TThere are different ways to level this playing field. At one extreme, the FDA could increase regulation of cigarettes to match that of nicotine products. This is politically challenging, as the FDA discovered in the 1990's , The FamAt another extreme, the FDA could regulate other nicotine products more permissively, as cigarettes have been regulated. The FDA has that authority, but would face criticism for facilitating a common addiction. For instance, the FDA could approve non-prescription nicotine nasal sprays and slow acting inhalers for indefinite use by adults. These nicotine replacement therapies have excellent safety records and low potential for addicting non-smokers, although a few ex-smokers become addicted. The widespread misconception that nicotine prohibition is practical and desirable makes it politically difficult for the FDA to pursue this path.A recent Lancet editorial called for the criminalization of tobacco products . HoweverThe first problem is that historically, smokers accept personal and public hazards that make the dangers we associate with tobacco look quaint. After Christopher Columbus failed to control his crew's tobacco use, monarchs from England to China tried to contain the weed by execution, disfigurement, exile, and onerous taxation . TobaccoThe second problem is that risk-tolerant smokers are no small group. Smoking persists where Mormon and Islamic prohibitions discourage it ,49. If 7The third problem is a common genetic predisposition to nicotine addiction . The modThe fourth problem is that so much money is involved. When state and local taxes raised New York City cigarette prices to US$7 per pack, tax revenues and black marketing both increased . During The fifth problem is that government taxation compromises tobacco control efforts. Historically, States willingly trade citizens' health for wealth. Seventeenth century monarchs who opposed smoking relented as tobacco tax revenue accumulated. The States demonstrated the same perverse values with petitions to protect their MSA payments when an Illinois court threatened Altria with a US$12 billion bond . The StaThe sixth problem is the lack of a popular mandate for prohibition. Tobacco control advocates in the United States are rightfully pleased with smoking's declining prevalence, reduced teen smoking, rising cigarette prices, spreading restrictions on public smoking, and fire-safe cigarette initiatives. However, none of this demonstrates popular support for prohibition. Citizens will balk at limiting the supposedly personal choice to smoke in private. Midwesterners have already rejected modest cigarette tax hikes . FurtherThe seventh problem is that nicotine use could have a favorable risk-benefit profile for some informed users. Nicotine causes a mild euphoria without intoxication, in contrast to more tightly regulated drugs: it does not destroy relationships as intoxicating drugs routinely do. Many smokers may use nicotine to treat various problems -60 incluNicotine accounts for very few of the long-term hazards of smoking. A smokeless tobacco proponent has likened nicotine's risks to the risks of consuming caffeine . Fetal eIn summary, however desirable tobacco prohibition may be, it is hopelessly impractical – unless smokers, governments, and producers have an equally satisfying alternative.Legal and regulatory pressures have prevented the development of meaningful alternatives to cigarettes. Directed to approve drugs as "safe and effective" for specific indications, and to ignore tobacco, the FDA has had little reason to approve chronic, addictive nicotine. Although the FDA approves more dangerous drugs for specified indications, without an indication there is no benefit to weigh against any nicotine risk. Consequently the FDA requires prescriptions for slow acting inhalers and nasal spray because of a small risk of inconsequential addiction in ex-smokers. Unfortunately, seemingly risk averse regulation of nicotine forces public health policies to rely on difficult smoking prevention, cessation, use restrictions, and treatment. The net effect is a risky public health policy, and very slow development of new nicotine delivery systems . AnotherTort threats have also delayed product improvement efforts within the industry. In 1963 an industry lawyer, anticipating condemnation in the 1964 Surgeon General's report, suggested competing on safe nicotine delivery, but was overruled . ProductIn the mid 1960's, motor vehicle accidents were also taking a terrific health toll in the United States. While alternatives to driving existed, no one expected private automobiles to disappear. Instead, systematic efforts transformed traffic safety. Haddon's matrix illustrated how complimentary strategies could work together to reduce the morbidity and mortality of driving . Table 1italicized) [For tobacco control, smoking replaces accidents as the focal event (Table licized) . As withlicized) ,97. but licized) . AvailabFor the reasons outlined above, the health benefits of addictive pharmaceutical grade nicotine products would likely outweigh the harms. If so, the FDA could improve public health by regulating nicotine much as the government has regulated cigarettes.A permissive nicotine regulatory policy would allow sales of pharmaceutical grade nicotine delivery systems to adults without a prescription. The FDA could suggest warnings appropriate for classes of products and delivery