Short bowel syndrome (SBS) is defined as the malabsorptive state that often follows massive resection of the small intestine. Most cases originate in the newborn period and result from congenital anomalies. It is associated with a high morbidity, is potentially lethal and often requires months, sometimes years, in the hospital and home on total parenteral nutrition (TPN). Long-term survival without parenteral nutrition depends upon establishing enteral nutrition and the process of intestinal adaptation through which the remaining small bowel gradually increases its absorptive capacity. The purpose of this article is to perform a descriptive systematic review of the published articles on the effects of TPN on the intestinal immune system investigating whether long-term TPN induces bacterial translocation, decreases secretory immunoglobulin A (S-IgA), impairs intestinal immunity, and changes mucosal architecture in children with SBS.The databases of OVID, such as MEDLINE and CINAHL, Cochran Library, and Evidence-Based Medicine were searched for articles published from 1990 to 2001. Search terms were total parenteral nutrition, children, bacterial translocation, small bowel syndrome, short gut syndrome, intestinal immunity, gut permeability, sepsis, hyperglycemia, immunonutrition, glutamine, enteral tube feeding, and systematic reviews. The goal was to include all clinical studies conducted in children directly addressing the effects of TPN on gut immunity.In vitro studies, 3) mortality, 4) villous atrophy, 5) duration of dependency on TPN after bowel resection.A total of 13 studies were identified. These 13 studies included a total of 414 infants and children between the ages approximately 4 months to 17 years old, and 16 healthy adults as controls; and they varied in design and were conducted in several disciplines. The results were integrated into common themes. Five themes were identified: 1) sepsis, 2) impaired immune functions: Based on this exhaustive literature review, there is no direct evidence suggesting that TPN promotes bacterial overgrowth, impairs neutrophil functions, inhibits blood's bactericidal effect, causes villous atrophy, or causes to death in human model.The hypothesis relating negative effects of TPN on gut immunity remains attractive, but unproven. Enteral nutrition is cheaper, but no safer than TPN. Based on the current evidence, TPN seems to be safe and a life saving solution. In the late 1960's, the introduction of total parenteral nutrition (TPN) as an alternative nutrition provided a life saving solution to children with chronic bowel obstructions, fistulas, loss of mucosal body surfaces, short bowel syndrome, and other clinical problems that precluded enteral diet by mouth or tube feeding for long periods of time. Intravenous administration of TPN became an essential fluid to meet nutritional needs and to avoid progressive starvation-induced malnutrition, which changed the outcome of patients from dying . As a reHowever, its use has been shown to associate with an increased incidence of infection . A numbeThis paper presents a descriptive systematic review of published research articles on the effects of the long-term TPN on gut mucosal immunity in children with SBS; specifically, it addresses whether TPN: 1) promotes bacterial translocation, 2) impairs intestinal mucosal immunity by decreasing S-IgA levels, 3) inhibits neutrophil and cytokine functions in blood, 4) promotes atrophy of the mucosal villi, 5) hyperglycemia, and 6) causes death. It is hoped that these findings will expand the knowledge of pediatric nurses, and have an impact on clinical practice by being included in the pediatric parenteral nutritional guidelines. Since the review of literature did not reveal any systematic reviews of TPN and mucosal immunity on children with SBS, the aim of this descriptive systematic review of individually published scientific studies is to impart a better understanding of the effects of TPN on gut mucosal defense and barrier function.Short bowel syndrome, one of the major indications for diagnostic categories using long-term TPN , is a clAfter resection, the residual or remaining small bowel undergoes intestinal adaptation, a process characterized by mucosal hyperplasia, villus lengthening, increased crypt depth , and bowel dilation. The process of adaptation is complex and includes both structural and functional changes. The earliest sign can be detected within 24-48 hours, and process may continue for months, possibly years. In the early phase, mucosal hyperplasia and villus hypertrophy occur. Oral nutrients and hormones stimulate this intestinal adaptation. The main clinical challenge in SBS lies in managing the many nutritional problems that occur as a result of malabsorption secondary to the reduced absorptive surface area ,21.duodenum, the jejunum, and the ileum. The duodenum begins at the pylorus and ends where it joins the jejunum. The end of jejunum and beginning of the ileum are not distinguished by an anatomic marker[The normal length of intestine in full-term newborn is estimated at 200 cm-250 cm , and theic marker. The ileThe primary function of the small intestine is to absorb nutrients into the circulation . During 12. The ileocecal valve forms an important barrier, slowing transit from the small intestine to the colon and limiting bacterial colonization of the small intestine [The tight junctions of the jejunum are highly porous, so that exposure of the mucosa to hyperosmolar nutrient solutions leads to efflux or flow of water and electrolytes into the lumen (interior space of intestine). This fluid is then reabsorbed in both the ileum and colon. The distal ileum has specialized transport carriers for the absorption of bile salts and vitamin Bntestine . Ileocecntestine .mucosa, the innermost layer, which comprises epithelium, lamina propria, and muscularis layers; (2) The submucosa consists largely of loose connective tissue with collagen and elastin fibrils. Glands, nerve trunks and blood vessels are also present in the submucosa; (3) The next layer, the muscularis, typically consists of two substantial layers of smooth muscle cells; an inner circular layer and an outer longitudinal layer; and (4) The serosa is the outermost layer of intestinal tract [Histological organization of the intestinal wall is divided into four major layers. These major layers include: (1) the al tract ,29-31.The mucosa is considered an "architectural marvel." The epithelial layer of mucosa regulates absorptive, secretory, and protective barrier functions. This thin epithelial layer composed of columnar absorptive cells, goblet cells, undifferentiated crypt epithelial cells, panet cells, enteroendocrine cells, tuft cells, cup cells, and intraepithelial lymphocytes . This muBeneath the mucosal epithelium is the connective and supportive tissue called the lamina propria. The lamina propria contains various immunocompetent cells, including plasma cells, mast cells, macrophages, and lymphocytes that produce not only immunoglobulins but also cytokine mediators .A complex intestinal mucosal defense system plays an important role to prevent invasion of gut bacteria or absorption of endotoxin. Peristaltic waves and contractions sweep the intestinal contents in a steady distal flow toward the colon, which is a major importance in reducing the growth of bacteria in the proximal intestine . During Mucus, an epithelial secretion, also helps reducing bacterial overgrowth. Mucus has special properties that enable to trap bacteria in an intraluminal location while moving organisms distally in bolus fashion. Carbohydrate serves as a nutrient for some bacteria, thus attracting them to a mobile colonization site that is continuously replaced. Enzymatic digestion also significantly reduces bacterial overgrowth. Pancreatic juice has antibacterial activity. Intestinal secretions from the stomach, intestine, and pancreas are a significant deterrent to growth due to their ability to dilute the bacterial mass ,32.in vitro study, bacteria instilled into an intact lumen of a normal human stomach were promptly killed in less than 15 minutes at a pH of less than 3.0, but remained viable in the achlorhydric stomach for at least 1 hour [2 (H2) receptor blockade in healthy adults, however, has been shown to increase the number of gastric bacteria [Gastric acidity acts as an initial line of defense against ingested bacteria. Gastric juice with pH less than 4.0 is bactericidal for most organisms, although not immediately . In one t 1 hour . Chronicbacteria .The intestinal tight junctions between epithelial cells and permeability have been reviewed . PermeabThomson and colleagues -42, citiThe permeability of the intestine can be increased by variety of factors, such as psychological stress, fasting, and certain drugs. During fasting or malnutrition, intestinal secretion of ions and fluid increased, permeability to ions and macromolecules is increased and associated mucosal atrophy reduces intestinal absorption of nutrients. Some drugs also alter intestinal permeability. Nonsteroidal anti-inflammatory drugs (NSAIDs) increase intestinal permeability in both animals and humans. Glutamine, the major source of the small intestinal mucosa, may be partially normalize this NSAID-induced permeability ,45.12 lymhocytes and more antibody (secretory IgA) than any other site in the body including spleen [The gastrointestinal track or gut associated lymphoid tissue (GALT) has an important role in gut immunity. The gut is considered the largest lymphoid organ in the body housing more than 10g spleen . The lamg spleen ,47.S-IgA contains antibodies directed to biologically active antigens such as viruses, bacteria, enterotoxins, and enzymes normally restricted to the intestinal tract. A reduction in S-IgA level results in a greater frequency of GI infections and impaired reticuloendothelial or macrophage function, which predispose the child to systemic bacteremia ,46,48.After surgical intervention, sympathetic response is activated creating a positive relationship between the severity of injury and the plasma concentration of adrenalin, noradrenalin and dopamine . FollowiIn the systemic level, counter-regulatory hormones, such as adrenocorticotropic hormone (ACTH), antidiuretic hormone (ADH), catecholamines and stress hormone, cortisol are released. These cytokines and systemic hormones working together cause the multiorgan system failure. They also induce hypercatabolism, which is characterized by protein breakdown within skeletal muscle, accelerated breakdown of branched-chain amino acids and increased release of glutamine and alanine into systemic amino-acid pool. Glutamine, conditionally essential amino acid, is critical as an energy source for enterocytes, immune cells, and rapidly growing tissues. Overall, physiological stress response results with increased intestinal permeability and bacterial translocation, which promotes sepsis or multiorgan failure and potantiate hypercatabolism and protein-calorie malnutrition .During illness, stress increases the concentration of counterregulatory hormones (glucagons, epinephrine, cortisol, and growth hormone) and cytokines. Counterregulatory hormones increase serum blood sugars by increasing hepatic glucose production and by decreasing peripheral glucose uptake . GlucoseThe most important ongoing complications in children with SBS reported in the literature