Daphnia pulex - the first completely sequenced crustacean genome. This is of particular interest because the pancrustaceans (hexapods+crustaceans) have more optical designs than any other major clade of animals, allowing us to test specifically whether the high amount of disparity in pancrustacean eyes is correlated with a higher rate of duplication and retention of vision genes.Duplication and divergence of genes and genetic networks is hypothesized to be a major driver of the evolution of complexity and novel features. Here, we examine the history of genes and genetic networks in the context of eye evolution by using new approaches to understand patterns of gene duplication during the evolution of metazoan genomes. We hypothesize that 1) genes involved in eye development and phototransduction have duplicated and are retained at higher rates in animal clades that possess more distinct types of optical design; and 2) genes with functional relationships were duplicated and lost together, thereby preserving genetic networks. To test these hypotheses, we examine the rates and patterns of gene duplication and loss evident in 19 metazoan genomes, including that of Using protein predictions from 19 metazoan whole-genome projects, we found all members of 23 gene families known to be involved in eye development or phototransduction and deduced their phylogenetic relationships. This allowed us to estimate the number and timing of gene duplication and loss events in these gene families during animal evolution. When comparing duplication/retention rates of these genes, we found that the rate was significantly higher in pancrustaceans than in either vertebrates or non-pancrustacean protostomes. Comparing patterns of co-duplication across Metazoa showed that while these eye-genes co-duplicate at a significantly higher rate than those within a randomly shuffled matrix, many genes with known functional relationships in model organisms did not co-duplicate more often than expected by chance.Overall, and when accounting for factors such as differential rates of whole-genome duplication in different groups, our results are broadly consistent with the hypothesis that genes involved in eye development and phototransduction duplicate at a higher rate in Pancrustacea, the group with the greatest variety of optical designs. The result that these genes have a significantly high number of co-duplications and co-losses could be influenced by shared functions or other unstudied factors such as synteny. Since we did not observe co-duplication/co-loss of genes for all known functional modules (e.g. specific regulatory networks), the interactions among suites of known co-functioning genes (modules) may be plastic at the temporal scale of analysis performed here. Other factors in addition to gene duplication - such as cis-regulation, heterotopy, and co-option - are also likely to be strong factors in the diversification of eye types. Genomic complexity is driven, to a large extent, by gene duplication, retention, and divergence ,2. This We define Pancrustacea as disparate in eye morphology because the group has the highest number of distinct optical designs of any animal group. At the broadest level, there are eight recognized optical designs for eyes in all Metazoa . Four ofDrosophila and other insects. For instance, there are several genes commonly expressed in the Drosophila compound eye, stemmata and Bolwig's organ patterning  that ar. -25. Droson , and thC. elegans does not possess conventional eyes, even though many other non-arthropod protostomes do. If, as a result of losing eyes during evolution, the lineage of C. elegans has a lower rate of eye-gene duplication, this could result in an underestimate of eye-gene duplication rate for the entire clade. Similarly, the pancrustaceans used here could have more eye-genes than other arthropods. In fact, Daphnia pulex does have a large number of genes compared to other arthropods, perhaps because of its asexual/sexual life history . These hypotheses could be examined using the approaches developed here, once additional genome sequences become available.Comparisons between eye-gene duplication rate in pancrustaceans and non-arthropod protostomes clearly supported our hypothesis, even when taking the conservative approach of not counting arthropod-specific genes. The observed difference in gene duplication rate between these two clades does not depend on the denominator used in rate calculations, and is significantly different for both developmental and phototransduction genes Tables , 4. DespCompared to rate differences between pancrustaceans and non-arthropod protostomes, rate differences between pancrustaceans and vertebrates were more variable. That is, using different denominators in our rate calculations led to different results . An important consideration in these comparisons is that vertebrates are known to have undergone multiple whole-genome duplications, which raised the overall estimated rate of gene duplication and accumulation for the group. This is evident in total gene duplications that we counted (80673 in vertebrates vs. 33113 in pancrustaceans) but is not reflected in our other distance measures (denominators): both clades show similar genetic distance (as measured by average ortholog distance - 1047 and 814 respectively) as well as similar clade ages (as estimated by a molecular clock - 470 and 450 mya). The high overall rate of gene duplication and accumulation in vertebrates may therefore explain why, counter to our hypothesis, vertebrates show a significantly higher rate of eye development gene duplication than pancrustaceans.D. pulex lineage , a phenomenon also known in other crustaceans . Phototstaceans ]. The mostaceans . If so, The consideration of pleiotropy also highlights another avenue for future research. If pleiotropy does result in a weaker correlation between eye disparity and gene duplication rate, gene choice must influence the final results. Therefore, future research might focus on a broader sampling of genes, especially to the extent that analyses conducted here could be fully automated to allow the analysis of very large datasets. For example, a recent study focusing on the insects found higher numbers of gene duplications in dipterans than other insects by examining 91 fly eye-genes . IntegraDaphnia pulex) within Arthropoda remains somewhat uncertain . Searches first retrieved the top 15 hits, this number was raised in subsequent searches until D. pulex models outside the group of interest were obtained. Redundant sequences were determined by examining the visual scaffold model on JGI and then removed by hand. The gene family for each D. pulex gene was assigned by inclusion in a maximum likelihood tree using UniRef50 and UniRef90 sequences. These trees were estimated using an in-house pipeline of shell and perl scripts that merge existing bioinformatic tools. The bait sequence from FlyBase was used to perform a similarity search using blastp [http://www.uniprot.org/. In most cases, we used two blast search strategies for each bait gene: 25/10 . In cases when there was either not enough resolution or no outgroup hits obtained; more hits were taken from the Uniref90 or Uniref50 databases, respectively  test [With a protein sequence for each gene of interest from FlyBase used as a "bait" sequence, g blastp  of non-rSH) test  implemenSH) test ,72 by coDaphnia pulex (confirming the automated genome assemblies with cDNA sequencing) and the ostracod crustacean Euphilomedes carcharodonta. Euphilomedes carcharodonta were collected at the University of Southern California's Wrigley Marine Lab on Catalina Island, California by free diving, collecting sediment with an aquarium net, and sorting with a dissecting microscope. Daphnia pulex were obtained from stock collections at Indiana University. We first isolated Pax-6 fragments using degenerate PCR primers to highly conserved regions in the paired and homeo domains of published Pax-6 sequences. After sequencing an initial Pax-6 fragment, we designed specific primers for 5' and 3' RACE, often using nested primers and the Gene Racer kit (Invitrogen). Primers and cycling conditions are given in Additional File In phylogenetic analyses of Pax-6, we utilized previously unpublished sequence data from blastall searches were executed against 19 genomes obtained from JGI and NCBI (Table blastall searches were modified and repeated to allow more returns in cases where all five best hits fell within the in-group for the gene of interest in initial phyogenetic analyses. Sequence alignment and maximum likelihood estimates of gene trees were performed as described above. Genes were identified phylogenetically as monophyletic ingroups sharing conserved domain architecture using Pfam databases and NCBI's Conserved Domain Database [With protein sequence for each gene of interest from FlyBase, initial Database ,78 (See Database  using NODatabase . RearranCaenorhabditis elegans-specific duplications were added to Lophotrochozoa-specific duplications to get a total duplications value for "non-arthropod protostomes." Ciona intestinalis, Branchiostoma floridae, and Nematostella vectensis were not included in this analysis. To estimate total numbers of gene duplications and losses during animal evolution, we used EvolMAP software [We estimated rates of gene duplication while normalizing for overall evolutionary time using three different denominators. Total numbers of duplications of genes along each branch of a species tree were estimated using data from the reconciled tree analysis Figures , 2. For software , which esoftware  for detasoftware ,43.To examine co-duplication, two methods were used. First, data matrices were created representing the number of losses or duplications for each gene along each branch of a species tree using the data from reconciled tree analysis Figures , 2. GeneAuthor contributions: ASR and THO designed research; ASR, MSP, DCP, CV, AES, JMS, ARO, and THO performed research; ARO contributed new reagents/analytic tools; ASR, MSP, DCP, CV, AES, JMS, ARO, and THO analyzed data; and ASR, MSP, DCP, JMS, and THO wrote the paper. All authors read and approved the final manuscript.Results of phylogenetic analyses on the 22 individual gene families used in this study.Click here for filePairwise correlation values of between duplication and loss of the 22 gene families examined.Click here for fileEuphilomedes carcharodonta and Daphnia pulex.PCR conditions for the amplification of Pax6 homologs from Click here for file