system constituents. Manufacturers would accurately label contents, and would be legally liable for undisclosed harms caused by the delivery system and constituents other than nicotine, as with any pharmaceutical product. The FDA could prohibit inherently risky delivery systems, and would undertake a full, traditional review of nicotine systems that give users a faster or higher peak arterial level of nicotine than cigarettes. The FDA would also review additives intended to provide antidepressant or other effects familiar to smokers ,100. YouStates could tax cigarettes and nicotine at different rates, in theory recovering expenses related to each product. This tax policy would discourage cigarette use, encourage switching to nicotine, and maintain some State revenue as cigarette sales decline. A satisfying, safe, legal, and affordable alternative to cigarettes would discourage black markets. The FDA and Federal Trade Commission could permit advertising of nicotine as an alternative to smoking, and monitor relative harm claims. Nicotine manufacturers would pay for monitoring of adverse health effects from their products until remaining health questions are answered.The FDA could implement a permissive policy in a series of simple, informative steps. First, the FDA could immediately approve non-prescription sales of the existing nicotine spray and inhaler, and similar competitors, with restraints consistent with the Synar amendment, the MSA, and the FCTC. In particular, it should require disclosure of known nicotine risks, such as addiction, invite comparisons to smoking risks, and forbid marketing to minors.Data collected during this first step will inform subsequent steps. Economists have shown that the price of nicotine gum and patches affects demand for cigarettes . This meIf experience with the current nicotine spray and inhaler is reassuring, the FDA can take a second step, to approve clearly addictive nicotine inhalers for non-prescription use. These fast acting inhalers will deliver nicotine in a powder  or aerosSeveral trends could develop that encourage smokers to switch to addictive nicotine inhalers, while limiting recruitment of non-smokers. Smokers will appreciate the healthier alternative to cigarettes, especially as they develop smoking related illnesses, while warning labels will discourage casual experimentation by non-smokers. Smoke-free workplace regulations would spread with less controversy, as fast acting inhalers give smokers a reasonable alternative. Employers might voluntarily forbid smoking to reduce health care and workman's compensation costs; reduce workplace fires; and increase productivity by eliminating both smoking breaks and withdrawal symptoms. Physicians might strongly encourage smokers to switch for their own health, primarily, but also for the health of family members. The government might permit more aggressive advertising of nicotine inhalers to further undermine cigarette smoking. Governments will probably find nicotine taxation irresistible, especially if their tobacco revenue declines. Nicotine taxation will raise the cost of experimenting with inhalers beyond some teenager's means. Communities might press smokers to switch so that fire departments, health care providers, and research funding agencies could shift resources to many other pressing problems. Ultimately, palatable inhaled nicotine products could finally allow governments to ban tobacco cigarettes.The public health benefits of these policy shifts could be substantial. We can estimate the long term relative public health burdens of different policies as the product of the risks posed by a delivery system, relative to cigarettes, and the fraction of the adult population using it. Using this formula, the current burden is more than 20% use × 100% risk = 0.2. Healthy People 2010 calls for 12% use × 100% risk = 0.12, about half of the current burden. If nicotine accounted for a surprising 10% of smoking risks, eliminating smoking by quintupling the prevalence of inhaled nicotine use – universal addiction – would cause 100% use × 10% risk = 0.1, a slight improvement over HP2010. A slightly less pessimistic scenario would be that all past and present smokers become inhaled nicotine addicts, but their risk is only twice that of smokeless tobacco users. This scenario is no more than 70% use × 4% risk = 0.028, about a quarter of the HP2010 burden. The most likely scenario is that nicotine risks are about 2% of smoking risks, and that policy changes will slowly shift the probabilities that smokers will attempt and succeed in quitting, that smokers will switch to nicotine indefinitely or use both nicotine and cigarettes, that ex-smokers relapse to nicotine use, and that non-smokers begin using nicotine. If the risks of inhaled nicotine are this low and if at least one smoker can switch to addictive inhaled nicotine for every 50 ex-smokers and non-smokers who start using nicotine, then public health will benefit from permissive nicotine regulation. One public health risk is that more people might begin or continue smoking cigarettes in the belief that inhaled nicotine will provide an easy escape path, but suffer irreparable harm before switching. We should quantify these probabilities through surveillance of the public's behavior and health consequences following each step toward more permissive nicotine regulation.Although S2461 was defeated, the continuing carnage