are: recurrent sepsis, catheter related sepsis, TPN-associated cholestasis, metabolic disturbances, hyperglycemia, electrolyte imbalances, hypertriglyceridemia, gastric hypersecretion, diarrhea, and organ dysfunction ,55-57.5 colony forming units/mL) [Bacterial overgrowth is defined as the presence of potentially pathogenic microorganism (PPM) in high concentration (≥ 10nits/mL) ; that isnits/mL) . BacteriSepsis in infants and children is defined as the systemic response to a possible infection. Evidence of bacteremia or an infectious focus is not required. The term septicemia is used when organisms or their toxic products are identified in the bloodstream, in other words, a pathogen is recovered from blood cultures or identified [Bacterial translocation is a phenomenon where intestinal pathogenic microorganisms, which are normally resident within the lumen of the intestinal tract, travel from the gut lumen into local mesenteric lymph nodes, and from there to distant sites, such as upper gastrointestinal tract, thereby causing sepsis and septicemia ,60-62. Sentified .Most studies of the pathogenesis of sepsis in animals have been carried out shown that gut mucosa is particularly susceptible to injury through a variety of mechanisms, such as decreased mucosal blood flow, increased oxygen demand, decreased oxygen delivery, and reperfusion injury . Sepsis It is also widely believed that these infectious complications are related to the central venous access devices because the most recurring infection was reported as line infection ,60,65,66D-lactic acidosis Syndrome." The episode of this syndrome causes patients develop slurred speech, ataxia, and altered affect. These patients appear "drunk." This condition is resolved after placing patient on a diet with restricted carbohydrate intake and metronidazole [Bacterial overgrowth frequently complicates, especially when the ileocecal valve is absent and the dysmotility is present in the remaining bowel loops. In the colon, unabsorbed carbohydrates undergo bacterial fermentation to D-lactate, short-chain fatty acids (acetate, propionate and butyrate), which can not be metabolized by D-lactate dehydrogenase; and these small molecules are absorbed from colon, "carbohydrate salvage" ,77. Absonidazole .In this review, a descriptive systematic, non-quantitative literature review was conducted using methodological guidelines -80. A sysynthesis of literature. This form of literature synthesis involves tabulation of study characteristics and results to summarize their findings in an area, and it also leads to new conclusions and knowledge as a result of systematically pulling together the fragmented results from single studies[A descriptive systematic review is also called a e studies,84.To select and assess the methodological quality of the research studies to be included in this review, the author used a list of standardized criteria or the guidelines Table  proposedTo what extent does long-term TPN affect the intestinal immune system of infants who undergo bowel resection with no enteral nutritional support?For the purpose of this paper, searches of the following resources were used to do a comprehensive and exhaustive literature search: 1) Electronic bibliographic databases, 2) Reference lists from relevant primary and review articles, 3) The Internet, 4) Hand-searched journals, and 5) Unpublished studies, such as Thesis and Dissertation Abstracts .Current Contents, HealthStar, OVID Full text journal articles), Cochran Library, Dissertation Abstracts, Thesis, PubMed, Web of Science, Evidence-Based Medicine, Evidence-Based Nursing, Journal Citation Reports, MD Consult, Academic Search, CancerLit, and Journal Collection Databases (Academic/IDEAL, Blackwell Science, Elsevier, and SpringerLink).A literature search was conducted using the following online databases: OVID Databases  database, and one unpublished thesis relevant to this review was included [In this systematic review, 12 published studies using quantitative methods met the clinical trials criteria. In addition, one unpublished study was included in this review. These 13 studies included a total of 414 infants and children between the ages approximately 4 months to 17 years old, and 16 healthy adults as controls. One of the 13 clinical trial samples was not counted because the same sample was used for secondary analysis ,60.in-vitro studies [Although high quality, well-designed study, or the preferred research design for a review would be the one that randomly allocates  the participants with the condition of interest to alternative therapeutic interventions, but when prospective randomized clinical trials (PRCT) are not possible to find or not available, NHS Center for Review and Dissemination Guidelines suggest that the next best available evidence should be considered based on the hierarchy of study designs, which are from highest to lowest level: 1) prospective experimental studies , 2) experimental study without randomization , 3) observational study with control group, a) cohort, b) case-control studies, 4) observational studies without control group, a) cross-sectional, b) before-and-after study, c) case series, and 5) expert opinion ,91,92. I studies ,51,67, o studies , and fou studies -98. The The descriptive methodological and characteristics of the samples are given in Table To collate and present the extracted data, a coding sheet was used to collect information or study characteristics that included both demographic and methodological from the retrieved primary studies ,99. The In vitro studies, 3) mortality, 4) villous atrophy, 5) duration of dependency on TPN after bowel resection. Table Five themes were identified: 1) sepsis, 2) impaired immune functions: Six of the 13 trials reported bacterial overgrowth, sepsis, or septicemia ,94,96,98E. coli, Klebsiella, Candida species and enterococci. Eight patients had nine episodes of septicemia caused by coagulase-negative staphylococci. The researchers concluded that there was an association between septicemia and elevated serum bilirubin level, indicating TPN related cholestasis.In the third study , the purDahlstrome  investigin-vitro control studies, investigators elucidated that long-term TPN in infants suppresses specific mechanisms of immune functions [in vitro challenge in five enterally fed infants (age<6 months) and 6 healthy adults. They found that in infants, after 2 hours of incubation with a physiological dose of TPN (1 μ of TPN in 1 ml of blood) there was a significant decrease (P < 0.05) in tumor necrosis factor alpha (TNF-α) production.In three unctions ,51,67. Iin vitro controlled-clinical trials focused on the cellular mechanism of neutrophil dysfunction, and the whole blood bactericidal activity against coagulase negative staphylococci in the infants receiving TPN [in vitro-controlled whole blood model to measure the host bactericidal activity against coagulase-negative staphylococci. When researchers added coagulase-negative staphylococci to whole blood drawn from control patients receiving enteral feeding, the median killing of coagulase-negative staphylococci was 65%. In contrast, blood from infants receiving long-term TPN failed to kill this organism as effectively. Infants on long-term TPN had the lowest levels of both whole blood and intracellular killing of coagulase-negative staphylococci due to a defect in neutrophil function. These investigators reported that there was a negative linear correlation between the duration of TPN in days and killing of coagulase-negative staphylococci (P = 0.002). For every additional week of TPN, there was a 7% reduction in killing of coagulase-negative staphylococci. These investigators also noted that there was a significant positive correlation between neutrophil count and killing of coagulase-negative staphylococci (r = 0.80, P = 0.001).The other two ving TPN ,67. ThesA total of 34 TPN complicated deaths were reported in five of the 13 clinical trials. Of these 34 deaths, 7 children died from SBS, 12 from sepsis, respiratory infections, and low immune deficiency, and other 15 from liver failure ,96-98.There were only three studies, retrospective and case study that showed the dependency on TPN in infants who had undergone resection of the small intestine -97. As rAlthough most children had difficulty being weaned from TPN in other studies, Bines and her co-workers  were ablIn addition, two other studies used enteral formulas while children were on TPN. Andorsky and colleagues  fed the In two separate clinical trials, researchers ,95 examiSystematic reviews focus on empirical studies and seek to summarize past research by drawing overall conclusions from many separate investigations that address related or identical hypotheses . The purNumerous experimental studies suggest that long-term TPN has harmful effect. Evidence in experimental studies about TPN's effects on mucosal immunity and villous atrophy is very convincing ,14; howeIn this review, bacterial translocation was reported only in one study ; it statIn a large critical review, Lipman  examinedPierro and colleagues  used thrThe importance of enteral stimulation on the mucosa-associated lymphoid tissue (MALT) system was studied in neonates who died soon after birth. Histological examination of the biopsies showed that infants who received enteral stimulation showed clear evidence of B cells and T cells within the mucosa; whereas, parenterally fed infants who died had a villous atrophy. Gut and bronchus samples were obtained and related to time of death of infants who died of sudden infant death syndrome between two weeks and 90 moths after birth. IgA plasma cells first appeared in the gut and later in the bronchi as the system matured. Plasma cells increased rapidly over time as IgA plasma cells predominated by three weeks in the gut and six weeks within the bronchi .In this review, none of these studies have tested whether TPN decreases S-IgA levels in intestinal mucosa causing increased bacterial translocation. Although there was only one study focused on the changes in IgA production during TPN in 8 healthy human volunteers , the resin vitro studies on infants and children demonstrate that TPN inhibits functions of neutrophils, cytokines, and bactericidal activity of phagocytosis [In this review ocytosis ,51,67. Iocytosis . Glucoseocytosis .There is a large body of evidence demonstrates that there is a positive correlation between high serum glucose levels and increased infection rates in acutely ill patients in critical care medicine. Insulin therapy seems to be beneficial in sepsis patients . RassiasSince its introduction, TPN has been a life saving nutrient to severely malnourished patients. It was believed that "if some nutrition is good, more is better" . Even inTwo randomized prospective clinical trials ,119 of aHyperglycemia is commonly seen in stressed patients during administration of TPN or other glucose-containing solutions. Stress may also induce insulin resistance in adipose tissue, liver and heart . HoweverSome studies reported that the complications during TPN were associated with the residual or the remaining bowel length in patients with SBS. The longer the residual small bowel, the shorter the duration of TPN, and the fewer infections ,96. The It has been suggested that enteral stimulation is a required component to protect gastrointestinal and respiratory immunity via increased