Gastroesophageal reflux disease (GERD) may cause, trigger or exacerbate many pulmonary diseases. The physiological link between GERD and pulmonary disease has been extensively studied in chronic cough and asthma. A primary care physician often encounters patients with extra esophageal manifestations of GERD in the absence of heartburn. Patients may present with symptoms involving the pulmonary system; noncardiac chest pain; and ear, nose and throat disorders. Local irritation in the esophagus can cause symptoms that vary from indigestion, like chest discomfort and abdominal pain, to coughing and wheezing. If the gastric acid reaches the back of the throat, it may cause a bitter taste in the mouth and/or aspiration of the gastric acid into the lungs. The acid can cause throat irritation, postnasal drip and hoarseness, as well as recurrent cough, chest congestion and lung inflammation leading to asthma and/or bronchitis/ pneumonia. This clinical review examines the potential pathophysiological mechanisms of pulmonary manifestations of GERD. It also reviews relevant clinical information concerning GERD-related chronic cough and asthma. Finally, a potential management strategy for GERD in pulmonary patients is discussed. Gastroesophageal reflux disease (GERD) is a condition in which the esophagus becomes irritated or inflamed because of acid backing up from the stomach. The inner lining of the stomach resists corrosion by this acid. The cells that line the stomach secrete large amounts of protective mucus. The lining of the esophagus does not share these resistant features, and gastric acid can damage it. Normally, the lower esophageal sphincter prevents reflux of acid. With GERD, however, the sphincter relaxes between swallows, allowing stomach contents and corrosive acid to regurgitate up and damage the mucosa of the esophagus. GERD affects nearly one third of the adult population to some degree, at least once a month. Almost 10% of adults experience GERD weekly or daily. Not just adults, even infants and children can have GERD.  demonstrNo one knows the exact cause of gastro esophageal reflux. The following are several contributing factors that weaken or relax the lower esophageal sphincter, making reflux worse:Lifestyle — Use of alcohol or cigarettes, obesity, poor posture (slouching).Medications — calcium channel blockers, theophyllines, nitrates, antihistamines.Diet — Fatty and fried foods, chocolate, garlic and onions, drinks with caffeine, acid foods such as citrus fruits and tomatoes, spicy foods, mint flavorings.Eating habits — Eating large meals, eating soon before bedtime.Other medical conditions — Hiatus hernia, pregnancy, diabetes, rapid weight gain.Historically, pulmonary manifestations have been recognized as a potential consequence of GERD. A major advance in the understanding of extra-esophageal manifestations comes from the recognition that a significant number of patients with asthma or chronic cough, particularly if it is nocturnal, have gastroesophageal reflux as a trigger. Extra-esGERD can cause various pulmonary manifestations: Chronic cough, bronchial asthma, bronchitis, pneumonia and interstitial fibrosis . Out of et al.[According to the studies in the literature, pathological GERD can be found in 30% to 80% of patients with asthma. On the other hand, patients with esophagitis are more likely to have asthma than patients without esophagitis. In the ProGERD study, the occuet al. showed tet al.,[et al.[The prevalence of GERD-associated cough ranges from 10% to 40%, depending on the patient population, type of diagnostic test used and whether more than one etiology of cough is ascertained. An epidemiological association between GERD and chronic cough has been reported in patients of all age groups. Patientet al., GERD waset al., With the.,[et al. found thGERD can be a compounding factor in the control of asthma. Not only is the asthmatic patient more likely to have GERD as compared to the general population, but also GERD is recognized as a potential trigger in many cases of severe asthma. BronchiaThere are 2 different mechanisms by which GERD can cause chronic cough: (i) acid in the distal esophagus stimulating a vagally mediated esophageal tracheobronchial cough reflex and (ii) micro- or macro-aspiration of esophageal contents into the larynx and tracheobronchial tree. One study proposed that gastro esophageal reflux causes esophago-tracheo-bronchial reflex. Using dual-probe 24-hour esophageal pH-monitoring, it has been observed that reflux occurred simultaneously in 78% and within 5 minutes in 90% of cough episodes. It is prMany patients with asthma report GERD symptoms, including heartburn, regurgitation and dysphagia. Some patients may have clinically silent GERD, especially in the context of difficult-to-treat asthma. A high dThe most common causes of chronic cough are postnasal drip, asthma and GERD. One should be able to predict cough due to GERD in the following categories of patients: Those not taking ACE inhibitor, nonsmokers, those with normal chest x-ray, those with negative broncho-provocative test for asthma, and those with persistent cough despite effective treatment for postnasal drip. A study The patient's history is an extremely important part of the diagnosis of GERD-associated asthma. The diagnosis is important to consider, however, because significant improvement in symptoms and in asthma control occurs with appropriately treated GERD. Certain Esophageal tests that may be helpful in diagnosis include the barium esophagogram, gastroesophageal scintigraphy, and prolonged esophageal pH–monitoring. Esophageal pH- monitoring is considered the gold standard for the diagnosis of GERD and is the only esophageal test that can directly correlate acid reflux episodes with wheezing or other symptoms of bronchospasm. Esophageal pH–monitoring in patients with suspected GERD-related manifestations may represent a more accurate approach. However,Treatment for GERD is aimed at reducing the abnormal backflow, or reflux of acid, into the esophagus; preventing injury to the esophagus or helping it to heal if injury has already occurred; preventing GERD from recurring; and preventing complications of GERD.Treatment for people who have symptoms of GERD begins with making lifestyle modifications. Sleeping with an elevated head in patients with documented nighttime reflux episodes, smoking cessation, weight reduction and low-fat diet (< 45g/d) have all been found to be useful measures. Avoidance of food and beverages with a pH of < 5 and/or capability of relaxing the lower esophageal sphincter — such as alcohol, chocolate, mint, onions, tea, cola, citrus fruits — is also highly recommended; and patients should avoid food and beverages 2 to 3 hours before going to the bed. Medicatiet al.[Proton pump inhibitors are the most effective acid-suppression medications available and are the cornerstone of therapy for GERD and other acid-mediated conditions. Once-a-day PPI therapy improves chronic cough in patients with GERD, and the effect of PPI in ameliorating both cough and reflux symptoms continues after treatment ceases. There is evidence that 2 months with PPI is sufficient to reduce cough in patients with GERD. found thet al. concludeet al.31 Based et al. The empiet al. A recentet al.et al.[With regard to medical therapy of GERD and asthma, studies using PPIs have had more encouraging results than those using antacids or H2 receptor antagonist. The latter have yielded inconsistent results on asthma symptoms and peak expiratory flow rates (PEFRs). Numerous clinical trials–41 have et al. performeet al. many flaet al. using PPIn a cohort of difficult-to-control asthmatics, PPI and prokinetic therapy for 7 weeks resulted in complete improvement of symptoms in 86%, moderate improvement in 11% and lack of improvement in only 4% of the patients. Symptomset al.,[Unlike asthma and cough, in which the esophago-bronchial reflex may play an important role, direct aspiration of gastric contents into the lung is thought to be the major patho-physiological mechanism in other respiratory disorders.  Epidemioet al., but suchet al., However,et al., Recentlyet al., showed tet al., Recentlyet al., In contret al., The roleet al.,Fundoplication surgery is the most common surgery used to treat GERD. It may be used to treat GERD symptoms that have not been well controlled by medications. In fundoplication surgery, the fundus of the stomach is wrapped around the esophagus and sewn into place to strengthen the lower esophageal sphincter. Some relatively new nonsurgical procedures used to treat GERD are still undergoing trials to determine their long-term safety and effectiveness. These include Stretta radiofrequency procedure, in which radiofrequency energy is delivered through an endoscope to tighten the lower esophageal sphincter; and EndoCinch procedure, in which an endoscopic sewing device is used to make a series of sutures that adjusts the lower esophageal sphincter so that it blocks acid reflux more effectively.Many uncontrolled studies have been carried out to investigate the effect of anti-reflux surgery on asthma outcome. The results of these studies suggest that surgery could improve asthma symptoms and reduce medication use in 80% to 90% of the asthmatics; and pulmonary function, in approximately 25%. The only


Ghrelin is a powerful orexigenic gut hormone with growth hormone releasing activity. It plays a pivotal role for long-term energy balance and short-term food intake. It is also recognized as a potent signal for meal initiation. Ghrelin levels rise sharply before feeding onset, and are strongly suppressed by food ingestion. Postprandial ghrelin response is totally macronutrient specific in normal weight subjects, but is rather independent of macronutrient composition in obese. In rodents and lean individuals, isoenergetic meals of different macronutrient content suppress ghrelin to a variable extent. Carbohydrate appears to be the most effective macronutrient for ghrelin suppression, because of its rapid absorption and insulin-secreting effect. Protein induces prolonged ghrelin suppression and is considered to be the most satiating macronutrient. Fat, on the other hand, exhibits rather weak and insufficient ghrelin-suppressing capacity. The principal mediators involved in meal-induced ghrelin regulation are glucose, insulin, gastrointestinal hormones released in the postabsorptive phase, vagal activity, gastric emptying rate, and postprandial alterations in intestinal osmolarity. Ghrelin is a 28-amino-acid gastrointestinal peptide with appetite-stimulating, growth hormone-releasing and adipogenic properties –3. It waOne of the most important actions of ghrelin is its regulatory role for long-term energy homeostasis and short-term food intake . There iThe postmeal inhibition of gastric ghrelin production is proportional to energy load and is profoundly influenced by the meal's macronutrient content , 14. In Despite the well-established stimulatory effect of ghrelin on appetite and eating behavior, little information is available regarding its relationship with fasting and postprandial energy expenditure in normal weight and obese humans. In rodents, ghrelin infusion promotes weight gain, both by increasing food intake and by decreasing