will motivate similar legislative proposals. Tobacco policies must preserve the possibility of permissive nicotine regulation. Bad legislation could solidify the position of cigarettes, delay safer products, prohibit disclosure of relative harms, or otherwise interfere with market forces that ought to benefit nicotine addicts . The FDANicotine use will remain common indefinitely. Pharmaceutical grade nicotine is the safest known substance that could replace cigarettes, but inherently addictive products are required to compliment traditional tobacco control policies effectively. The FDA could apply restrictions, similar to those on cigarettes, to an increasingly addictive portfolio of nicotine products, requiring honest portrayals of absolute and relative risks, and expect a significant reduction in cigarette smoking and related illness with modest recruitment of new nicotine addicts at each step. A brief and focused research effort could solidify the already substantial justification for such permissive nicotine regulation. Meanwhile, federal legislation must not obstruct free market ideals of competitive innovation and informed consumption.The author(s) declares that he has no competing interests.The pre-publication history for this paper can be accessed here:


Surgeons seem to love publishing case series, which are of limited usefulness. How can we encourage them to do randomized clinical trials? Lancet, chastised much of current surgical research and, in particular, questioned the usefulness of the case series as a predominant form of communication among surgeons [In 1996, Richard Horton, editor of the Randomized controlled trials (RCTs) have become the pillar of clinical research. Such trials attempt to obtain an unbiased randomization of patients with respect to known and unknown baseline conditions and to assess the effects of an intervention. However, only a minority of surgical studies involve a valid randomization scheme. The case series remains a favored method of clinical investigation in surgery.Case series are easy to perform, require less resources in terms of personnel and funds, can be performed at a single center, and, for many surgeons, represent a means to illustrate their surgical method and skills. In many instances, case series also serve as valuable intellectual background for future clinical or scientific work. For example, consider Dennis Burkitt's report on jaw tumors in African children, Alfred Blalock's initial efforts in cardiac surgery, or, more recently, Starzl and colleagues' observations, in a small collection of patients, of donor leukocyte chimerism, whereby recipients acquire tolerance to foreign donor cells. In all three cases, the authors' work led to powerful shifts in our understanding of the biology and treatment of disease ,3,4. AllThere are many reasons why RCTs in surgical patients may be more difficult to perform than those in non-surgical patients. One of the most important—though least understood—is that the complexities of human disease in surgical patients makes them a more difficult group to study. Surgical patients are often heterogeneous in many more ways than non-surgical patients. So it would be inherently easier, for example, to study a new medication for generally healthy young adults with essential hypertension than a surgical technique for older patients with hepatic failure needing transplantation.In addition, while there may be value in studying patients from multiple centers, there may be important differences in the skill levels of different surgeons, either between centers or across the country. For example, the skill levels of surgeons in trials of carotid endarterectomy may be greater than those across the surgical community as a whole. This makes the applicability of some surgical RCTs to the wider community less certain than trials of medical therapies.So when it comes to surgical research, for both researchers and funding agencies, it is easier to grapple with a difficult, but ultimately soluble, basic science question than to face the uncertainty of clinical research. Investigators understand these implicit issues and trim their sails accordingly.Nonetheless, too much surgical work is conducted in the less rigorous format of the case series. What can and should be done to improve the rigor of surgical investigation? It would seem that improvements are required from within and beyond the surgical world.First, as Horner observed, and several eminent surgeons have since agreed, reforms must begin within the field itself ,5,6,7. BSecond, surgeons must establish firm and friendly relations with biostatisticians so that the latter may play a strong role in helping to develop adequately powered studies that can answer critical questions raised by new therapies and techniques. This is an especially acute need in an accelerating age of targeted therapies and disease biomarkers.Third, surgeons must re-engage in the clinical research enterprise and resume leadership roles in local and national clinical trials that involve surgical patients. In the United States, for example, an important step in this regard has been the establishment of the American College of Surgeons Oncology Group, which invites surgeons from all sectors, including private practice, to become active participants in well-designed, multi-institutional trials . SimilarFinally, similar to the pressures faced by their colleagues