levels of mucosal IgA ,121. EntIn Weber's study , 21 infaÂ® (Novartis Nutrition, Bern, Switzerland) supplemented with arginine, ribonucleic acid, and omega-3 fatty acids versus standard diet on outcome. Their results showed that early postoperative enteral infusion of nutrients is safe and well tolerated and stimulates an early return of bowel function.However, Daly et al.  performeimmunonutrition has been coined to describe molecular compound that, while being dietary components, such as glutamine, arginine, ω-3 fatty acids, ribonucleic acid, also influence immunologic response mechanisms when added to standard TPN solutions or enteral nutrition [There has been a considerable amount of interest in recent years in the use of alternative specific gut substrates. The term utrition -130. GluIn the late 1980s, glutamine has been the most extensively studied substrate in animal research, especially in rat TPN model. It has been suggested that parenteral or enteral glutamine supplements could dramatically effect on the maintenance of GALT; and glutamine-enriched TPN significantly attenuates the mucosal hypoplasia associated with prolonged TPN following massive bowel resection -134. TheIn the following years, many controlled trials and case series were conducted to demonstrate the benefits of immune enhancing substrates to the intestinal adaptation process in patients with SBS during long-term TPN -147. TheHowever, Scolapio and colleagues ,151 cond® (Novartis Nutrition, Bern, Switzerland) containing glutamine, arginine, dietary nucleotides and fish oil; and Immune-Aid (McGaw, Irvine, CA) containing glutamine, arginine, nucleic acids, and omega-3 fatty acids. Some studies indicate that these immune-enhancing diets demonstrated successful results or benefits in studies of sepsis, immunity, altered intestinal permeability, inflammation, including reduced mortality and the length of hospital stay [Currently, two commercially prepared immune-enhancing enteral formulas are available in the market, IMPACTtal stay -156.Some studies also show that the use of breast milk in SBS contains high levels of immunoglobulin A (IgA), nucleotides, leukocytes, and other components that bolster the neonate's immune system. In a stOverall, findings are inconclusive. Immunonutrition may be species or patient specific.The successful management of infants and children with SBS is demanding and requires a multidisciplinary approach. The care of patients with SBS can become so complex that many patients with SBS almost lose their primary physician and become patients of multiple specialists. Long-term care is, therefore, best delivered by a variety of healthcare professionals, including the gastroenterologists, surgeon, nurses, nutritional support team, care coordinators, microbiologist or infection control team, clinical chemist, pharmacists, social workers, and psychologist with expertise in infant feeding difficulties. Patients cared for in this way have better outcomes ,148,157.Infants go through a critical developmental phase in feeding at about 6â€“12 months, and those who do not receive oral feeds at that time will loose the window of opportunities for sucking and swallowing coordination, and will have long-term eating difficulties ,158. In The management of neonates and children with SBS continues to provide a major challenge for practitioners . A group2 blockers and proton pump inhibitors are effective for reducing gastric fluid secretion, and therefore, will also reduce fluid losses during this period [Hypersecretion is seen in infants and children during the initial first 6 months after surgery and a start of enteral feeding. The Hs period ,161.13C-xylose in children ages 3 to 12 years for breath hydrogen test [13C-xylose is a safe, nonradiactive isotope that has recently been developed (Martek, Columbia, MD). This study results showed that all patients with bacterial overgrowth had positive breath test results (100% sensitivity).The diagnosis of bacterial overgrowth is determined by aspiration of the jejunum and demonstration of increased bacterial contents by culture. Since this procedure is invasive and infeasible in some sick infants, breath hydrogen test can be used easily as noninvasive diagnostic test . The bregen test . 13C-xylFor infants prone to overgrowth, routine scheduled antibiotic treatment may be useful. D-Lactic acidosis has been reported in children with bacterial overgrowth, causing metabolic acidosis, drowsiness, and confusion. This diagnosis should be considered in a child with SBS who presents with metabolic acidosis, high serum anion gap, normal lactate level, and without urinary ketones . TreatmeOptimal timing of the initiation of enteral nutrition has not been established . PatientAlthough the optimal formula for feeding infants and children with SBS is not well established, elemental amino acid based hydrolyzed formulas are suggested by some clinicians and have been shown to be well tolerated . DietaryA prospective, randomized, cross-over trial compared two protein hydrolysate formulas given by continuous nasogastric infusion to six malnourished infants with SBS aged 1-13 months . AlthougA controlled trial compared two feeding regimens, continuous intragastric feedings and intermittent oral feeding, in nine infants with protracted diarrhea and malnutrition and two infants with surgically created short bowel . ContinuIn infants under 1 year of age with SBS who might have dilated gut, poor motility, or bacterial overgrowth, increased epithelial permeability to food antigens occurs frequently and may result in the development of allergic reaction to any protein in the formula. In order to reduce the risk of allergic injury to the gut, a hypoallergenic formula, such as Nutramigen, Pregestemil(Mead Johnson Laboratories, Evansville, IN), and Alimentum (Ross laboratories, Columbus, OH), should be used during the first year of life. To further decrease the risk of allergic reaction in highly susceptible infants, an amino acid based formula should be used. Neocate (SHS Inc, Rockville, MD, USA) is the only one listed in this category for infants. Elecare (Ross Laboratories), which is quite similar to Neocate, was formulated for use in children over 1 year of age .Initially slow introduction of continuous enteral feeding via a nasogastric or gastrostomy tube feeding is beneficial to reduce emesis, diarrhea, and maximally saturate carrier proteins. Rationales for this that constant saturation of mucosal digestive enzymes and transport carriers should be optimize absorption. Regardless of the length of small intestine, some oral, even if only 1 or 2 ml per day, and a few ml in continuous fashion should be offered from an early stage .A diluted infusion is initiated continuously at low volumes and increased to 0.67 cal/mL for infants less than 1 year of age or to 1.0 cal/mL in older children. Once the final concentration is achieved in low volumes, enteral feeding rates are advanced, and TPN rates are decreased isocalorically every 1 to 3 days as tolerated . A markeMonitoring patients on enteral feeding is required routine assessment of gastrointestinal, metabolic, mechanical, and growth parameters . ToleranA literature review conducted by Metheny and colleagues  found thEnsuring correct placement of the feeding tube is also necessary. Although auscultation is the most common method of confirming placement, it has been found unreliable. The best evidence for confirming correct tube placement is X-ray . MethenyAccording to American Gastroenterological Association (AGA) , and AmeFour major categories of complications exist: (1) mechanical or technical; (2) infectious; (3) metabolic; and (4) nutritional . MechaniStaphylococcus epidermidis, Staphylococcus aureus, and other skin flora are the most common pathogens isolated in patients with systemic infections. Enterococcus and enteric flora are the next most frequently isolated organisms. Catheter-related infections are treated based on the type of infection and pathogen [Catheter related infections are the most common complications associated with central line TPN . Localizpathogen .TPN is a life saving alternative nutritional support to severely malnourished surgical patients. Its use is indicated to prevent the adverse effects of malnutrition in patients who are unable to tolerate nutrients by oral or enteral routes. Evidence from a large systematic reviews involving 82 randomized control clinical trials on both adult and paediatric population showed that: 1) TPN did not have a significant impact on survival, 2) TPN did not affect either the total or infectious complication rates, 3) TPN had no effect on the duration of hospitalization of surgical patients . Based oShort bowel syndrome (SBS)Total parenteral nutrition (TPN).Immunoglobulin A (IgA)Secretory immunoglobulin A (S-IgA),Gut-associated lymphoid tissue (GALT)Gastrointestinal (GI)Mucosa-associated lymphoid tissue (MALT)Necrotizing enterocolitis (NEC)Migrating motor complex (MMC),2)Histamine2 (HNitric oxide (NO),Nitric oxide synthase Tumor necrosis factor (TNF)Interleukin-1 (IL-1)Interleukin-6 (IL-6)Adrenocorticotropic hormone (ACTH)Antidiuretic hormone (ADH),Free fatty acids (FFA),Pathogenic microorganism (PPM)Neutrophil polymorhonuclear (PMN)Prospective randomized clinical trials (PRCT)Intensive Care Unit (ICU)Nasogastric (NG)Gastrostomy tube (GT)Nasojejunal (NJ)Jejunostomy tube (JT)American Gastroenterological Association (AGA)American society for parenteral and enteral nutrition (ASPEN)Percutaneously inserted central catheters (PICCs)The author(s) declare that they have no competing interests.The pre-publication history for this paper can be accessed here:


High-throughput genotyping of single nucleotide polymorphisms (SNPs) generates large amounts of data. In many SNP genotyping assays, the genotype assignment is based on scatter plots of signals corresponding to the two SNP alleles. In a robust assay the three clusters that define the genotypes are well separated and the distances between the data points within a cluster are short. "Silhouettes" is a graphical aid for interpretation and validation of data clusters that provides a measure of how well a data point was classified when it was assigned to a cluster. Thus "Silhouettes" can potentially be used as a quality measure for SNP genotyping results and for objective comparison of the performance of SNP assays at different circumstances.We created a program (ClusterA) for calculating "Silhouette scores", and applied it to assess the quality of SNP genotype clusters obtained by single nucleotide primer extension ("minisequencing") in the Tag-microarray format. A Silhouette score condenses the quality of the genotype assignment for each SNP assay into a single numeric value, which ranges from 1.0, when the genotype assignment is unequivocal, down to -1.0, when the genotype assignment has been arbitrary. In the present study we applied Silhouette scores to compare the performance of four DNA polymerases in our minisequencing system by analyzing 26 SNPs in both DNA polarities in 16 DNA samples. We found Silhouettes to provide a relevant measure for the quality of SNP assays at different reaction conditions, illustrated by the four DNA polymerases here. According to our result, the genotypes can be unequivocally assigned without manual inspection when the Silhouette score for a SNP assay is > 0.65. All four DNA polymerases