energy expenditure and fat catabolism . This efThe present review aims to shed light on the underlying mechanisms of postprandial ghrelin regulation. In addition, a significant body of clinical and experimental data will be discussed, elucidating the macronutrient-specific effect of several isocaloric test meals on postprandial ghrelin levels. We searched PubMed and other electronic databases for high quality articles written in English, using the following search terms: ghrelin, macronutrients, carbohydrate, protein, fat and postprandial response. A recent study in humans demonstrated that ghrelin levels can be suppressed by sham feeding , as well as by actual feeding, indicating the importance of cephalic response to nutrient intake and supporting the role of vagal activity for the control of postprandial ghrelin secretion . The vagInsulin and glucose are thought to be dynamic modulators of plasma ghrelin concentrations in rodents and humans –25. HypeThe effect of acute hyperinsulinemia on plasma ghrelin concentrations is still a matter of debate. Clinical data regarding the existing interrelation between ghrelin and insulin are rather conflicting. According to the study of Flanagan et al., using a stepped hyperinsulinemic eu-, hypo- and hyperglycemic glucose clamp, insulin may suppress circulating ghrelin independently of glucose, although glucose might have an additional synergistic effect . In the The exact mechanism of insulin affecting plasma ghrelin concentrations remains to be established. Insulin might inhibit ghrelin synthesis or secretion, either directly or indirectly. Studies in rats have shown that insulin-induced hypoglycemia increases, instead of decreasing, ghrelin mRNA levels in the gastric fundus, providing no evidence for a direct inhibitory action of insulin on ghrelin synthesis . HoweverContrary to the findings mentioned above, Caixás et al. reported that, unlike food intake, the subcutaneous administration of a short-acting insulin analog was not able to suppress ghrelin levels . In concTaking everything into consideration, glucose and insulin are unlikely to explain the entire postprandial ghrelin response, since ghrelin remains suppressed long after the normalization of glucose and insulin levels, and furthermore, because lipids tend to suppress ghrelin in the absence of substantial increases in glucose or insulin . Other hIt would be also interesting to examine the role of leptin in postprandial ghrelin regulation, since ghrelin and leptin are part of a dynamic peripheral feedback system that regulates body weight and energy homeostasis by modulating satiety. Data concerning the relationship between ghrelin and leptin at fasting and postprandial state are rather contradictory. In vitro studies have previously shown that leptin inhibits ghrelin production from the gastric mucosa, and leptin levels in humans appear to be inversely related to ghrelin concentrations . On the In an interesting study by Foster-Schubert et al., three different macronutrient preloads of equal caloric content, volume and energy density  were compared for their relative efficacy to suppress total and acylated (bioactive) ghrelin levels . Total gThe faster gastric emptying after CHO ingestion, compared with lipids or proteins, can partly explain the strong and rapid postprandial ghrelin suppression in the first phase . The rapNot all types of dietary CHO are likely to have the same effect on postprandial ghrelin levels. In a study comparing high-glucose with high-fructose meals, the mean postprandial suppression of ghrelin was markedly attenuated and significantly less pronounced after consuming the high-fructose meals . FructosCHO-enriched test meals, containing both simple and complex carbohydrates, have been used in various clinical studies investigating the differential response of postprandial ghrelin to meals of different macronutrient composition. In all of them, and particularly in normal weight subjects, CHO ingestion provoked a significant postprandial ghrelin decline by approximately 30% from baseline values within 2 hours after meal onset , 40–42. Dietary protein is considered to be the most satiating macronutrient . The higIn a study of three isoenergetic meals  consumed by healthy young women, acylated ghrelin fell significantly after ingestion of both balanced and high-protein meals, while ghrelin persisted at significantly lower levels than baseline for a longer duration, following the high-protein meal . Apart fDespite the accumulating evidence supporting the satiating and ghrelin-suppressing capacity of dietary protein, there have been a few studies suggesting that protein ingestion stimulates, instead of suppressing, ghrelin levels , 47, whiIngested lipids appear to suppress the orexigenic hormone ghrelin less effectively than do CHO or protein . The relIn a study by Pavlatos et al., total ghrelin levels did not decline significantly after a fat-rich meal in normal weight women, as opposed to an isoenergetic protein-rich meal . In a siThe potential impact of varying fatty acid composition  on postprandial ghrelin response has been only scarcely investigated. In a relevant double-blind crossover study, researchers assessed two high-fat test meals, one with a high saturated to unsaturated fat ratio (70/30) and the other with a low ratio (55/45), and concluded that increasing saturated fat consumption had no deleterious effects on fasting and postprandial plasma ghrelin concentrations .BMI, body fat and indices of central fat distribution are inversely associated with fasting plasma ghrelin concentrations. A large number of clinical studies have shown that obese subjects tend to display lower total and acylated ghrelin levels in the fasting state compared with normal weight individuals . This fiAnother important aspect of ghrelin regulation in obese subjects is the blunted postprandial ghrelin response. This means that obese subjects have low ghrelin levels preprandially, but postprandial ghrelin secretion is not sufficiently suppressed, suggesting a severe defect in ghrelin-induced satiety mechanisms, which makes them feel still hungry, even though they have just completed their meal. Pavlatos et al. have shown that neither a protein- nor a fat-rich meal was able to elicit a significant acute ghrelin response in obese women . In the ad libitum energy intake, as shown by the study of Erdmann et al. [ad libitum energy intake at a buffet meal only in the obese group, and not in the lean, indicating that obese people are highly sensitive to the appetite-stimulating effects of ghrelin, even when the circulating ghrelin is low [The fact that lean subjects display higher fasting ghrelin levels than obese does not necessarily mean that they also consume greater amounts of food. On the contrary, food intake is most likely to be increased in obese subjects, because of the blunted postprandial ghrelin response, as described above. Besides, the effect of ghrelin on hunger and satiety sensations is not necessarily translated into alterations in n et al. . An addin is low . As a reAdditional factors that can in part explain the suppressed basal ghrelin levels in obese subjects include hyperleptinaemia and increased circulating levels of IL-1b (interleukin 1b), since both leptin and IL-1b are thought to inhibit ghrelin secretion . HyperleThe macronutrient-specific effect of meals on postprandial ghrelin levels has interesting implications only in normal weight individuals. In the obese population, the macronutrient effect appears to become blunted and is therefore of minor importance. This hypothesis is further corroborated by a recent study by Heinonen et al., where obese individuals with metabolic syndrome elicited no differences in plasma ghrelin or feelings of hunger and satiety, after consuming two high-CHO meals producing different insulin responses (whole-grain rye bread and wheat bread) . DespiteDiet-induced weight loss, contrary to gastric bypass surgery where ghrelin levels remain dramatically decreased, has shown to elevate fasting ghrelin levels and normalize postprandial ghrelin response . This meZimmermann et al. addressed the interesting question, whether acute ethanol ingestion affects ghrelin secretion . GhrelinAs far as smoking is concerned, in an interesting study by Kokkinos et al., acute cigarette smoking induced no significant suppression of post-smoking ghrelin in habitual smokers, possibly desensitized to any possible effect of smoking on ghrelin, through prolonged nicotine exposure . On the Many clinical studies have used isoenergetic test meals  in order to examine the relative efficacy of each macronutrient to suppress postprandial ghrelin. Even though the overall concept in these studies is common, the experimental design  is slightly or moderately different. This discrepancy may be in part responsible for heterogeneity in findings. Trying to delineate the central message behind all these divergent data, carbohydrate appears to be the most effective macronutrient in terms of postprandial ghrelin suppression, possibly because of its glucose-elevating and insulin-secreting effect. However, recent data indicate that CHO ingestion may provoke a delayed ghrelin rebound in the later postabsorptive period, questioning the role of CHO-rich meals in weight loss dietary approaches. Besides, all types of dietary CHO are not equally effective. Fructose-enriched meals display a poor ghrelin-suppressing capacity, promoting increased caloric intake, weight gain and obesity under conditions of chronic consumption. The most satiating macronutrient appears to be dietary protein. Protein induces prolonged ghrelin suppression and elevation of gut-derived anorexigenic hormones that delay gastric emptying regardless of the type of protein consumed. However, the influence of solid forms of protein  on postprandial ghrelin levels may require assessment over a longer period of time than 3-4 hours, since slow gastric emptying delays postprandial ghrelin nadir. As far as fat is concerned, it appears to be the least potent ghrelin-suppressant, even in normal weight subjects. Some studies have shown that fat decreases ghrelin concentrations, but later or more weakly than other macronutrients. At the same time, other studies report that fatty meals have absolutely no effect on postprandial ghrelin levels. In obese subjects, postprandial ghrelin response is blunted, and the macronutrient effect on ghrelin levels appears to be rather neutral. However, weight loss restores ghrelin response and leads to a significant improvement of ghrelin-mediated appetite regulation. From now on, it would be interesting to evaluate the long-term effect of macronutrient-enriched diet manipulations on fasting and postprandial ghrelin levels. Additional parameters that could possibly influence ghrelin response and should be further investigated are food form and viscosity , portion size and meal duration.