elsewhere in academia, surgeon clinician-investigators must be nurtured, protected, and valued by their colleagues and medical administrators. The financial health of academic medical centers relies heavily on the generation of clinical revenue, which in many centers falls disproportionately on the shoulders of surgeons. New paradigms for revenue generation and funding of clinical research are needed.Beyond the walls of the academic medical center, there also needs to be greater recognition of the value of scientifically sound surgical research and clinical investigation. However, the National Institutes of Health (NIH), the major source of biomedical funding in the United States, continues to convey a less welcoming attitude toward surgical research than toward other types of clinical or basic science,9.At the NIH, the principal instrument for performing peer review and making grant funding decisions is the study section, composed of about 10–20 members with expertise in a given field. There are few study sections devoted to surgically oriented clinical research and only two study sections (from among more than 100) in which surgeons make up even a reasonable minority of the committee members . In compFunding agencies need to recognize the importance of the surgical endeavor to modern medicine.Surgical research is also impeded by processes affecting other types of research as well. The number of researchers under 35 years of age receiving a first RO1 grant, the main NIH mechanism for external funding, in any field, is below 4%. The average age of initial funding for US physicians is about 44 years, and shows a trend toward advancing age that has progressed significantly in the past two decades. Thus, the NIH appears to reward experience and proven results very heavily, which may stifle innovation and likely serves as an innate barrier for younger physician-investigators contemplating research careers .To help correct for this worrisome trend, the NIH created the “K” award system—career development grants designed to help starting researchers gain the experience needed to compete for RO1 grants. However, nearly 40% of the clinicians who receive KO8 awards never apply for RO1 funding , which sFinally, outside the US, surgeons face similar, if not greater problems. This bodes poorly for countries where the cost of evaluating new therapies and technologies may be an unaffordable luxury. These challenges to the surgical research enterprise are therefore global issues and should merit the attention of surgeons, medical institutions, and funding agencies in all countries.http://nihroadmap.nih.gov) designed to provide “new pathways to discovery.” Clear, careful, scientific surgical investigation must be part of this roadmap, although it is not specifically mentioned. Outreach efforts to include surgeons in a variety of study sections should be made to ensure that important insights into the pathophysiology and treatment of disease, with which surgeons are concerned on a daily basis, are not overlooked. Additional efforts are needed to improve funding for clinical research, both for individuals at early stages of their careers and for multi-disciplinary clinical research and clinical trials. Locally, and individually, surgeons must join efforts to improve the clinical research enterprise by including training in clinical investigation at an early stage in medical school and during surgical residency training, fostering the careers of young surgeon-investigators through committed, protected time, participating in local and national clinical research groups, and recognizing that development as a clinical researcher takes time—many years in fact.What can be done? On the national and international level, funding agencies need to recognize the importance of the surgical endeavor to modern medicine. Recently, in the US the NIH unveiled a “roadmap” (These efforts may help ensure that surgical research is a vital part of the future of medicine and that it leads to the kind of high-quality work that shapes and remodels the face of medicine. To foster these efforts, surgeons must change and adapt to the currents of modern medical research. If this is successful, the case series will become the occasional rather than the common form of surgical communication. And surgeons, other clinicians, and, most importantly, basic scientists will be better able to take advantage of the new avenues of biomedical science opening before us.But the case series will always represent one important tool for early studies or uncommon conditions. It remains true that while the method one uses influences the answer one receives, it can be just as important to ask the right questions, which can be asked even in a series of one patient . And sur