performed satisfactorily in our Tag-array minisequencing system."Silhouette scores" for assessing the quality of SNP genotyping clusters is convenient for evaluating the quality of SNP genotype assignment, and provides an objective, numeric measure for comparing the performance of SNP assays. The program we created for calculating Silhouette scores is freely available, and can be used for quality assessment of the results from all genotyping systems, where the genotypes are assigned by cluster analysis using scatter plots. High-throughput single nucleotide polymorphism (SNP) genotyping assays generate large amounts of data, which usually is presented as scatter plots of signals corresponding to the two SNP alleles. A robust SNP genotyping assay is characterized by large distances between the three clusters that define the genotypes and small distances between the data points within each cluster. Numeric quality measures for the scatter plots would allow objective and automatic assessment of the success of a SNP assay."Silhouettes" were introduced in 1987 as a general graphical aid for interpretation and validation of cluster analysis [In this study we created a program (ClusterA) to calculate numeric Silhouettes for assessing the quality of genotype clusters obtained in SNP genotyping assays. We show the utility of Silhouettes and the program by applying it to our "in-house" developed four-color fluorescence minisequencing system for SNP genotyping in a microarray format . Single We created a program, denoted ClusterA, for calculating numeric "Silhouettes" for clustered data, such as for example the three clusters of signal ratios commonly obtained in SNP genotyping assays. Figure Allele2/  is plotted, since this is the major determinant for genotype assignment in our system. The logarithm of the sum of the signals from both alleles  plotted on the y-axis is only used to set the cut-off values for failed genotype calls. Figure Here, we applied ClusterA to calculate "Silhouettes" for comparing the quality of the genotype clusters obtained in our "in-house" Tag-array minisequencing system. For each scatter plot, the mean of the average silhouette widths for the three genotype clusters were used to define a "Silhouette score" for each SNP assay. Thus the Silhouette score condenses the cluster quality for each SNP assay into a single measure that ranges from 1.0 to -1.0. When calculating the Silhouette score, the distance between data points can be measured either in one dimension, for example on the x-axis, or in two dimension using vectors, as illustrated in Figure The examples in panels E, F and G of Figure Here we exemplify the use of Silhouette scores by comparing the performance of the TERMIPol, Therminator, KlenThermase and ThermoSequenase DNA polymerases in the Tag-array minisequencing system . Twenty-Figure In the comparison between the enzymes, KlenThermase displayed the highest average Silhouette score, ThermoSequenase had the highest median Silhouette score and also obtained the highest Silhouette score most frequently Table . In addi. We believe that the ClusterA program for calculating Silhouette scores created in the present study is a useful and general tool for any genotyping system, where the genotypes are called by cluster analysis with the aid of scatter plots.We conclude that "Silhouette scores" for assessing the cluster quality is well suited for comparing the performance of SNP assays. Here we used a one-dimensional calculation of the Silhouette scores, by measuring the distances between the data-points along the x-axis only. A two-dimensional Silhouette calculation using vectors should be applied when genotypes are assigned by scatter plots with the fluorescence signals corresponding to the two alleles on the y- and x-axis. Both options are available in the ClusterA program that also calculates mean, variance and F-statistic for the input data set. The program is freely available through our website Genomic DNA was extracted from blood samples from 16 volunteer blood donors using the Wizard genomic DNA purification kit (Promega, Madison, WI).2-groups to form 80 subarrays per slide, each with 60 cTags as duplicate spots. Finally the microarray slides were scanned, and the fluorescent signals were measured.Twenty-six SNPs, selected to be located in unique PCR amplicons, were included in the test panel. For information on the single nucleotide polymorphisms and oligonucleotides used, see the Cyclic minisequencing reactions were performed in solution with 10 nM of each of the 52 tagged minisequencing primers using 0.1 μM ddATP-Texas Red, ddCTP-Tamra and ddGTP-R110 and 0.15 μM ddUTP-Cy5 (Perkin-Elmer Life Sciences, Boston, MA), and 0.064 U/μl of one of the four DNA polymerases in 15μl of 0.02% Triton-X, 4.1 mM MgCl2 and 33.6 mM Tris-HCl pH 9.5. The cyclic extension reactions were performed on a Thermal Cycler PTC-225 (MJ Research, Watertown, MA) with an initial 96°C for 3 min followed by 55 cycles of 95°C and 55°C for 20 s each. The DNA polymerases were; TERMIPol (Solis BioDyne, Tartu, Estonia), Therminator (New England BioLabs Inc., Beverly, MA, USA), KlenThermase (Gene Craft, Lüdinghausen, Germany), or ThermoSequenase . A custom made reaction rack holding the arrayed slides with a silicon grid to give 80 separate reaction chambers was used during capture of the minisequencing reaction products on the Tag-arrays.® instrument (Perkin-Elmer Life Sciences, Boston, MA). The excitation lasers were: Blue Argon 488 nm for R110; Green HeNe 543.8 nm for Tamra; Yellow HeNe 594 nm for Texas Red and Red HeNe 632.8 nm for Cy5. The fluorescence signal intensities were determined using the QuantArray®analysis 3.1 software (Perkin-Elmer Life Sciences, Boston, MA). The QuantArray file was exported to the SNPSnapper v4.0 software ) for genotype assignment. Raw data as fluorescence signals and signal ratios are provided as supplementary material, see Allele1+SignalAllele2) plotted on the y-axis, and the fluorescence signal fraction, obtained by dividing the fluorescence signals from one allele by the sum of the fluorescence signal from both SNP alleles , on the x-axis [The fluorescence signals were measured from the microarray slides using a ScanArray ExpressLL planned the experiments, guided the laboratory work and performed the analysis of results, interpreted the data and drafted the manuscript. AA carried out the laboratory work and part of the data analysis and provided input to the manuscript. MJ programmed the ClusterA program and took part in the interpretation of Silhouettes. ACS initiated the study, supervised it, and coordinated the manuscript writing process. All authors have read and approved the final manuscript.Lists the dbSNP identification numbers and the sequences of the PCR and minisequencing primers.Click here for fileIncludes the raw fluorescence signals and the fluorescence signal intensity ratios for the two experiments as a tab delimited text file.Click here for file


Mortality statistics are key inputs for evidence based health policy at national level. Little is known of the empirical basis for mortality statistics in China, which accounts for roughly one-fifth of the world's population. An adequate description of the evolution of mortality registration in China and its current situation is important to evaluate the usability of the statistics derived from it for international epidemiology and health policy.The Chinese vital registration system currently covers 41 urban and 85 rural centres, accounting for roughly 8 % of the national population. Quality of registration is better in urban than in rural areas, and eastern than in western regions, resulting in significant biases in the overall statistics. The Ministry of Health introduced the Disease Surveillance Point System in 1980, to generate cause specific mortality statistics from a nationally representative sample of sites. Currently, the sample consists of 145 urban and rural sites, covering populations from 30,000 – 70,000, and a total of about 1 % of the national population. Causes of death are derived through a mix of medical certification and 'verbal autopsy' procedures, applied according to standard guidelines in all sites. Periodic evaluations for completeness of registration are conducted, with subsequent corrections for under reporting of deaths.Results from the DSP have been used to inform health policy at national, regional and global levels. There remains a need to critically validate the information on causes of death, and a detailed validation exercise on these aspects is currently underway. In general, such sample based mortality registration systems hold much promise as models for rapidly improving knowledge about levels and causes of mortality in other low-income populations. Data on the causes, levels and patterns of mortality are critical to support the development of evidence-based health policy. Cause of death statistics represent the longest historical series of data on the health of populations, in some cases extending back well over 150 years , large sPrior to 1950 vital registration hardly functioned in China, and even then only yielded reports on causes of death for the cities of Beijing and Nanjing. The repIn 1976, a nationwide mortality survey was undertaken yielding information on the causes of about 20 million deaths .In brief, the system functions as follows. When a person dies, family members report the death to the registration office of the nearest township hospital in order to get a death certificate, then to the police station to deregister permanent residence and obtain ratification for the burial procedure. Staff in the vital registration office fill in the death certificate based on information from family members, and available medical records or documents. One copy of the death certificate is kept in the registration office, and another is sent to the county Center for Disease Control (CDC), where the cause of death is coded. At the time of inception of the coding system in 1987, a specially designed Chinese Classification of Diseases List consisting of over 500 specific diseases or injuries was used, which could be translated into codes from the Ninth Revision of the ICD [The county CDC then uses a prescribed summary tabulation format to submit monthly reports of deaths by age, sex and cause to the Center of Health Information and Statistics, a Department within the Ministry of Health. This is known as the Ministry of Health – Vital Registration (MOH-VR) system, the annual compilations of which are submitted to the World Health Organization and published by WHO. Since 1996, several cities have started producing local tabulations for internal use.Quality control measures, including training of staff and development of guidelines and regulations for death registration vary between different areas. Registration in cities is better than in counties, and is better in eastern than western areas. An annual review of data is conducted, and areas that reported implausibly low death rates are excluded from statistical tabulations produced by the MOH-VR system.A good test to assess the quality of the vital registration data is to examine trends in cause-specific death rates . For exaAn additional limitation of the data system is that birth registration was not included. This undoubtedly contributed to the implausibly low death rates reported for infants in both the