Hendra and Nipah viruses are related emergent paramyxoviruses that infect and cause disease in animals and humans. Disease manifests as a generalized vasculitis affecting multiple organs, but is the most severe in the respiratory and central nervous systems. The high case fatality and person-to-person transmission associated with the most recent NiV outbreaks, and the recent re-emergence of HeV, emphasize the importance and necessity of effective therapeutics for these novel agents. In recent years henipavirus research has revealed a more complete understanding of pathogenesis and, as a consequence, viable approaches towards vaccines and therapeutics have emerged. All strategies target early steps in viral replication including receptor binding and membrane fusion. Animal models have been developed, some of which may prove more valuable than others for evaluating the efficacy of therapeutic agents and regimes. Assessments of protective host immunity and drug pharmacokinetics will be crucial to the further advancement of therapeutic compounds. Pteropus are considered to be the natural reservoir for both viruses as demonstrated by seroconversion and isolation of HeV- and NiV-like viruses from bat tissues and secretions [Henipavirus genus within the family Paramyxoviridae [Hendra virus (HeV) and Nipah virus (NiV) are closely related highly pathogenic paramyxoviruses that have emerged independently in the past 15 years and continue to emerge in new locations. Flying foxes in the genus cretions . Additiocretions . It is aoviridae . HeV hasoviridae -8. NiV foviridae , 10). Inoviridae -15. Signoviridae , and higoviridae , 17. In NiV and HeV are classified as zoonotic biosafety level 4 (BSL-4) viruses and infectious virus can only be studied at a handful of laboratories worldwide. Both viruses have also been included among the various pathogenic agents of biodefense concern and each are classified as priority pathogens in category C by the Centers for Disease Control and Prevention (CDC) and the National Institute of Allergy and Infectious Diseases (NIAID). The category C agents include high emerging pathogens with the potential for causing morbidity and mortality with major economic and health impacts. Henipaviruses in particular, could be engineered for mass dissemination because of their availability from natural sources and their relative ease of propagation and dissemination. Currently there are no specific antiviral therapies or vaccines available for treating or preventing NiV or HeV infection resulting from a natural outbreak, laboratory accident or deliberate misuse.There have been two fatal cases of HeV infection in humans, the first presented with severe respiratory disease and upon autopsy the patient’s lungs had gross lesions of congestion, hemorrhage and edema associated with chronic alveolitis and syncytial cell formation. The second fatal case presented thirteen months post-exposure and exhibited leptomeningitis with lymphocytes and plasma cells and foci of necrosis in various parts of the brain parenchyma (reviewed in ). Multinin vivo. Ephrin-B3 ligand is expressed in certain neural subsets and may play an important role in HeV and NiV infection in the CNS. All paramyxoviruses F glycoproteins, including HeV and NiV, are typical class I fusion proteins that utilize two internal heptad repeats to mediate membrane merger  glycoprotein, which mediates pH-independent membrane fusion between the virus and its host cell (reviewed in ). Followiewed in ). BecausHeV and NiV are currently classified as BSL-4 agents and consequently human efficacy studies for testing potential therapeutic products are not easily achievable. In 2002, the U.S. Food and Drug Administration (FDA) implemented the Animal Efficacy Rule for the development of therapeutic products under these circumstances. Specifically, FDA can rely on evidence derived from animal studies in the evaluation of product effectiveness when particular criteria are met, such as a well-understood mechanism for both the pathogenicity of the agent and the underlying mode of action of the product. Importantly, the therapeutic effect must also be demonstrated in more than one animal species. Once these criteria have been met, human clinical trials could commence, most likely in populations at high risk of natural infection by HeV and NiV.50 HeV)  or with either an intranasal (6x105 pfu NiV) or intraperitoneal (107 pfu NiV) challenge of NiV [50 HeV) [50 HeV) [50 NiV) clinically manifested as ruffled fur and abnormal (less fearful) behavior [A strong epidemiological association existed in Malaysia between human NiV infection and close direct contact with pigs, especially sick and dying animals. No direct association with flying foxes was made. An epidemiological link has been noted between HeV infection in people and horses dying from HeV disease. Again, neither HeV disease nor seroconversion has ever been identified in wildlife carers who came into close and regular contact with sick and injured bats. While both horses and pigs have been experimentally infected with HeV and NiV, respectively , 44, neie of NiV , althoug[50 HeV) . Guinea [50 HeV)  and deve[50 HeV) , 38. Howbehavior  with gro50 values for hamsters inoculated by intraperitoneal and intranasal routes were 270 pfu and 47,000 pfu, respectively. Notably, the brain was the most severely affected organ in terms of vascular and parenchymal lesions. Neurons in the vicinity of vascultis showed numerous cytoplasmic eosinophilic inclusion bodies and both viral antigen and RNA were found extensively throughout neurons. Ultrastructural analysis revealed cytoplasmic inclusions composed of defined herringbone nucleocapsids typical of paramyxoviruses. However, although HeV and NiV clearly cause systemic disease in humans, NiV was not detected in serum samples from infected hamsters. Moreover, the pathology seen in the lungs and kidneys of NiV-infected hamsters differed from that seen in HeV-infected horses and NiV-infected humans. HeV infection of hamsters has not been reported.A golden hamster animal model for acute NiV virus infection has been established where, importantly, encephalitis and neuron infection were demonstrated similar to those seen in NiV-infected humans . The LD550 NiV [50 of low passage, plaque purified HeV [Experimental HeV infection of cats has been performed and findings from those studies were similar to those of horses and humans, namely generalized vascular disease with the most severe effects seen in the lung , 44. NiV50 NiV , or oronfied HeV , 42, 46 fied HeV . Histolo50 NiV remained clinically well throughout the study period and no febrile responses were recorded following NiV inoculation via a parenteral route. Challenged bats developed a sub-clinical infection characterized by episodic viral shedding in urine, limited presence of virus within selected viscera and seroconversion. No gross abnormalities were identified on post-mortem examination of animals at various times post exposure; all bat tissues were negative upon immunohistochemical labeling for NiV antigen.Experimental NiV infection in Pteropid bats has been attempted . All bat50 . A lower dose of 50 TCID50 failed to produce infection as defined by fever, illness, detection of virus, viral antigen or viral genome, histopathological lesions or seroconversion. Infection first manifested as fever and this was followed by inappetance and severe depression. A mild increase in respiratory rate in some animals was attributable to fever with one ferret showing tremors and hindlimb weakness. Gross pathology included subcutaneous edema of the head, hemorrhagic lymphadenopathy of submandibular, retropharyngeal and sometimes visceral lymph nodes, numerous diffuse pin-point hemorrhagic nodules scattered throughout the pulmonary parenchyma and petechial hemorrhages of the renal cortex. Histopathological lesions included focal necrotizing alveolitis, vasculitis, degeneration of glomerular tufts, and focal necrosis in a range of other tissues, including lymph nodes, spleen, adrenal cortex, bladder and ovary. Lesions in each case were associated with significant quantities of viral antigen, as determined by immunohistochemical staining  (reviewed in ), for thiewed in ). Becausiewed in -56 with in vivo experiments. Consequently, the optimal target and time frame for drug intervention are not known. Theoretically, therapeutics that target the mucosa should reduce viral loads in the lung. Intravenous agents should decrease viral loads and reduce systemic spread. Although targeting the CNS may prove difficult, a significant reduction in viral loads peripherally may enable the host to generate a protective immune response.HeV and NiV are transmitted oranasally, spread systemically and infected individuals succumb to disease within 7 to 10 days. Clinical and experimental data demonstrate that multiple organ systems are affected and that the lungs and brain are major sites of virus replication. Viremia has been documented in both clinical and experimental infections; however, the kinetics and duration vary. For both viruses, acute and late-onset encephalitis has been documented clinically. The initial sites and duration of henipavirus replication upon infection are largely unknown, mainly due to a lack of extensive in vitro or in vivo.To date, all novel henipavirus therapeutic agents that have been identified target and interfere with HeV and NiV virus entry and these candidates can be divided into two categories: those used for prevention of disease, including various vaccines, and those used for treatment post-exposure which includes antibodies, fusion inhibitors and soluble receptor molecules. The scope of this review will be limited to those agents tested using infectious HeV or NiV The use of safe and efficacious vaccines has been crucial to prevention strategies for several important viral pathogens in humans. Presently, there are some sixteen FDA-approved vaccines routinely used to prevent infection by virulent human pathogens and the majority comprise live-attenuated virus preparations (reviewed ). AlthouThe first successful experimental henipavirus vaccine utilized recombinant vaccinia viruses that encoded NiV F or G glycoprotein. In these studies, vaccination protected against NiV challenge in golden hamsters and importantly, protection lasted five months post-challenge suggesting protection potentially from late-onset disease symptoms . AlthougRecombinant subunit