The assumption that lifestyles formed early in life track into adulthood has been used to justify the targeting of health promotion programmes towards children and adolescents. The aim of the current study was to use data from the Northern Ireland Young Hearts Project to ascertain the extent of tracking, between adolescence and young adulthood, of physical activity, aerobic fitness, selected anthropometric variables, and diet.n 245) and females (n 231) were assessed at age 15 y, and again in young adulthood [mean (SD) age 22 (1.6) y]. At both timepoints, height, weight and skinfold thicknesses were measured, and physical activity and diet were assessed by questionnaire and diet history method respectively. At 15y, fitness was assessed using the 20 metre shuttle run, while at young adulthood, the PWC170 cycle ergometer test was used. For each measurement made at 15y, subjects were ranked into 'low' , 'medium'  or 'high'  categories. At young adulthood, similar categories (L2, M2, H2) were created. The extent of tracking of each variable over time was calculated using 3 × 3 matrices constructed using these two sets of categories, and summarised using kappa (κ) statistics.Males (Tracking of diet and fitness was poor (κ ≤ 0.20) in both sexes, indicating substantial drift of subjects between the low, medium and high categories over time. The tracking of physical activity in males was fair (κ 0.202), but was poor in females (κ 0.021). In contrast, anthropometric variables such as weight, body mass index and sum of skinfolds tracked more strongly in females (κ 0.540, κ 0.307, κ 0.357 respectively) than in males (κ 0.337, κ 0.199, κ 0.216 respectively).The poor tracking of fitness and diet in both sexes, and physical activity in females, suggests that these aspects of adolescent lifestyle are unlikely to be predictive of behaviours in young adulthood. In contrast, the fair to moderate tracking of anthropometric variables, particularly in females, suggests that attempts to reduce the ever increasing incidence of overweight and obesity in adults, should probably begin in earlier life. Numerous epidemiological studies in adults have identified environmental and physiological risk factors that are associated with increased risk for cardiovascular disease (CVD). Among the many that have been identified , the majAlthough the clinically relevant effects of CVD are often not manifest until middle age or later, it is now generally well accepted that the disease is likely to have its antecedents in childhood -4. In adHowever, if health promotion interventions in younger life are to have any hope of success, it must be assumed that physiological and behavioural risk factors exhibited in early life track into adulthood. Tracking has been defined as the maintenance of relative position in rank of behaviour over time, such that subjects who rank highly for unfavourable risk profiles at a young age are likely to maintain their ranks through into adulthood ,11.The aim of the current study was to use data from the Northern Ireland Young Hearts Project to ascertain the extent of tracking, between adolescence and young adulthood, of selected modifiable risk factors for CVD. Specifically, we investigated the tracking of physical activity, aerobic fitness, selected anthropometric variables, and diet.n 251;12-year old girls, n 258; 15-year old boys, n 252; and 15-year old girls, n 254) randomly selected from post-primary schools. At that time, the resulting cohort represented a 2% sample of each of the two age populations in Northern Ireland. In 1992/93, a follow up study (YH2) was undertaken, in which subjects from the original 12 year old cohort were reassessed using procedures identical to those used in YH1. The response rate in the follow-up study was 90%. Between October 1997 and October 1999, all YH1 subjects were invited to participate in the third, hospital-based screening phase (YH3), and a 48.2% response rate was achieved. Reasons for the low response rate in YH3 have been described in full elsewhere [The Young Hearts Project (YH) is an ongoing longitudinal study evaluating the prevalence of CVD risk factors in young people living in Northern Ireland. Sampling procedures and methods used in the first two, school-based screening phases of the YH study have been described fully elsewhere . Brieflylsewhere , attemptn 245) and females (n 231) for whom there were complete data sets at age 15y (either from YH1 or YH2) and at young adulthood [mean (SD) age 22.0 (1.6)y]. Ethical approval for each phase of the study was obtained from the Medical Research Ethical Committee of The Queen's University of Belfast, and written informed consent was obtained from all subjects prior to participation.The analyses reported in the current paper are restricted to males . Two measurements were taken at each site and the average was recorded. The sum of the four skinfolds thicknesses was then calculated for each subject.Each subject's height, weight and skinfold thicknesses were measured at all study timepoints. Standing height was measured to the nearest millimetre using a Harpenden portable stadiometer (Holtain, UK), and body weight was measured to the nearest 0.1 kg using an electronic balance . For both measurements, subjects wore light indoor clothing and no shoes. Body mass index (BMI) was then calculated as weight (kg)/ [height (m)]At all study timepoints, dietary data were obtained using the diet history method . This coAt age 15y, habitual physical activity was assessed by self-report questionnaire, and scored according to the method of . This meAs the school-based questionnaire was not relevant to the young adult subjects, a modification  of the B2max (ml/kg/min), the number of laps completed by each subject in this maximal endurance test was entered into a sex-specific regression equation, based on data obtained in the Northern Ireland Fitness Survey [Aerobic fitness at age 15y was assessed by the 20 metre shuttle test (20MST). In order to estimate maximal aerobic capacity, or VOs Survey .2max was assessed using the Physical Work