MOH-VR data and the DSP system Table , since tMore importantly, the coverage of the MOH-VR system is biased towards the more urban and better-off populations of eastern China. Death rates from infectious disease are lower in the MOH-VR system than those reported by the more representative DSP system, which includes populations in poor rural areas Figure . SimilarIn order to improve the usability of data from the vital registration system, the Peking Union Medical University/Chinese Academy of Medical Sciences put forward a proposal in 1978 to develop a sample based Disease Surveillance Point (DSP) system. The system was designed primarily to collect data on births, causes of death, and the incidence of infectious diseases. A pilot study was carried out in East Town and Tongxian counties of Beijing in 1978. The Ministry of Health then instructed departments of health at province and county levels to recognize disease surveillance as an important public health task, and set up disease surveillance points under the technical guidance of the Chinese Academy of Preventive Medicine. By 1989, there were 71 DSPs scattered throughout 29 provinces in the country, with a standard working procedure for data collection, management, analysis and dissemination. However, the system was not representative of the national population . In 1990Based on the principle that the characteristics of the population under surveillance should be similar to that of the general population in different geographic areas, a multi-stage cluster probability sampling was designed with stratification at three levels.The first level of stratification was according to 7 geographic regions  and 3 municipalities (Beijing, Tianjin and Shanghai) in China. The second level was based on the urban and rural location of primary sampling units. Within rural areas, a third level of stratification was based on a classification of rural sites into four socio economic strata, based on the 1982 Census returns about average levels of variables such as literacy rates, GDP per capita, and dependency ratios. Also, urban areas were re-classified according to population size into big cities, with over 1 million population, middle sized cities with 0.5 -1 million population and small cities with 0.2–0.5 million population.The primary cluster unit in urban areas was the city, and in rural areas, the county. Probability proportionate to population size sampling (PPS) was used to select a city or county, using 1982 Census data. In the second stage cluster, in selected cities or counties, the unit of sampling was a 'neighborhood' (Jiedao) within cities, or 'townships' (Xiang) in rural areas. Both the 'Jiedao' and the 'Xiang' represent a community with a primary government, with a population ranging from 30 000 – 100 000. PPS sampling was again used for selection of units at the second stage, such that the probability of selection was according to population size of the neighborhood or township The resuA population of about 10 million resides in the areas covered by the system (a little under 1% of the Chinese population). Based on national data on public health indicators used for stratification, the selected DSP sites are representative of the national population, and theSince 1990, the system has covered natality, mortality, and the incidence of 35 notifiable diseases. In this section, we describe the process of mortality registration within the DSP system, and comment on aspects regarding quality control of data, particularly with respect to completeness of reporting, and the use of the data for public policy.In each DSP site, there is at least one township hospital, and the 'Disease Prevention Unit' in these hospitals is responsible for vital registration. The detailed working procedure for mortality registration is described in the guidelines for surveillance in the DSP. In urbaIn rural areas, about 80% of adult deaths occur at home, with few occurring at the township hospital, or other tertiary hospitals in the vicinity. Even for those deaths that occur at home, there is often clinical evidence available from recent consultations with medical staff at township or other hospitals. The procedure for collection and compilation of cause of death data is as follows:• For deaths occurring at home, a village health worker reports the event to the Prevention Unit at the township hospital. A staff member from the Unit visits the household, and completes a death certificate based on a description of symptoms from family members, and available documents from recent contact with health services.• For deaths occurring in the township hospital, the DSP staff collect the death certificate from the hospital, completed by the physician who attended the death.• For deaths occurring in other hospitals, relatives of the deceased submit physician-certified death certificates to the Prevention Unit at the township hospital.• In the event of a childhood death, or deaths in women of maternal age, the Maternal and Child Health Unit at the township hospital undertakes the investigation of the cause of death, and screens death certificates for such deaths from other hospitals for accuracy.• Data cleaning and compilation is done at the county or provincial level, and following computerization, an electronic data-file is transferred to the Chinese Academy of Preventive Medicine.• ICD coding of the underlying cause of death and subsequent tabulation and publication of results is done at the central level in Beijing. Annual reports of deaths by causes, age and sex have been published in Chinese by the Chinese Academy of Medical Science since 1990, and a public access website for these data is currently under development.There are instances where the above procedures are not strictly adhered.to. In situations where there is a delay in the household investigation by the Prevention Unit staff, family members or neighbours visit the unit to deregister the residential status of the deceased, and obtain the necessary documentation for corpse disposal and other legal purposes. Such instances can promote improper assignment of cause of death in individual cases, since the respondents in these cases may not be familiar with the disease and related conditions experienced by the deceased.Within the DSP system, there are two methods employed for controlling data quality. The first is an internal procedural check system, which evaluates timeliness of death registration, completeness of entries in the registration form, and the accuracy of data entry. Errors detected from these checks are corrected through re- enquiry, and enhance the usability of the datasets.At a second level, the datasets are evaluated using statistical measures. The completeness and accuracy of population enumeration in the DSP has been evaluated using the standard United Nations Age Sex Accuracy Index , and theWhile, there is no mechanism for evaluating the completeness of death registration in the MOH-VR system, the DSP evaluates completeness of both birth and death registration. This is done through independent resurveys, and statistical techniques based on "capture – mark – recapture" methods are used to estimate the completeness of registration . These sResults from three such surveys conducted in 1992, 1995 and 1998 are presented in Table In this paper, we have described in detail for the first time in English, the Disease Surveillance Points System that operates in China, and provides critical information on the health of one-fifth of the world's population, from a sample of less than 1 % of the Chinese population. This is a remarkable achievement, and perhaps the most cost-effective system of data collection to inform health policies and programs ever devised. Yet, the performance, even the existence of the system is not widely appreciated outside of China, despite its obvious implications for rapidly improving knowledge about causes of death in several other low income populations.Undoubtedly, complete vital registration of deaths with full medical certification is the most appropriate means to monitor the health of populations, but to establish, and particularly to maintain such a system will be outside the realms of possibility for most developing countries for decades to come. Meanwhile, novel, affordable and sustainable approaches to data collection on mortality that is representative of populations are required. The Chinese DSP system described in this paper has many advantages. Almost all countries conduct censuses at least every ten years, and hence the socio-demographic information on which to select a representative sample of surveillance sites is available. Adequate information can be obtained from relatively small samples (≈1% in China and India), and substantial progress has been made with the development of 'verbal autopsy' instruments and procedures to have sufficient confidence in the utility of cause of death data that they produce, at least for broad causes of death. While this may not be sufficient for specific disease or injury control programmes, field experience in Tanzania suggests that the data are useful for determining the need for priority health programs .The data from the DSP have been used to monitor the emergence of tobacco-caused mortality in China, and to The authors are currently undertaking such a validation study based on a sample of 2900 deaths in six cities and 3500 deaths in nineteen rural counties in China. In the urban sites of the study, two arms of the project are being implemented. Firstly, medical records of the sampled deaths are being reviewed to develop a reference 'gold standard' diagnosis of the underlying cause of death, using the international form of medical certificate of cause of death. For the same deaths, the diagnosis from the routine registration system is compared and validated against this reference diagnosis, to assess the validity of the routine system. Secondly, for each of these deaths, a verbal autopsy interview was conducted to derive a cause of death, and this will be compared with the reference diagnosis to establish the validity and operational characteristics of the verbal autopsy procedure to be used in the DSP system.In rural areas, the same standard procedures for verbal autopsy are being introduced, and diagnoses from these standard procedures will be compared with the diagnoses from the routine registration system to measure the reliability of cause of death ascertainment in rural China.It is envisaged that the results from these studies, as well a proposed under-reporting survey in 2005, will enable correction of datasets from mortality registration systems in China to improve knowledge of cause specific mortality at the population level. The research will also provide the evidence base to strengthen mortality registration in China by identifying structural weaknesses and areas for development, which will minimize undercount and misclassification of deaths in the future. In addition, this evaluation research will build capacity that will result in long term improvements in data quality from the DSP, given the recent changes in the funding, management and coordination of activities within the system. In particular, opportunities to build on existing networks, such as the family planning services system, will need to be more effectively exploited in future to accelerate the implementation of vital registration nationwide.