immunogens represent a viable avenue of vaccine development for the henipaviruses. These vaccines can be quickly implemented, are quite simple, and can be administered with no risk of infection. Recently, recombinant, soluble, oligomeric versions of the G glycoprotein of both HeV and NiV (sG) were generated as potential subunit vaccines . Recombivs mucus membranes). Finally, it will be important to determine the role of cell-mediated type 1 immune responses in the strength and longevity of protective immunity.In order for a henipavirus vaccine to be considered for human use, not only does it need to be successful in two animal models, a natural route of infection should be used in protection studies, the mechanism and limits of protection should be elicited, and the highest standards should be used for reagents production to assure safety and success in human trials. Additionally, for HeV and NiV, we believe it would be ideal to have one vaccine that protects against both viruses. All of the above vaccine trials offer critical data towards these goals, but further refinement of reagents and protocols are necessary. Firstly, the selection of adjuvant will be important in human use. Secondly, a correlation between vaccine dose and protection will need to be established to ensure that vaccine preparations induce protective immune responses well above sub-optimal levels. Correlations of immunity will also be critical, including antibody subclasses such as IgA, IgG and IgM and their location , a more cost-effective and highly sucessful treatment compared to the original polyclonal product . Passivein vitro, its mechanism of neutralization and the fact that is a fully human antibody, m102.4 could provide a valuable post-exposure or post-infection therapeutic for disease caused by HeV or NiV in humans. Pharmacological assays are currently under development and will be discussed below.A major advancement in antibody technology has been the development of the phage display platform of combinatorial antibody libraries  as well There have been considerable advances in mechanistic models of how several viral envelope glycoproteins function in driving the membrane fusion reaction (reviewed in -78). A kOver the past decade, targeting the viral membrane fusion process for antiviral drug development has received much attention, primarily lead by work on HIV-1 (reviewed in ). Importin vitro in a dose-dependent fashion [in vivo and was found to be unusually short , especially when compared to the half-life of Enfuvirtide in humans. As pure carnivores, cats have a unique metabolism designed to convert protein into sugar and this feature may have played a role in the rapid clearance of peptides from the bloodstream. For these reasons the peptide plasma half-life is currently being reevaluated in another animal species, ferrets. Once pharmacological studies have been completed and adequate peptide plasma half-life has been demonstrated, these peptides will be administered in vivo to examine their efficacy in treatment of ongoing NiV-mediated disease. It is hoped that, as was seen with enfuvirtide, the fusion inhibiting peptides might reduce viral load and or systemic spread in vivo.For the henipaviruses, second generation heptad-derived peptides were synthesized with chemical modifications of either the N- or C-terminus of the peptide by capping and/or “pegylation”, the coupling of polyethylene glycol (PEG) to proteins. Specific chemical modifications of the NiV F heptad-derived peptide were chosen, especially pegylation, to help improve plasma half-life and thus enhance therapeutic success. Pegylation is currently considered one of the most successful techniques to prolong the residence time of protein drugs in the bloodstream -98. Like fashion . The plain vitro [in vitro . Although only recently identified as a functional receptor for the henipaviruses, the ligand ephrin-B2 and its cellular receptor EphB4, have attracted much attention due to their roles in cancer biology [in vitro . The potential passive therapeutic efficacy of soluble ephrin-B2 ligand or EphB3 protein in an animal model remains to be determined. However, their mechanism of action could be viewed as similar to that of a G glycoprotein-specific or receptor-specific antibody. A summary of all prevention and post-exposure henipavirus therapeutics is shown in Fig. (In addition to using virus-neutralizing human mAbs or peptides as a post-exposure therapeutic modality for HeV and NiV infection, a soluble virus receptor which binds G and prevents attachment of the virion to the host cell may also represent a feasible therapeutic strategy. It has been demonstrated that soluble ephrin-B2 ligand can block infectious henipavirus infection in vitro  and more biology , 101. Cu biology , 102. Inin vivo, and we believe that soluble ephrin-B2 ligand and EphB3 studies will commence in the not too distant future. To aid drug measurement ex vivo, new assays that exhibit extraordinary sensitivity have been developed. Differential henipavirus serology and neutralization assays using Bio-plex array systems were recently described [ex vivo and set the stage for evaluating the efficacy of each in henipavirus animal challenge models.A complete understanding of plasma half-life and toxicity are required for the development of any human use therapeutic. The pharmacological properties of virus-specific human mAbs and heptad-derived peptides are currently being evaluated escribed . These ain vitro and will require in vivo half-life studies and disease prevention efficacy studies in at least two animal models. The complexities of such studies include optimizing drug delivery in vivo and site-specific targeting of post-exposure antivirals. Although many avenues of henipavirus therapeutic research have been successful, it will most likely take at least five to ten years before any therapeutic agent undergoes all necessary testing, gains FDA approval and is used routinely during an outbreak.The increased understanding of henipavirus pathogenesis has lead to the development of suitable animal models of disease and potential therapeutics. The successful vaccine candidates have been trialed in one animal model but still need to be evaluated in an independent animal model. Additionally, as discussed, while providing important proof of concept data, further understanding of protection mechanisms and refinement of reagents will be critical if any vaccine candidate is to progress further. Most of the post-exposure therapeutic agents have only been tested in vitro cell culture systems have been developed for examining various henipavirus gene-specific functions. Consequently, protein-based mechanisms, such as attachment and fusion, F cleavage, interferon interference and virus particle formation have been further elicited [in vivo and efficacy studies still represent the most challenging phases of drug development for HeV and NiV, especially under BSL-4 restrictions.An understanding of the molecular biology of HeV and NiV has expanded rapidly in recent years. Although this review has been limited to those agents tested against infectious virus, numerous elicited . Importaelicited , althoug


Pneumonia is a leading cause of death among children under five years of age. The Integrated Management of Childhood Illness strategy can improve the quality of care for pneumonia and other common illnesses in developing countries, but adherence to these guidelines could be improved. We evaluated an intervention in Benin to support health worker adherence to the guidelines after training, focusing on pneumonia case management.We conducted a randomized trial. After a health facility survey in 1999 to assess health care quality before Integrated Management of Childhood Illness training, health workers received training plus either study supports  or "usual" supports. Follow-up surveys were conducted in 2001, 2002 and 2004. Outcomes were indicators of health care quality for Integrated Management-defined pneumonia. Further analyses included a graphical pathway analysis and multivariable logistic regression modelling to identify factors influencing case-management quality.We observed 301 consultations of children with non-severe pneumonia that were performed by 128 health workers in 88 public and private health facilities. Although outcomes improved in both intervention and control groups, we found no statistically significant difference between groups. However, training proceeded slowly, and low-quality care from untrained health workers diluted intervention effects. Per-protocol analyses suggested that health workers with training plus study supports performed better than those with training plus usual supports . Both groups tended to perform better than untrained health workers. Analyses of treatment errors revealed that incomplete assessment and difficulties processing clinical findings led to missed pneumonia diagnoses, and missed diagnoses led to inadequate treatment. Increased supervision frequency was associated with better care .Integrated Management of Childhood Illness training was useful, but insufficient, to achieve high-quality pneumonia case management. Our study supports led to additional improvements, although large gaps in performance still remained. A simple graphical pathway analysis can identify specific, common errors that health workers make in the case-management process; this information could be used to target quality improvement activities, such as supervision . Streptococcus pneumoniae and Hemophilus influenzae could prevent many pneumonia cases, adequate management of cases that do occur is essential to reduce pneumonia mortality. Evidence suggests that children with pneumonia often do not receive potentially life-saving antibiotics IMCI/usualsupports) were estimated with predicted probabilities from the logistic regression models at baseline and follow-up time points for each of the health worker groups, with confounders held constant.We evaluated 17 factors  as potential confounders of the health worker group-outcome association by entering factors into models one at a time. Factors thought to be in the causal pathway between the intervention and correct treatment (e.g. correct diagnosis) were not considered. Factors that changed model estimates by >20% without causing model instability were considered confounders and retained in the final model . Effect communes in the 1999 survey into three parts: four IMCI pilot