Capacity at a heart rate of 170 beats per minute (PWC170) cycle ergometer test [2 in ml/kg/min), and this was used to estimate VO2max at the age-adjusted maximum heart rate [As it was not feasible to conduct the 20MST at young adulthood , VOter test . PWC170 art rate .All data were analysed using SPSS version 11.0.1 (SPSS Inc, Chicago, USA). Means and standard deviations were used to summarise the data for physical characteristics, aerobic fitness, physical activity scores and energy and macronutrient intakes of males and females at age 15y and young adulthood.Tracking of each of these variables over time was assessed by determining the extent to which subjects who were placed into low, medium and high categories at age 15y, maintained their ranking in young adulthood. Owing to the fact that different techniques were used to measure physical activity and aerobic fitness at each timepoint, a method based on ranks, rather than actual measurements, was employed for assessing the tracking of these variables. Tracking of the other variables was also assessed using the rank based method because of its relative simplicity, and its ability to show the numbers of subjects making the transition between low, medium and high categories . For exa2max assessed at young adulthood was significantly lower than at age 15y. At age 15y, males and females were 4.8% and 8.4% heavier than the British age-specific reference population, while at young adulthood, they were 7.9% and 9.9% heavier respectively. Details of the reference populations are described in Annex 1 of the 'Dietary Reference Values for Food Energy and Nutrients for the United Kingdom' [The physical characteristics, aerobic fitness and physical activity levels of the Northern Ireland Young Hearts cohort at age 15y and at follow-up (young adulthood) are summarised in Table Kingdom' .P 0.04), total fat  and total carbohydrate  than at age 15y. In contrast, intakes of protein  reported by males at young adulthood were significantly greater than at age 15y. Similar patterns were observed for females, with the exception that % energy derived from total carbohydrate did not change significantly between age 15y and young adulthood.The energy and macronutrient intakes reported by the Young Hearts cohort at age 15y, and at follow-up, are presented in Table 2max) was poor, but was greater than the κ value obtained for the females (κ 0.150 vs κ 0.076). A similar pattern was observed for physical activity scores (κ 0.202 vs κ 0.021).Table The extent of tracking of energy and macronutrient intakes reported at age 15y and at young adulthood is presented in Table The current paper describes the extent of tracking for a range of behavioural and biological risk factors for CVD, between adolescence (15y) and young adulthood (22y) in 245 males and 231 females from Northern Ireland.In relation to nutrient intakes, the poor tracking between 15 and 22 years revealed in this study reflects previous findings in this cohort between 12 and 15 years . This suWhile adolescence is widely regarded to be a time of transition, it could also be argued that young adulthood is an equally important time of change, especially with regard to dietary habits. This is a time when people in this age group are likely to move out of home, go to university, start a family or to encounter other environmental or psychosocial factors that influence food intakes. Thus it is possible that the poor maintenance of ranks that we have observed in this study has arisen simply because we attempted to assess tracking between two very unstable periods of time in the life-cycle.It is also possible that at least part of the explanation for poor dietary tracking in a cohort of this nature lies in the unsuitability of the diet history method for this purpose. Although the diet history method has shown good validity at the group level in adolescents, it is prone to significant problems of precision at the individual level . MoreoveBoth physical fitness and physical activity are now accepted as independent risk factors for several chronic diseases. The identification of low levels of fitness and/or activity at an early stage in the lifecourse might, therefore, enable early remedial strategies, provided that a degree of tracking for these risk factors is demonstrable. The results of the present study fail to provide such evidence, with poor tracking being demonstrated for fitness in both sexes, and poor tracking in females and only moderate levels of tracking in males for activity. By and large, these results are in keeping with the handful of other studies which have examined tracking of fitness and activity in this age group ,27. WhilIn contrast to diet and physical activity/fitness, anthropometric variables relating to body weight and adiposity showed stronger and consistent tracking, particularly in females. Good tracking of BMI from adolescence to young adulthood has been noted in previous studies ,24,33, wThe authors declare that they have no competing interests.CB conceived of the study, participated in its design and co-ordination and co-drafted the manuscript. PR supervised the collection of dietary data and co-drafted the manuscript. AG and GC carried out the statistical analysis. LM participated in the design and co-ordination of the project. JS conceived of the study and participated in its design and co-ordination. All authors read and approved the final manuscript.