We compared the effect of a novel NO-releasing derivate, NCX 4040, with that of aspirin and its denitrated analog, NCX 4042, in In vitro cytotoxicity was evaluated on a panel of colon cancer lines (LoVo, LoVo Dx, WiDr and LRWZ) by sulforhodamine B assay. Cell cycle perturbations and apoptosis were evaluated by flow cytometry. Protein expression was detected by Western blot. In the in vivo experiments, tumor-bearing mice were treated with NCX 4040, five times a week, for six consecutive weeks.in vitro studies, aspirin and NCX 4042 did not induce an effect on any of the cell lines, whereas NCX 4040 produced a marked cytostatic dose-related effect, indicating a pivotal role of the -NO2 group. Furthermore, in LoVo and LRWZ cell lines, we observed caspase-9 and -3-mediated apoptosis, whereas no apoptotic effect was observed after drug exposure in WiDr or LoVo Dx cell lines. In in vivo studies, both NCX 4040 and its parental compound were administered per os. NCX 4040 induced a 40% reduction in tumor weight. Conversely, aspirin did not influence tumor growth at all.In the in vitro and in vivo antiproliferative activity, indicating its potential usefulness to treat colon cancer.NCX 4040, but not its parental compound, aspirin, showed an  One of the most important approaches for reducing cancer incidence is chemoprevention. This is especially relevant for colon cancer, which represents the third leading cause of cancer mortality in developing countries and for which diagnostic tests and clinical treatments are not satisfactory .Recently published reviews -4 underlDespite a multiplicity of studies conducted on this compound family of drugs, little is known about the molecular targets that are responsible for their tumor-preventing properties and which are important, not only from a mechanistic point of view, but also because of their clinical implications in identifying individuals or subsets of patients who are sensitive to these drugs. Originally, it was believed that the regular use of these compounds acted exclusively through the inhibition of COX-1 and COX-2 activity involved in the production of prostaglandins . There iIn an attempt to reduce gastrointestinal toxicity, conventional NSAIDs have been coupled with a nitric oxide (NO)-releasing moiety ,16. The in vitro [Interestingly, the activity of some NO-NSAIDs is considerably higher than that of the parental drugs. For example, NCX 4016 has been found to be much more effective than the parent aspirin in reducing aberrant crypt foci, a precancerous lesion, in a rat model of colorectal cancer  and in iin vitro . Similarin vitro ,23, in ain vitro and in vivo human colon cancer models and investigated the mechanisms of action underlying its antitumor activity.In the present work, we compared the effect of a novel NO-releasing derivate, NCX 4040, with that of aspirin and its denitrated analog in The studies were performed on colon adenocarcinoma cell lines: 2 (LoVo and WiDr) were obtained from the American Type Culture Collection (Rockville, MD), 1 (LRWZ) was isolated in our laboratory and derived from a patient with a confirmed diagnosis of colon adenocarcinoma, and 1 was doxorubicin-resistant (LoVo Dx) derived from the above-mentioned LoVo cells. Cell lines were maintained as a monolayer at 37°C and subcultured weekly. Culture medium was composed of DMEM/HAM F12 (1:1) supplemented with fetal calf serum (10%), glutamine (2 mM), non-essential aminoacids (1%) , and insulin (10 μg/ml) . Cells were used in the exponential growth phase in all the experiments.Aspirin, NCX 4040 (NO-releasing aspirin), NCX 4042  Fig. ,  assay was used according to the method by Skehan et al . BrieflyThe cells were treated according to the previously described Western blot procedure.  COX-1 anTotal RNA was isolated from cells by direct lysis and quantified spectrophotometrically. After quantification, RT reaction was performed with 1 μg of each sample using Gene Amp Gold RNA PCR Core Kit . The same cDNA was used for RT-PCR amplification of COX-1 and COX-2. PCR reactions were carried out in a final volume of 25 μl containing 2 μl of cDNA template, 1 unit of Klen Taq, 0.2 mM dNTP and 0.4-μM amounts of each forward and reverse primer, using a thermal cycler . 5-μl aliquots of the amplified DNA fragments were separated on an ethidium bromide-stained 2% agarose gel.After a 24-h exposure to 10 μM of NCX 4040, cells were harvested and stained in a solution containing RNase  and NP40 . After 30–60 min, samples were analyzed by flow cytometry using a FACS Vantage flow cytometer. Data acquisition (10,000 events were collected for each sample) was performed using CELLQuest software. Data were elaborated using Modfit (DNA Modelling System) software and expressed as fractions of cells in the different cycle phases. Samples were run in triplicate, and each experiment was repeated three times.Apoptosis was evaluated by flow cytometric analysis according to the previously described TUNEL assay procedure . BrieflyIn situ cell death detection kit, fluorescein; Roche Diagnostic GmbH, Mannheim, Germany).In LoVo cells, positivity to TUNEL assay was also evaluated by fluorescence photomicroscope (Zeiss, Axioscope 40) according to the manufacturer's instructions  were used to visualize chromatin condensation and/or fragmentation typical of apoptotic cells.Antitumor efficacy was evaluated on 6–8-week old CD-1 male nude (nu/nu) mice weighing 22–24 g . All procedures involving animals and their care were conducted in conformity with institutional guidelines, which are in compliance with national  and international laws .NCX 4040 and aspirin were homogeneously suspended in 0.5% carboxymethyl cellulose (CMC) containing 10% of DMSO for antitumor efficacy studies. All the drugs were administered orally.in vivo experiments were performed on WiDr, LoVo and LoVo Dx lines. Tumor cells were resuspended (5 × 106 viable cells) in 0.2 ml of serum-free medium and injected into the hind leg muscles of mice.Since LRWZ line showed a low tumorigenicity when cells were injected into nude mice, th day after the tumor cell implant, when a tumor mass of about 300 mg was evident in all the animals. The dose of aspirin and NCX 4040 was chosen on the basis of previous observations obtained using the parental NCX 4016 compound [T – C, where T and C are the median times for treated and control tumors, respectively, to achieve equivalent size.Each experimental group included at least 6 mice. 10 mg/kg of aspirin and NCX 4040 were administered orally five days a week for 6 consecutive weeks starting on the 6compound . Toxicitcompound . The antThe significance of results was analyzed by the Mann-Whitney non parametric test. Differences were considered significant at P values < 0.05 (two-sided).50) ranging from 5.4 μM in LoVo cells to 24 μM in LoVo Dx. The same treatment scheme also induced the highest cytocidal effect, with a modulation for the different cell lines and a 50% lethal concentration (LC50) in LoVo cells (9.6 μM) about four- and threefold lower than that observed in WiDr and LRWZ, respectively. In LoVo Dx, no cytocidal effect was observed at any of the concentrations tested.The effect of aspirin, NCX 4040, and its structural analog, NCX 4042, on cell growth was determined after different exposure schemes and using various concentrations. Aspirin and NCX 4042 did not exhibit an effect on any of the cell lines Fig. . In partFlow cytometric analysis of apoptosis performed after exposure to various concentrations of NCX 4040 showed 90% of apoptotic cells at 24 h starting from a 10-μM concentration in LoVo line, whereas apoptosis was not detected at the same time in any other cell line at any of the concentrations tested , these proteases were activated only in LoVo and in LRWZ cells, where apoptosis was found. In particular, in LoVo cells caspase-3 and -9 activation was already evident after 3 h, whereas in LWRZ, activation took a longer time to occur (at least 10 h for caspase-9 and 12 h for caspase-3). The temporal activation in these 2 cell lines corresponds to the early or late induction of apoptosis Fig. .in vitro data, which demonstrated that LoVo line is the most sensitive to NCX 4040 exposure.The administration of aspirin in mice bearing all 3 cell lines did not influence the growth of tumors Fig. . In contin vitro on a panel of human colon cancer lines with different genetic profiles representing clinical tumor heterogeneity, and in vivo on xenografted immunosuppressed mice.Our study evaluated the antitumor activity of the novel NO-aspirin NCX 4040 2 group in the mechanism of action underlying its in vitro activity. Extensive evidence indicates that nitric oxide is capable of initiating apoptotic cell death in some cell types as a consequence of DNA-induced damage [Neither aspirin nor the denitrated analog, NCX 4042, influenced the growth of any of the cell lines studied, whereas NCX 4040 produced an important cytocidal effect. These findings suggest a pivotal role of the -NOd damage ,30, but In LRWZ cells, a relevant apoptosis was also observed but after a longer exposure time and at higher NCX 4040 concentrations, after which the irreversibly damaged cells underwent apoptotic death through the activation of caspases-9 and -3.Moreover, two NO donors were used to better clarify the role of the aspirin component. These compounds, at concentrations similar to those utilized for NCX 4040, showed a very modest cytotoxic activity (data not shown), confirming the importance of the aspirin component for the antineoplastic effectiveness of the NCX 4040 molelcule.In addition, WiDr and LoVo Dx cell lines, which proved to be resistant to NCX 4040-induced apoptosis, were characterized by baseline expression of COX-2 protein. This finding, which is consistent with data recently published for breast and colorectal cancer cells, would seem to support the hypothesis that a high level of COX-2 protein protects cells from apoptosis, especially that induced by NO-compounds ,32.in vitro studies showed a high tumorigenic capacity, making it possible to evaluate in vivo the antitumor efficacy of NCX 4040. The long-term administration of this drug was well tolerated by the mice and reduced the tumor mass of LoVo, LoVo Dx and WiDr lines compared to untreated mice. Analysis of growth curves highlighted differences in the degree of drug sensitivity between cell lines. NCX 4040 was very effective in delaying tumor growth in LoVo tumors, whereas WiDr, and especially LoVo Dx lines, regrew after the end of treatment. These results confirm the in vitro data and seem to indicate that, as observed in vitro, the ability of NCX 4040 to induce apoptosis has a key role in the antitumor efficacy of this drug.With the exception of LRWZ, all the lines evaluated in in vitro and in vivo, whereas the parental compound aspirin showed no activity, which suggests that the NO-releasing derivate could prove useful in the clinical management of colon cancer, possibly in combination with conventional antineoplastic drugs.In conclusion, NCX 4040 was effective in reducing the growth of several human colon cancer cell lines both COX, cyclooxygenase; NO, nitric oxide; NO-NSAID, nitric oxide-releasing nonsteroidal antiinflammatory drug releasing; NSAID, nonsteroidal antiinflammatory drug; SRB, sulforhodamine B; TUNEL, terminal uridine nick end labeling.Manlio Bolla is an employee of NicOx SA.in vitro experiments. CL, MS and GZ also participated in the study design and carried out the in vivo experiments. All authors read and approved the final manuscript.AT was responsible for study design, data analysis, and drafting the manuscript. WZ, DA and MB participated in the study design and acted as scientific advisors. AT, PU, FF and MR performed the