communes in the intervention area (baseline for the IMCI/study supports group), four IMCI pilot communes in the control area , and eight non-IMCI-pilot communes (baseline for the no-IMCI group). For details, see Figure The above effect sizes require an estimate of baseline (pre-IMCI) outcome values for each of the health worker groups. These values were estimated by dividing the 16 To examine specific causes of common errors in the case-management process (Objective 2), we used a simple graphical pathway analysis. In quality improvement methodologies, this is conceptually similar to a "root-cause" analysis . We begaTo identify the factors that influenced case-management quality (Objective 3), we studied the 70 children with uncomplicated pneumonia seen by IMCI-trained health workers whose treatment quality was defined. We assessed three health facility factors, 26 health worker factors and 21 child/consultation factors for their association with recommended treatment and "recommended or adequate" treatment. A forward-stepwise modelling approach was used to construct multivariate logistic regression models ,25; corrAltogether 1577 ill-child consultations were observed in the four health facility surveys , treatment quality improved over time for both primary outcomes, although differences in improvements between the study supports area and usual supports area were not statistically significant Figures  and 4. HResults of the per-protocol analysis are presented in Additional file For recommended treatment, improvements in the IMCI/study supports group were 20.4%-points greater than the IMCI/usual supports group, although this result was of borderline statistical significance (p = 0.08) ; 9.0% received adequate but not recommended treatment ; and 47.2% received inadequate treatment . The next two sections present in-depth analyses that explore reasons for correct treatment and errors in the management of the 70 children with uncomplicated pneumonia and defined treatment quality.This analysis Figure  revealedThe 70 children with uncomplicated pneumonia and defined treatment quality were seen by 44 IMCI-trained health workers . To screen hypotheses in an exploratory analysis of which factors influence correct treatment for pneumonia, we used logistic regression modelling to examine 44 factors for their association with treatment quality.Unfortunately, several factors of particular interest could not be studied because of a lack of variability: pre-service training , health facility type , job aids  and health worker knowledge (mean score of a knowledge assessment based on case scenarios was 97%). By exclusion, these factors were unlikely to confound the associations reported below.For recommended treatment . We also demonstrated a statistically significant 19.2%-point effect of the study supports for adequate or recommended treatment, and a similar but borderline-significant (p = 0.08) trend for recommended treatment. These results suggest that to improve treatment quality, a one-time training input has less impact than training coupled with continued support, as in our study.We found diverse results for improvements in case-management quality for different important conditions in Benin. Improvements were seen with IMCI training for all outcomes studied  . HoweverThe in-depth examination of errors by IMCI-trained health workers via graphical pathway analysis allowed us to pinpoint problems in how health workers applied the guidelines, and thus gives a view into the decision-making process we have not previously seen in the published literature. In 40% of the 70 non-severe pneumonia cases, all aspects of care  were adequate. In the remaining 60% of cases with problems, we found that errors were not uniformly distributed throughout the algorithm, but were grouped in several specific points; identifying these error points led to specific recommendations for improvement.For example, not surprisingly, missing the pneumonia diagnosis preceded virtually all major errors (no antibiotic prescribed). Of the 29 missed diagnoses, one third could be attributed to incomplete assessment , and two thirds could be attributed to health workers' misinterpreting clinical signs and symptoms or incorrectly processing clinical data into a diagnosis. Another example was that incomplete documentation, which could confuse pharmacists and caretakers, was relatively common. The analysis also revealed that some potentially important problems, such as under-dosing antibiotics, were rare. These results could direct supervision and other efforts to focus on complete assessments, correctly processing clinical data into diagnoses and full documentation of prescribed medicines.Multivariate modelling showed that study supports, supervision visits, longer consultation duration and a greater number of IMCI classifications were associated with at least one measure of treatment quality, although only supervision was associated with both outcomes.Supervision, a key component of our intervention, was associated with health care quality in a dose-response relationship. This finding agrees with other studies  and suppLonger consultation duration was associated with better adherence to IMCI guidelines, but the direction of causality is unclear. Better-performing health workers could be taking more time with patients. Alternatively, given ample time to spend with patients, health workers might perform better. Though not significant in multivariate modelling, univariate results showed that lower caseloads were associated with better health care quality, possibly supporting the latter explanation. A recent time-motion study of IMCI-trained physicians in Brazil found that caseload was inversely associated with consultation time, with the association being strongest at caseloads over 50 per day, and that quality of care was highest in the areas where health workers spent, on average, more time with each patient . RegardlOur multivariate analyses revealed that an increasing number of IMCI classifications (diagnoses) were associated with better pneumonia treatment quality. This finding differs somewhat from other analyses in this cohort (unpublished data). Taking all consultations together-not just pneumonia cases-we found that children with more IMCI classifications and more-complex cases generally received poorer quality care, in a linear fashion.One explanation for the different finding among the subgroup with pneumonia might be that antibiotics for pneumonia have many uses and might be more often overused than other IMCI medications . Figure Finally, knowledge of pneumonia case management was very high among IMCI-trained health workers, despite fairly poor care being delivered. This finding is a striking example of the knowledge-practice gap that has been observed in other settings  and mighFirst, the sample of pneumonia cases was relatively small, and the intervention was not fully implemented for all health workers. Second, with health workers being trained over several years, our cross-sectional surveys did not allow us to evaluate a single cohort of health workers over time. However, a re-analysis of these data by time since training still provided fairly robust evidence that performance did not deteriorate up to three years after training.Third, the study was initially planned as a group-randomized trial, and due to implementation problems, the data presented are from per-protocol analyses that stratified subjects by intervention exposure-an analytic approach recommended by some experts . Fourth,Fifth, our use of group randomization with only two groups was unlikely to have prevented bias from unknown factors and did not result in groups with equal baseline quality of care. Moreover, the robustness of the statistical results might have been affected. Sixth, the observation of consultations could have influenced health worker practices, perhaps overestimating quality somewhat , althougFinally, incomplete documentation of prescriptions was a considerable problem. A sensitivity analysis, which assumed adequate treatment quality for missing information, showed some differences from the main analysis . This issue raises an important question for researchers studying quality of care-especially for those doing direct observation studies. Should we ask health workers about missing prescription information and potentially introduce bias toward better quality, or should we remain silent observers and potentially accept uncertainty in our measures of quality?Our results add to a growing body of literature indicating that carefully designed interventions can improve health worker performance in low-resource settings, but that considerable attention must be paid to supporting health workers beyond one-time investments in training. The difficulties we encountered with training and supervision underscore the challenges of scaling up even the most basic components of a quality improvement intervention.Though quality of care for the condition studied here remained relatively low, with no group treating more than 56% of children correctly, care did improve over time, and gains in quality were sustained. Considerable attention in future research must be paid to attributes of interventions that are scaleable and that lead to quality improvement within the context of programmes in real-world settings. Additionally, we have shown how a simple method  can identify specific, common errors that health workers make in the case-management process; this information could be used to target quality improvement activities, such as supervision.The authors declare that they have no competing interests.FO, ML, MD and AR conceived of and planned the study and interventions; FO, ML and AR conducted surveys and collected data; DO and AR performed analyses and wrote the initial draft of the manuscript; all authors worked on interpreting the results and finalizing the manuscript.Per-protocol analysis: effect of study supports and IMCI training on the quality of pneumonia management. Table presents results for effectiveness of study supports and IMCI training on quality of care according to 5 different measures of quality of pneumonia case management. Effects are a percentage point improvement in the proportion of cases with correct management. MS Word table in landscape orientation.Click here for file