In view of the disturbed esophageal peristaltic activity and abnormal esophageal motility in gastroesophageal reflux disease, (GERD), we investigated the hypothesis that these changes result from a disordered myoelectric activity of the esophagus.The electric activity of the esophagus (electroesophagogram, EEG) was studied in 27 patients with GERD (16 men, 11 women, mean age 42.6 ± 5.2 years) and 10 healthy volunteers as controls (6 men, 4 women, mean age 41.4 ± 4.9 years). According to the Feussner scoring system, 7 patients had a mild (score 1), 10 a moderate (score 2) and 10 a severe (score 3) stage of the disease. One electrode was applied to the upper third and a second to the lower third of the esophagus, and the electric activity was recorded. The test was repeated after the upper electrode had been moved to the mid-esophagus.The EEG of the healthy volunteers showed slow waves and exhibited the same frequency, amplitude and conduction velocity from the 2 electrodes of the individual subject, regardless of their location in the upper, middle or lower esophagus. Action potentials occurred randomly. In GERD patients, score 1 exhibited electric waves' variables similar to those of the healthy volunteers. In score 2, the waves recorded irregular rhythm and lower variables than the controls. Score 3 showed a "silent" EEG without waves.The electric activity in GERD exhibited 3 different patterns depending on the stages of GERD. Score 1 exhibited a normal EEG which apparently denotes normal esophageal motility. Score 2 recorded irregular electric waves variables which are presumably indicative of decreased esophageal motility and reflux clearance. In score 3, a "silent" EEG was recorded with probably no acid clearance. It is postulated that the interstitial cells of Cajal which are the electric activity generators, are involved in the inflammatory process of GERD. Destruction of these cells appears to occur in grades that are in accordance with GERD scores. The EEG seems to have the potential to act as an investigative tool in the diagnosis of GERD stages. Reflux esophagitis is a multifactorial disease that entails the reflux of gastric contents into the esophageal lumen . EsophagAbnormal esophageal motility results in an increased exposure of the esophageal mucosa to gastric contents. Progressing severity of the reflux disease is associated with failing primary peristalsis -9. The fPrevious studies have shown that the esophagus possesses electric activity presenting as slow waves (SWs) followed or superimposed by fast activity spikes or action potentials (APs) ,13. ActiIn view of the disturbed esophageal peristaltic activity and abnormal esophageal motility in GERD, we hypothesized that these findings result from a disordered myoelectric activity of the esophagus. This hypothesis was investigated in the current study.Twenty seven patients with GERD (16 men, 11 women, mean age 42.6 ± 5.2 SD years, range 36–48) were enrolled in the study. The diagnosis was confirmed by 24-hour pH monitoring, endoscopy and esophageal motility test. According to Feussner's scoring system , seven pThe study also included 10 healthy volunteers (6 men, 4 women, mean age 41.4 ± 4.9 SD years, range 35–50) who had no reflux esophagitis. They had no gastrointestinal complaints in the past or at the time of enrollment in the study.Physical examination of both the patients and healthy volunteers had normal findings. The results of laboratory work comprising blood count, renal and hepatic function tests as well as electrocardiogram were unremarkable. The studied subjects gave an informed consent after having been fully informed about the nature of the study, the tests to be done and their role in the study. The study was approved by the Review Board and Ethics Committee of the Cairo University Faculty of Medicine.The electric activity of the esophagus was recorded in the patients with GERD and the healthy volunteers. We used a monopolar silver-silver chloride electrodes of 0.8 mm diameter (Smith-Kline Beecham, Los Angeles, CA, USA) introduced through a 6F catheter (Rubber Industries Ltd., London, UK) with the electrode protruding by 1 cm from the catheter tip. The catheter was attached to the esophageal mucosa by negative pressure suction of 50 to 100 mmHg which was maintained during the test.Two electrodes were introduced into the esophagus by means of an endoscope and fixed to the esophageal mucosa by suction; one electrode was applied to the upper third, and the second to the lower third of the esophagus, and the electric activity was recorded. The upper electrode was then transferred to the middle third of the esophagus and recordings of the electric activity from the middle and lower third electrodes were performed. Signals from the electrodes were fed into an AC amplifier with a frequency response within ± 3 dB from 0.016 Hz to 1 kHz; they were displayed on a recorder at a sensitivity of 1 mV/cm. A metal disc applied to the abdominal skin served as the indifferent electrode. A strain gauge respiration transducer was attached to the thoracic wall for respiratory artefacts. We allowed the esophagus a 30 minute period to adapt to the electrodes applied to its wall, before we started a 120-min recording session for each subject.The results were analyzed statistically using the Student's t test. Differences assumed significance at p < 0.05 and values were given as the mean ± standard deviation (SD).No adverse side effects were encountered during or after the performance of the tests and all the subjects were evaluated. There was no difficulty in applying the electrodes to the esophageal mucosa. We found that a suction pressure of 50–60 mmHg was sufficient to keep the electrode fixed to the esophageal mucosa during the testing period in most of the subjects; in few cases we had to increase the suction pressure to 100 mm to keep the electrodes in position. Applying the aforementioned pressures, we encountered