The birth of agriculture, some 10,000 years ago in the Middle East's Fertile Crescent, revolutionized human culture and society. Refined farming techniques led to increased yields and freed humans from the demands of constant foraging. Along with that freedom came social complexity, division of labor, improved standards of living, and a measure of leisure time. Agriculture also led to overpopulation followed by starvation, conflict over fertile farming land, and environmental damage. For the Maya and other civilizations, such consequences proved fatal.Many consumer and environmental groups believe that modern industrial agricultural practices like factory farming of animals and genetic engineering of crops threaten to bring similar ruin. But with 6 billion people living on the planet—a figure that's expected to increase 50% in just 50 years—many plant scientists believe that feeding a burgeoning population will require the tools of biotechnology. Plant breeders face the daunting challenge of developing high-yielding, nutritious crops that will improve the global quality of life without harming the environment or appropriating dwindling natural habitats for agricultural production. A major roadblock to feeding the world is a continuing decline in the genetic diversity of agricultural crops, which has in turn limited their yield improvement. (Domestication often involves inbreeding, which by definition restricts the gene pool.) Now Amit Gur and Dani Zamir of Hebrew University report a way to lift these productivity barriers by tapping into the natural diversity of wild plants.Traditional plant breeders improve the quality and yield of crops by crossing plants with desired traits to create a new, hopefully improved, hybrid strain. But traditional breeding is limited by the available gene pool of a cultivated plant species and eventually hits a wall—reshuffling the same genetic variation can boost yield only so much. With the advent of biotechnology, plant scientists were buoyed by the prospect of improving plants through genetic modification. But aside from a few successes with introducing single-gene herbicide- and pest-resistant traits, most plant traits have proved too complex to repay the incorporation of a single transgene—that is, a gene taken from a different species—with the hoped-for response. Biotech-based investigations and applications in plant science have also been hampered by consumer reaction against genetically modified organisms. Faced with these limitations, Gur and Zamir tried another approach—a back-to-nature approach. “Natural biodiversity is an unexploited sustainable resource that can enrich the genetic basis of cultivated plants,” they explain in the report. The distantly related wild cousins of cultivated plants can be seen as a “huge natural mutagenesis resource” with novel gene variants that can increase productivity, quality, and adaptability. Not only that, the genetic material of wild plants—every gene and regulatory element—has already been refined and tested by over a billion years of evolution and natural selection.To identify genomic regions in wild tomato species that affect yield, Gur and Zamir created a population of hybrid crosses of a wild tomato species and a cultivated tomato species; each line had a single genomic region from the wild tomato inserted into the cultivated plant. Rather than introducing a single wild tomato gene into the cultivated plants, the authors used a “pyramided” strategy that combined three independent yield-enhancing genomic regions from the wild species into the new plant line. Plants were grown over three seasons, during which they were exposed to different environments, including drought. By combining traditional phenotyping techniques—which characterize the plant's physical traits based on its genetic makeup—with genetic marker analysis, the authors identified a number of wild tomato genomic regions that increased yield.Their results demonstrate that an approach based on biodiversity—which takes advantage of the rich genetic variation inherent in wild relatives of cultivated crops—can produce varieties that outperform a commercially available hybrid tomato in both yield and drought resistance. Gur and Zamir attribute the improved performance to their unique pyramiding strategy. Their hybrid model—applying the tools of modern genomics to traditional plant breeding—offers plant breeders a powerful approach to improving the quality and yield of cultivated plants by taking advantage of the inherent biodiversity of the natural world. It's a strategy that may well apply to rice, wheat, and other vital staples of the world's food supply.


The relapsing nature and varying geographical prevalence of ulcerative colitis (UC) implicates environmental factors such as diet in its aetiology.In order to determine which foods might be related to disease activity in UC a new method of dietary analysis was developed and applied. Eighty-one UC patients were recruited at all stages of the disease process. Following completion of a 7 d diet diary, clinical assessment including a sigmoidoscopic examination  to 6 (very active disease)) was conducted. Food weights for each person were adjusted (divided) by the person's calorific intake for the week. Each food consumed was given a food sigmoidoscopy score (FSS) calculated by summing the products of the (adjusted) weight of food consumed and sigmoidoscopy score for each patient and occurrence of food and dividing by the total (adjusted) weight of the food consumed by all 81 patients. Thus, foods eaten in large quantities by patients with very active disease have high FSSs and vice versa. Foods consumed by <10 people or weighing <1 kg for the whole group were excluded, leaving 75 foods.High FSS foods were characterized by high levels of the anti-thiamin additive sulfite (Mann-Whitney, p < 0.001), i.e. bitter, white wine, burgers, soft drinks from concentrates, sausages, lager and red wine. Caffeine also has anti-thiamin properties and decaffeinated coffee was associated with a better clinical state than the caffeine containing version. Beneficial foods (average intake per week) included pork (210 g), breakfast cereals (200 g), lettuce (110 g), apples and pears (390 g), milk (1250 ml), melon (350 g), bananas (350 g), bacon (120 g), beef and beef products (500 g), tomatoes (240 g), soup (700 g), citrus fruits (300 g), fish (290 g), yogurt (410 g), cheese (110 g), potatoes (710 g) and legumes (120 g).The dietary analysis method described provides a new tool for establishing relationships between diet and disease and indicates a potentially therapeutic diet for UC. Ulcerative colitis (UC) is a chronic, relapsing mucosal disorder that extends in continuous fashion proximally from the rectum and is limited to the colon. The aetiology of UC includes a genetic component possibly involving an abnormal cell-mediated immune response to commensal enteric bacteria in the large intestine. The relapse/remission pattern of the disorder and substrate driven nature of microbial metabolism in the large bowel implicate environmental factors such as diet.Apart from nutritional repletion, dietary measures do not play a role in the management of UC. Nonetheless, attempts to link the cause of UC with specific foods date back at least 50 years. Many foIt has been proposed that sulfide, produced in the large bowel from either amino acid fermentation or sulfate reduction, may be a triggering factor in the inflammatory process of UC -16. ReceThe main source of inorganic sulfur, predominantly sulfate, in the diet are the S (IV) family of additives; the sulfiting agents. Sulfites have been used as food preservatives since the 17th century and are amongst the most widely accepted and versatile of additives. Sulfiting agents, denoted by E220–228 in Europe and generally recognized as safe (GRAS) substances in the USA, include sulfur dioxide, hydrogen sulfites, sulfites and metabisulfites. Sulfiting agents are cheap, easy to use and extremely effective at preventing microbial growth and reducing spoilage. They seHowever, there are some problems with sulfite use,20. In tA study of diet and disease activity in UC using a 7 d dietary diary, a full assessment of disease activity and a method of dietary data analysis that allows trends in food consumption not apparent using customary dietary software was therefore undertaken.Eighty-one UC patients were recruited and informed consent obtained. Ethical permission was granted by Tayside Committee on Medical Ethics, Dundee, UK (ref 007/00). As it was important to have a range of disease activities present, recruitment included patients at all stages of the disease. Patients were excluded if clinical examination or histology indicated Crohn's disease or indeterminate colitis, if there was a positive stool culture for pathogens or if the patient had antibiotic treatment within 3 months preceding the start of the study.All the UC patients were asked to complete a 7 d diet diary. The die7d diet diaries were coded and analyzed using Tinuviel, WISP v3.0 nutritional analysis software (Warrington, UK). Due to the variation in the sulfiting protocols and widespread use of sulfiting agents, current tables of food composition do not contain inorganic sulfur values and cannot be used to quantify intake. Instead of quantitating the intake of particular dietary components, foods and food groups were assessed in their entirety using the method described in the dietary data analysis section (below).Clinical assessment included history, physical examination and global clinical grading, plus full blood count, liver function tests and inflammatory markers. Patients were examined by rigid sigmoidoscopy or flexisigmoidoscopy and graded on a scale 0–6  according to the macroscopic appearances of the rectal mucosa at a distance 5–10 cm from the anal verge. The cliPatterns of dietary intake associated with disease activity became apparent through the study of the dietary diaries, e.g. high intakes of sulfite containing foods coupled with a modern processed, convenience diet was associated with a high sigmoidoscopy score. Traditional dietary coding (WISP) did not show any such clear associations between micro or macro nutrient intake and sigmoidoscopy score. Traditional dietary analysis was therefore thought to be missing important patterns in dietary data and a new method of dietary assessment was subsequently developed.This new method used the following procedure. To calculate the association of a particular food with clinical score, each food or food group consumed was given a food sigmoidoscopy score (FSS) calculated by summing the products of food weight and sigmoidoscopy score for each occurrence of the food or food group and dividing by the total weight of the food or food group contained in all diaries. In order for each diary to make equal contributions to the FSSs, the weight of each food was adjusted using the calorific intake for each person. This procedure was carried out separately for every food item recorded in the 7 d diet diaries but is explained below using the example of red wine.Red wine score = (Σv(i)s(i))/Σv(i) for i = 1 to 81     equation 1.Where: -i is the 7 d dietary diary number (n = 81).v(i) is the volume  of red wine recorded in 7 d dietary diary i.s(i) is the sigmoidoscopy score associated with 7 d dietary diary (i).Thus foods eaten in large quantities by patients with high levels of disease activity will have high scores and vice versa. The denominator in the above equation is the total volume of the food in question from all diaries  so the food scores can be equated with the effect of a typical portion of the food in question on the sigmoidoscopy scores of the patients. This procedure is repeated for every food item. Foods or food groups were excluded from the analysis if 10 or fewer people consumed them or if they made up less than 1 kg of the total intake of the entire population. The decision as to where food group boundaries lay was made depending on the size of the group and whether the differences between the foods were considered important for this study.Dietary data was exported from WISP to Microsoft EXCEL 98 (Macintosh version, 1998). A worksheet containing the core headings; Patient ID, food description, weight and patient sigmoidoscopy score was completed. The data was then sorted by food description and each food copied to a separate EXCEL file. Equation 1 was then used to calculate food sigmoidoscopy scores for each food in a manner similar to the example in table 2 see .2)) combined with t tables provided corresponding significance levels.Correlation values for scatter plots were obtained using the linear regression function in EXCEL. The equation t = r √((n-2)/(1-r2 = 0.25 (n = 81).Of the 81 patients recruited 43 were male and 38 female. The average age (range) of the males and females were respectively 53 (26–78) y and 47 (19–74). The distribution of sigmoidoscopy scores is shown in fig Table 3 see  shows thThe dietary diaries were assessed for completeness by comparing calorific intakes with expected values for the sexes. Expected  versus a2 = 0.07, p < 0.02).Foods for which regulations exist in the EU permitting sulfite addition are shown in table 4 see  29]. Ty. Ty29]. Decaffeinated coffee appeared better for the UC patient than the caffeine-containing counterpart. Decaffeinated tea is not shown on table 3 because it was only drunk by 9 people but had a FSS of 1.71 versus 2.01 for the caffeine-containing product. Whole fruit consumption appeared better than the corresponding juice (e.g. fruit juice scored 2.43 compared to citrus fruits at 1.96 and apples at 1.67).An average thiamin concentration (mg / 100 g) for each food or food group is also shown in table 3. There is a significant correlation (p < 0.005) between this thiamin value and the food's sigmoidoscopy score.Ulcerative colitis is considered to have a genetic component. Twin studies have shoOf all the dietary components studied in relation to UC risk and disease severity, milk has probably received the most attention. Andreson was the In order to ascertain whether dietary antigens may sustain the mucosal inflammatory response, two prospective controlled trials have investigated the effectiveness of bowel rest and total parenteral nutrition as primary therapy in the management of acute UC,36. NeitThe dietary analysis procedure proposed here has the potential to highlight trends in dietary data that would not be apparent using traditional dietary analysis software and could be useful in the study of other diseases with dietary associations. This system would highlight any possible dietary factors both positive and negative, not just sulfite. The proposed method is less reductionist than traditional coding as it assesses the risk of each food item or group rather than the risk from the foods' (quantitated) constituents. Part of the power of this study derives from the availability of a sigmoidoscopic grading (0–6) of the severity and extent of the disease. This grading provides the statistical variable that is normally obtained from a non-UC control group. Other alternative systems for analysis of disease risk for dietary components are; the use of disease occurrence odds ratios between the top and bottom quartiles of intakes, and assessing the correlation coefficients between disease activity and intakes. The odds ratio method loses data and data accuracy by characterizing intakes as high, high middle, low middle and low and then discarding the middle two quartiles. The correlation method is dependent on spread. The proposed system has neither of these disadvantages. The food sigmoidoscopy score calculation does rely on the assumption that the sigmoidoscopy score is an approximately linear scale, i.e. a sigmoidoscopy score of 6 is caused by the consumption of a double portion of a harmful food item of sigmoidoscopy score 3. This could be argued to be reasonable. Both the sigmoidoscopy grading and dietary analysis method are validated methodologies. The food sigmoidoscopy score is simply a mathematical function of these two variables. As all data is transformed according to the same simple rules any statistical treatment of the results is as valid as statistical treatment of the raw data.Whilst clinical activity indices were used to generate analogous scores to the food sigmoidoscopy scores, the results from these measurements are not included in this paper. Clinical activity index involves subjective measurements such as a feeling of well being. Thus, the food orders generated by these measurements were not thought to be as accurate as those generated by the sigmoidoscopy scores.The consensus of previous studies on diet and UC pointed to the modern, processed, highly refined, Western diets as being damaging. The results presented here linking diet with disease activity are broadly in agreement with this. Additionally they propose a new risk factor for UC, namely intake of sulfited foods.The involvement of diet in UC is controversial. Differences in dietary intake between patients and controls could be a result of changes in diet brought on by the symptoms of the disease process. While tet al. demonstrated that the thiamin status of adult humans depends on carbohydrate intake[Alternatively, the relevance of sulfite to UC may be because of its ability to degrade thiamin (particularly at colonic pH). Thiamin deficiency manifests itself in the nervous and cardiovascular systems. It is unlikely that it is the status of the patient that is important, but rather the amount of thiamin available to the gut microflora. An example of the importance of thiamin to the gut microflora is the requirement of the probiotic bacteria, lactobacilli, for thiamin. Thiamin status is influenced by a number of factors. Firstly, thiamin intake; in foods such as pork, fortified cereals and legumes which are good sources of thiamin, intakes were associated with improved clinical state. Traditional dietary analysis did not reveal a significant correlation between thiamin intake and sigmoidoscopy scores though no allowance is made in dietary coding software for the reduction in thiamin content caused by sulfite usage. Secondly, carbohydrate intake; Elmadfa te intake. CarbohyHowever, there was a sub group (n = 8) of this population who recorded an intake of either vitamin B complex or multivitamins. This sub group did not have a mean sigmoidoscopy score significantly lower that the general UC population. It is likely that vitamin B1 is a factor in the disease process but not the only nutritional one.An additional possible interpretation for the experimentally determined food order is the carbohydrate nature and content of the foods. Carbohydrates, such as the α-amylase resistant starch (RS) and prebiotics, escape digestion in the small intestine and provide an energy substrate for the colonic microflora. Both prebiotics  and resistant starch (potatoes, bananas, lentils and legumes) have been hypothesised to improve the colonic health of the host. For RS, resistance to digestion is a function of the morphology of the starch granules and their crystalline organisation, which is determined by the botanical source of the starch and the processing it has undergone before being eaten. PrebiotAny dietary advice provided to ulcerative colitis patients should be based on the FSS table. The table is of course imperfect because of experimental error, natural variation and the associations between foods. For example, milk and cereal are coded separately but are often consumed together. Thus the magnitude of the difference in the FSSs for these two foods is less than if they'd been independent variables. Suggestions have been made in this discussion as to the factors responsible for the FSS order and to distill these factors into the advice given in Table 5 see . This taThe list of dietary risk factors for colon cancer bears a A dietary analysis method is described that provides a new tool for establishing relationships between diet and disease. This method has been applied to the study of ulcerative colitis and points to sulfite and caffeine as being harmful, with thiamin and resistant starch being potentially therapeutic. For the first time, dietary guidelines for ulcerative colitis patients, including food portion sizes have been developed.Ulcerative colitis (UC); Food sigmoidoscopy score (FSS); European Union (EU); Odds ratio (OD) and Confidence interval (CI).The author(s) declare that they have no competing interests.JHC, EAM and LME contributed to the study design and the writing of the manuscript. EAM was the co-ordinator of the study and along with ST had responsibility for managing the patients and coding the diaries. LME developed the dietary data analysis protocol assisted by RC. CK and JHC performed the sigmoidoscopic examinations of the patients. All authors read and approved the final manuscript.Review of studies of diet and ulcerative colitis (UC).Click here for fileFood sigmoidoscopy score (FSS) calculation example for red wine (NB incomplete data set used).Click here for fileFoods consumed in order of food sigmoidoscopy scores (FSS).Click here for filePermitted levels of sulfite in the UK.Click here for fileProposed dietary advice for ulcerative colitis patients.Click here for file