While these characteristics of catechins have been well documented, actions of catechins as mediators on inflammation-related cardiovascular diseases have not yet been well investigated. In this article, we reviewed recent papers to reveal the anti-inflammatory effects of catechins in cardiovascular diseases. In our laboratory, we performed oral administration of catechins into murine and rat models of cardiac transplantation, myocarditis, myocardial ischemia, and atherosclerosis to reveal the effects of catechins on the inflammation-induced ventricular and arterial remodeling. From our results, catechins are potent agents for the treatment and prevention of inflammation-related cardiovascular diseases because they are critically involved in the suppression of proinflammatory signaling pathways.Tea polyphenols known as catechins are key components with many biological functions, including anti-inflammatory, antioxidative, and anticarcinogenic effects. These effects are induced by the suppression of several inflammatory factors including nuclear factor-kappa B (NF- In. InκB, wolerance –24. TherRecently, Tripathi et al. demonstrated that green tea extract (GTE) in combination with low dose CyA significantly prolongs graft survival as well as increase the production of immunosuppressive cytokines in the murine model of nonvascularized cardiac allografts. They concluded that the potential of GTE as an adjunctive therapy in combination with CyA to prolong allograft survival and to reduce CyA induced nephrotoxicity .κB related inflammation is enhanced by ischemia and reperfusion of myocardium; the activated NF-κB induces MMPs. Thus, decoy against NF-κB reduces myocardial inflammation induced by ischemia/reperfusion injury . In. In15]. ry cells .Nitric oxide (NO) is an important molecule that plays a pivotal role in inflammatory conditions of hearts, and many papers showed interesting data. Babu and Liu demonstrated that catechins regulate vascular tone by activating endothelial NO . Paquay κB, which is a central mediator for the development of inflammatory diseases. We have reported specific inhibition of NF-κB using a decoy in the myocardial ischemia [κB decoy suppresses many inflammatory factors including adhesion molecules, cytokines, and MMPs. Although catechins are not specific inhibitors of NF-κB, they have similar effects to the inhibitors such as suppression of adhesion molecules and other inflammatory factors. Therefore, catechins have the potential to suppress clinical inflammatory diseases.We have demonstrated that the catechin intake significantly suppresses the expression of inflammatory factors including adhesion molecules, cytokines, and MMPs. These key factors are known to be regulated by NF-ischemia , myocardischemia , and heaischemia . In thes


Regulation of cellular functions during dermal repair following injury is complex and critically dependent on the interaction of cells with the surrounding extracellular matrix (ECM). The ECM comprises various families of macromolecules that form the structural scaffold of the tissue, but also carry distinct biological activities. After injury to the skin, the defect is filled by a provisional matrix that is invaded by inflammatory cells, sprouting blood vessels and fibroblasts. In a later phase, the wound contracts, the tissue is replaced by mature connective tissue produced by activated fibroblasts, and a scar is formed. All cells involved communicate directly with the ECM by integrins and other matrix receptors. These transmit signals and induce adaptive responses to the environment by the embedded cells. The ECM or proteolytic fragments of individual ECM constituents exert defined biological activities influencing cell survival, differentiation of myofibroblasts, ECM synthesis and turnover, wound angiogenesis and scar remodeling. Extensive crosstalk exists between ECM and growth factors, and between growth factors and integrins. ECM-cell contact also enables direct transmission of mechanical tension, which then modulates many activities of all cellular players. Understanding this complex interplay is important to provide a basis for designing effective wound therapy and for strategic interference with mechanisms that have gone out of control in fibrotic conditions. The functioning of cells is critically dependent on their interaction with the surrounding extracellular matrix (ECM) . These cResearch over the past 20 years has increased our understanding of the composition of the ECM structures, and identified and characterized distinct families of matrix proteins (Appendix 1). These protein families include several subfamilies of collagenous proteins, adhesive glycoproteins, proteoglycans and matricellular proteins. A common denominator is their composition from a limited number of structural domains. It is important to note that these individual domains carry biological activities, modulate the activity of growth factors and cytokines, and harbor recognition sites for the interaction with specific cell membrane receptors . ECM proCollagens represent a group of diverse protein subfamilies containing at least 28 different collagen types that are encoded by more than 42 genes ,5. All mEndostatin, a fragment of collagen XVIII, is one of the best-characterized collagen fragments . It is aTogether with other large glycoproteins, such as, fibrillin and fibulin, elastin is a component of the elastic tissue. It is present in the dermis and several other connective tissues such as vessel walls. Several diseases are linked to mutations in components of the elastic tissue, including some forms of cutis laxa or Marfan syndrome . ElasticProteoglycans are composed of positively charged polysaccharide chains attached to a protein backbone. They can be very large and are hydrophilic, which endows them with high water-binding capacity, and confers viscoelasticity and compression resistance to tissues. Proteoglycans are found in basement membranes, on cells and in matrices surrounding mesenchymal cells. Perlecan, a heparan sulfate proteoglycan, has  a number of signaling functions via its heparan sulfate chains, which bind and modulate growth factors such as fibroblast growth factor (FGF)2, VEGF and platelet-derived growth factor . The impOne of the best-characterized glycoproteins of the ECM is fibronectin, a major product of dermal fibroblasts and myofibroblasts, which has a wide distribution in the dermis, in the dermal-epidermal basement membrane zone. Fibronectin is a large adhesive glycoprotein with a huge number of different functions. Besides its structural role, fibronectin is the main cell adhesion molecule, modulating many cellular activities. In humans, alternative splicing generates close to 20 fibronectin variants with different functions, of which the ED-A variant is an important regulator of fibroblast to myofibroblast differentiation . After iLaminins represent a protein family of 16 currently known members. Laminins are large trimeric ECM glycoproteins consisting of three different polypeptide chains . LamininMore recently a group of proteins named the matricellular proteins was identified. These proteins interact with other structural ECM molecules and cell surface receptors, and modulate cytokine activation. They are thought to function as important regulatory molecules rather than as structural components .The multi-domain protein thrombospondin represents a family of five members, of which thrombospondin-2 is the predominant member; it is expressed by fibroblasts and endothelial cells in murine skin. Thrombospondin-1 is found in platelets and is secreted by inflammatory cells ,27. ThroIn vitro, both proteins exhibit de-adhesive and anti-migratory activities. Wound closure is accelerated in both SPARC and hevin null mice [The secreted acidic and cysteine-rich protein family includes SPARC  and hevin, both expressed in skin, primarily by fibroblasts . In vitrull mice ,34. Howeull mice .The matricellular family of tenascin proteins comprises four members in mammals. Tenascins are large glycoproteins with structural similarity to fibronectin. Like SPARC and thrombospondin-1, they do not support cell adhesion. Tenascin-C is abundantly present in the provisional wound matrix, and its synthesis by fibroblasts is strongly enhanced by mechanical tension . Skin woMany ECM constituents actively modify the activity of growth factors and cytokines and some act as a reservoir from which growth factors can be rapidly released if required Table . The ECMProbably the best example is the binding of FGF-2 to the heparan sulfate proteoglycan, perlecan. This binding promotes FGF receptor activation and ultimately downstream signaling, which supports mitogenesis and angiogenesis . Other eThese modifications in the availability and activity of growth factors and cytokines by defined constituents of the ECM contribute to the versatile response of cells to a changing environment. This interaction is most obvious in situations in which large amounts of active cytokines are required, such as in wound healing and tissue remodeling. However, ECM modulation is equally important in situations when cellular activities must be turned down to reduce tissue damage and scarring, as in late stages of tissue repair or after inflammatory processes.Within the past few years, a new class of ligands, the matrikines and matricryptins, has been characterized as subdomains of various ECM proteins, which can signal to cells through receptors such as growth factor receptors . The EGFIn summary, the individual domains of the ECM can interfere with growth factor signaling either by direct interaction with growth factor receptors or indirectly by controlling the availability and activity of growth factors and cytokines. This versatility of ECM components in regulating cellular activities is further expanded by close cooperation between growth factor signaling and integrin-mediated cell-matrix interactions and mechanical tension (discussed in detail below).Although many receptors for ECM proteins have been identified, the integrin family probably constitutes the most abundant receptors mediating the interaction of cells with their surrounding ECM. Integrins have a relatively restricted, cell type-specific and context-dependent expression with defined substrate specificity. Ligands bind to the extracellular domains of integrins, and integrin cytoplasmic tails connect via linker proteins to the cytoskeleton. Integrins mediate cell adhesion, migration and survival, and also specific differentiation programs relevant to development, tissue maintenance and repair. By virtue of linking the outside environment with the cellular inside, they transduce signals and mechanical tension bidirectionally from the outside in and the inside out ,53.Integrin function is closely connected with integrin conformation . UsuallyIntegrin expression and activity can be further modulated by other transmembrane proteins such as tetraspanins or cell surface heparan sulfate proteoglycans. Most of these are expressed in a cell type-specific manner in different phases of skin repair and scarring .Activation of integrins, transmission of mechanical tension originating from tissue remodeling, and the regulated activity of growth factors play crucial roles in controlling cellular activities during dermal repair, scarring and fibrosis. Activation of TGF-β by αv integrins is a good example of such interplay. Binding of latent TGF-β to integrin αvβ6, which is abundantly expressed by keratinocytes in wound epithelium and in chronic wounds , inducesAngiogenesis can be controlled by the crosstalk between αvβ3 and FGF that results in modulation of FGF signaling ,66, and The mechanisms by which integrins modify cellular responses to growth factors can involve direct regulation of transcriptional programs, complex activation mechanisms or various signaling cascades. The data available also clearly demonstrate a close link between the ECM, the cytoskeleton and growth factor/cytokine release, and the cellular response. Obviously, most experiments have been based on monolayer culture systems, and only limited information is available on how cells interact with the ECM in a complex three-dimensional system such as the dermis. In those situations, the cellular architecture is modified and mechanical tension is generated, which modulates integrin functions and the cellular response to growth factors.Although there is increasing evidence that in certain specialized tissues, epithelial cells participate in fibrosis and scarring, in most tissues fibroblasts are the main effector cells for the production and deposition of new and sometimes excessive ECM. Fibroblasts were among the first cells grown in culture dishes, but specific markers are still lacking and their differentiation is only incompletely understood. They are generally defined by their morphology and by a characteristic pattern of gene expression. In tissue repair, they synthesize most ECM molecules and produce proteases required for the remodeling in later phases. Fibroblasts also secrete numerous growth factors and cytokines that act on other cell types thereby modulating tissue repair and scarring ,69.After tissue damage and the initial inflammatory reaction, fibroblasts are modulated from quiescent cells to migratory, proliferating and biosynthetically active cells. It is well established that they can be transformed into myofibroblasts, the contractile activated cell type, which is mainly responsible for scar contraction and excessive ECM deposition . The finThe exact origin of fibroblasts in wounds or fibrotic tissue remains unclear and is still a matter of debate. A certain fraction of fibroblasts within wounds and scars originates from resident cell populations. In normal wounds, the balance between growth factor activity and the integration of fibroblasts into an environment with stable mechanical tension ensures cell survival and gene expression patterns required for normal tissue remodeling . ChangesMany studies have considered alternative origins of fibroblasts in scarring and fibrosis. A significant percentage of fibroblasts in dermal wounds was derived from bone marrow in green fluorescent protein transgenic mice . There iAlthough this field is rapidly developing, the specific roles of these different fibroblast progenitor populations are incompletely understood. It is likely that the extent of matrix deposition and tissue destruction in wounds or inflamed tissue depends on the local environment, ECM composition, magnitude of integrin-mediated mechanical stress, and growth factors released by different populations of circulating progenitors Figure .Myofibroblasts have long been associated with wound repair and fibrosis . They arPreviously, myofibroblasts were characterized ultrastructurally by electron microscopy. They differ from normal fibroblasts by the expression of α-SMA, which is the most widely used marker for myofibroblasts. They are also characterized by expression of the ED-A splice variant of fibronectin and the upregulated synthesis of several ECM proteins and growth factors . InductiMyofibroblasts promote wound and scar contraction by establishing cell-cell and cell- ECM contacts and tractional forces. They form bundles of contractile microfilaments and extensive cell matrix contacts, which attach the microfilaments to the surrounding ECM. These contacts  have been defined ultrastructurally . FormatiIn the later stages of normal wound repair, myofibroblasts undergo apoptosis and disappear. Loss of mechanical tension, alterations in cell density or protease activity and/or changes in the pattern of secreted growth factors are thought to contribute to cell death. Understanding the exact cellular and molecular basis for clearance of myofibroblasts is essential because in pathological situations (e.g. scarring or fibrosis), myofibroblasts persist and are responsible for some of the clinical symptoms.Many other cell types either directly or indirectly contribute to scar formation. The early inflammatory infiltrate is initially activating a complex cascade of factors, which depend on one another and profoundly influence late events after most of the inflammatory cells have disappeared.In certain tissues, epithelial-mesenchymal transition plays an important role during development, but has more recently also attracted attention for explaining the development of fibrosis, for example, in kidney and possibly in lung. In response to growth factors and altered ECM contacts, epithelial cells can lose their epithelial polarity and adopt a mesenchymal phenotype .The microvascular pericytes are integral parts of all microvessels, and play an important role in regulating endothelial cells. Although the precise function of pericytes is not clear they crucially contribute to stabilize and mediate forces within the microvasculature . PericytAll adherent cells including endothelial cells, fibroblasts and myofibroblasts sense tension originating from the environment. Tension is transmitted via cell-ECM contacts , leadingObviously, fibroblasts and myofibroblasts are cells implicated in scarring, which is strongly influenced by mechanical tension. Both cell types firmly attach to ECM structures via matrix adhesions, which have been studied in detail in monolayer cultures (see previous sections). These include the focal complexes, focal adhesions and fibrillar adhesions. The major structures required to form such matrix contacts are the integrin receptors, which directly connect the ECM structures to the intracellular cytoskeleton network . FibroblMore recently, fibroblasts have been grown in three-dimensional ECM structures, in which cell shape is modified and they become bipolar. A clear distinction of the different attachment sites is more difficult, but these structures are equally important and establish a direct connection between the surrounding ECM and the cell nucleus ,88. UsinThe precise mechanisms by which different cell types transmit mechanical signals are not fully understood. They might involve stretch-activated ion channels, direct interactions between structural and signaling components, or activation of small GTPases .in vitro systems has demonstrated that tenascin-C expression is strongly upregulated at the transcriptional level by mechanical stress, involving specific cis-acting elements in the tenascin-C promoter [As outlined above, many cooperative interactions exist between integrins and growth factor signaling. In particular, fibroblast to myofibroblast conversion and α-SMA expression crucially depend on a combination of mechanical tension and TGF-β . Thus inpromoter ,93.These data clearly demonstrate that mechanical tension, which is generated during wound contraction, scar formation and in fibrotic tissue, can modulate the gene expression of fibroblasts and myofibroblasts embedded into this tissue at different molecular levels. Tension directly modifies gene transcription, induces signaling from integrins affecting small GTPases or induces/inhibits growth factor signaling, which then indirectly affects ECM protein synthesis by fibroblasts/myofibroblasts. By a combination of these mechanisms, mechanical tension induces an activated, contractile fibroblast/myofibroblast phenotype characterized by high levels of synthesis of ECM proteins, low protease activity and high production of fibrogenic cytokines.Interaction of cells with the surrounding ECM is an important modulator of all cellular activities Figure . Tight cThe interaction of these cells with different ECM constituents is mediated by several integrins that signal bidirectionally and transmit mechanical forces that are generated during pathological situations. Cell-ECM interactions mediated by integrins are closely linked to growth factor signaling and to complex modulation of the cellular cytoskeleton. This complex interplay controls the specific gene expression program at any given time. Detailed insight into the roles of different ECM proteins, the function of integrins, their modulation by forces, and their interplay with growth factors will help us to understand the orchestration of tissue architecture and the processes underlying excessive scarring and fibrosis.The authors declare that they have no competing interests.BE, RN and TK drafted the manuscript. TK was responsible for its design and coordination. All authors read and approved the final manuscript.Appendix 1: Composition of the extracellular matrix in skinStructural proteins: collagens, elastin, fibrillins, fibulins• Adhesive glycoproteins: fibronectin, vitronectin, matrilins, laminins• Proteoglycans: perlecan, syndecans, versican, decorin, biglycan, lumican• Matricellular proteins: thrombospondins, tenascins, SPARC/BM-40/osteonectin• Appendix 2: The collagen subfamilies• Fibril-forming collagens • Fibril-associated collagens with interrupted helices • Hexagonal network-forming collagens (VIII)• Basement membrane-associated collagens • Beaded-filament forming collagens (VI)• Transmembrane collagens • Multiplexins