neither migration nor detachment of the electrodes during the entire test. We met no mucosal bleeding, tears or ulcers during application or after removal of the electrodes either.Monophasic negatively deflected SWs were recorded from the 2 electrodes of each subject of all the studied individuals fig. . They haIn score 1 GERD, the electric waves' variables were similar to those of the healthy volunteers in all the subjects (p > 0.05, fig Score 2 GERD patients exhibited a different electromyographic pattern. The SWs had an irregular rhythm with varying but lower frequency, amplitude and conduction velocity compared to the normal controls fig. . The APsThe current study has demonstrated that the esophagus possesses an electric activity in the form of regular SWs and APs. The waves in the healthy volunteers were reproducible with identical frequency, amplitude and conduction velocity in the same subject. Previous studies have shown that the APs were coupled with increased esophageal pressure, while the SWs were not ,13, thesEffective esophageal motility is a critical determinant for esophageal clearance of refluxed gastric contents . A singlThe relationship between esophageal peristalsis and gastroesophageal reflux has been studied using stationary and ambulatory prolonged esophageal manometry. Various motor-disorders have been detected in GERD including a significantly high rate of incomplete primary peristalsis, changes in esophageal motility and an increased number of non-transmitted contractions ,19. ContPatients with abnormal GER but mild esophagitis, or none, had normal amplitude of contractions with increased prevalence of simultaneous contractions ,21. MeanThe electric activity in GERD exhibited different patterns depending on the stage of the GERD. In score 1 GERD, an electroesophagram similar to that of healthy volunteers was recorded. This apparently denotes that the motile activity of the esophagus in score 1 is normal and that the esophageal peristaltic activity can probably clear the esophagus of the refluxed gastric contents. The irregular and diminished esophageal electric wave variables displayed in score 2 GERD are presumably indicative of diminished motile activity and peristalsis of the esophagus with a resulting inhibited reflux clearance rate. The failure of adequate esophageal clearance is probably responsible for the clinical manifestations and investigative results encountered in score 2 GERD. With progress of the condition to score 3, there is probably no motor or peristaltic esophageal activity as evidenced by the absence of the esophageal electric waves. In such case, we presume that there is no esophageal clearance.We do not know the cause of the diminished esophageal electric activity in GERD. Is it due to the refluxed acid material or to the resulting esophageal inflammation? It may be argued that the refluxed acid content into the esophagus inhibits its motile and peristaltic activity. However, the current and earlier studies have demonstrated normal peristalsis in score 1 GERD in which acid reflux was manifest . FurtherWhat then could be the cause of the deranged electric activity and peristaltic movement in the more advanced stages of GERD?The electric waves seem to be generated from the interstitial cells of Cajal that are located at the level of the myenteric plexus and in the circular muscle layer of the esophageal wall -24. TheyIn the advanced stages of GERD it may be assumed that the inflammatory changes in the esophageal wall have involved the interstitial cells of Cajal. Destruction of these cells appears to occur in grades that are in accordance with the GERD socres. It seems that in score 1 GERD, the mucosal inflammatory changes of the esophagus, if present, have not involved the Cajal cells yet. With the more advanced stages of the disease as in scores 2 and 3, the Cajal cells are presumingly being gradually destroyed by the advancing esophageal inflammatory process. The diminished SW variables encountered in score 2 GERD seem to be due to partial involvement of Cajal cells in the inflammatory process; the cells are not yet completely destroyed and are still mediating electric and peristaltic activity. However the Cajal cells in score 3 GERD are believed to be extremely injured so that they cannot generate electric activity.There are various methods for the diagnosis of GERD. They include pressure measurements using water-perfused manometry catheters, external transducers or intraluminal transducers, pH-metry and others ,29. HoweThe electric activity in GERD expressed 3 different patterns depending on the stage of GERD. In score 1 GERD, a normal electroesophagogram was recorded which would denote normal esophageal motile activity and acid clearance. Score 2 GERD exhibited irregular and diminished esophageal electric waves' variables which are presumably indicative of decreased esophageal motility and reflux clearance. In score 3, a "silent" electroesophagogram was recorded with probably no acid clearance. A new theory of the pathogenesis of diminished electric activity in GERD is put forward. It is postulated that the interstitial cells of Cajal which are considered as the generators of the pacemaker activity and electric waves, are involved in the inflammatory process of GERD. Destruction of these cells appears to occur in grades that are in accordance with GERD scores. The electroesophagogram may serve as an investigative tool in diagnosing the various GERD stages, especially if they can be recorded percutaneously.gastroesophageal reflux disease = GERD(electroesophagogram = EEGslow waves = SWsaction potentials = APsstandard deviation = SDAS: Study design/ planningOES: Data collection/entry, Data analysis/statistics, Literature analysis/searchIS: Data collection/entry, Data analysis/statistics, Literature analysis/searchAS: Data collection/entry, Preparation manuscriptThe pre-publication history for this paper can be accessed here: