This review article combines four disparate observations about Neural Tube Defects (NTDs). They are the worldwide decline in the birth incidence that began prior to prenatal diagnosis; family recurrence risks; the effect of prenatal diagnosis and termination of affected pregnancies; and the effect of folic acid. NTDs are due to many different causes . The incet al [Family studies suggest the recurrence risk for first-degree relatives of affected individuals is approximately 1 in 30. For second-degree relatives (the children of the mother's sisters and brothers) the risk is approximately 1 in 220 . Howeveret al  report tet al [et al [The third aspect, prenatal diagnosis and termination of affected pregnancies, is one that should be discussed with all women in the reproductive age range and, more importantly, with patients who have a family history of an NTD. Folic acid taken orally on a daily basis is shown to lower the occurrence and recurrence of NTDs in their own offspring and in their relatives. The Medical Research Council  was the et al  have recl [et al  demonstrl [et al  and dietl [et al ,18.When recent trends in the birth incidence of NTDs are reported, they focus additionally on the effect that folic acid has on the early second trimester prevalence of affected fetuses. As for the epidemiological studies noted above, these reports include varying types of cases; some report only "spina bifida", others "spina bifida" and anencephaly, and still others mention these two types and encephalocele with or without hydrocephalus. Some studies report only deaths due to complications in these groups of patients as stillborns, or deaths in the neonatal time period; other reports study all affected newborns, and still others cover selective or spontaneously aborted fetuses. Those that include time intervals after the introduction of intrauterine diagnosis and selective termination do not take into consideration the variations in incidence at different gestational ages and at birth, whether stillborn or live . Creasy Our data and this review clearly demonstrate the effects of intrauterine diagnosis and selective termination prior to the recommendation for supplementation and fortification of foodstuffs with folic acid. Because the reason for termination of a pregnancy is not reportable in our state and the USA, we cannot determine the effect of folic acid on the prevalence of myelomeningocele and anencephaly in first and early second trimester fetuses. Studies of the effect of folic acid in reducing the birth incidence in communities with a low incidence, and active prenatal diagnosis associated with termination of affected fetuses, require longer-term studies than published to date. The differences in data discussed above need to be considered if one is to evaluate the effect of prenatal diagnosis and elective termination as well as the effects of fortification or supplementation with folic acid. We recommend that these variables be discussed with women of reproductive age, particularly if they are relatives of a patient with an NTD. Regardless of the uncertainties, we recommend supplementation of the diet of women, beginning three months prior to an anticipated pregnancy. We recommend all women of childbearing age take at least 400 mcg of folic acid daily when they begin sexual activity. Relatives of a patient with an NTD should take 4.0 mg daily, beginning three months prior to conception.The authors declares that he has no competing interests.


Absenteeism due to communicable illness is a major problem encountered by North American elementary school children. Although handwashing is a proven infection control measure, barriers exist in the school environment, which hinder compliance to this routine. Currently, alternative hand hygiene techniques are being considered, and one such technique is the use of antimicrobial rinse-free hand sanitizers.A systematic review was conducted to examine the effectiveness of antimicrobial rinse-free hand sanitizer interventions in the elementary school setting. MEDLINE, EMBASE, Biological Abstract, CINAHL, HealthSTAR and Cochrane Controlled Trials Register were searched for both randomized and non-randomized controlled trials. Absenteeism due to communicable illness was the primary outcome variable.Six eligible studies, two of which were randomized, were identified . The quality of reporting was low. Due to a large amount of heterogeneity and low quality of reporting, no pooled estimates were calculated. There was a significant difference reported in favor of the intervention in all 5 published studies.The available evidence for the effectiveness of antimicrobial rinse-free hand sanitizer in the school environment is of low quality. The results suggest that the strength of the benefit should be interpreted with caution. Given the potential to reduce student absenteeism, teacher absenteeism, school operating costs, healthcare costs and parental absenteeism, a well-designed and analyzed trial is needed to optimize this hand hygiene technique. Routifection" . This stfection" -5. The efection" ,7. Ignazfection" ,7. This fection" ,7. Thesefection" ,9.The elementary school environment is also negatively impacted by outbreaks of disease causing microorganisms ,11. Theset al. (1997) reported that proper handwashing compliance, with soap and water, in school-aged children ranged from 8 to 28 percent. Reported reasons for the observed inadequacy in compliance included insufficient time during the day, and the use of substandard washing facilities in hard to access locations of the school environment [Despite the scientifically proven evidence of the effectiveness of handwashing, and the increasing promotion of proper hand hygiene techniques, observational studies in school settings have indicated that handwashing practices are often lacking ,14. Guinironment ,14.In attempts to overcome the obstacles of routine handwashing in school environments, antimicrobial rinse-free hand sanitizers are being used as an alternative hand hygiene technique. The concern is that such programs may be carried out in the absence of evidence of effectiveness in the school environment. Thus, it is timely to review the evidence currently available for the effectiveness of antimicrobial rinse-free hand sanitizer programs in reducing absenteeism due to communicable illness. The aim of this systematic review was to determine whether antimicrobial rinse-free hand sanitizer interventions are effective in preventing illness-related absenteeism in elementary school children.A detailed written protocol was prepared and reviewed in advance (complete protocol can be obtained from the corresponding author).A comprehensive search was conducted to identify all relevant studies regardless of publication status. Six electronic databases were searched for studies published in any language. The databases included: Biological Abstracts (1990-May 2003), CINAHL (1982–2003), the Cochrane Controlled Trials Register (1981–2003), EMBASE (1980-May 2003), HealthSTAR (1975-May 2003), and MEDLINE (1966-May 2003). A detailed search strategy was developed for use in MEDLINE and an iterative process was completed to refine the MEDLINE search for each database. Descriptions of the database search strategies are presented in Appendix 1 . The interventions of interest were those that administered antimicrobial rinse-free hand hygiene programs compared with no intervention or placebo treatment arm in a school setting. The outcome of interest was the comparison of the number of absences due to communicable illnesses in children who received the antimicrobial rinse-free hand hygiene intervention with the number of such absences in those who received a placebo or no intervention. We included cluster randomized controlled trials (RCTs) and cluster non-randomized controlled trials regardless of publication status.et al. 2002 and Moher et al. 2003 indicate that the exclusion of trials in languages other than English (LOE) does not bias measures of effectiveness- however, both are cautionary, advocating language inclusive search strategies [All relevant citations, titles and abstracts, were imported into a reference database where duplicates were manually removed. Priority in downloading was given to MEDLINE. Reviews were excluded, but the bibliographies from such articles were examined for relevant studies. The screening was completed in an unblinded manner; there is inconclusive evidence that blinding introduced bias into the process . One indrategies ,19. Due Two reviewers  independently abstracted data from all studies meeting the eligibility criteria, excluding one abstract where EM independently abstracted pertinent information , using pTwo reviewers  independently assessed the quality of each of the included studies, excluding the abstract by Thompson 2004) previously mentioned , using t04 previoData synthesis and analysis was performed in accordance with the Cochrane Reviewers' Handbook . FirstlyA flow diagram of the search results is illustrated in Figure After review of the full text of these studies, 21 were excluded for the following reasons: no outcomes of interest (n = 1), inappropriate population (n = 1), inappropriate interventions (n = 9), inappropriate study design (n = 3), irrelevant subject matter (n = 5), and review (n = 2). Thus, a total of 4 trials fulfilled the inclusion criteria ,27,31,41Of the 6 remaining studies, 2 were crossover studies ,27, 1 waAll six trials were conducted in the United States, 4 with reported industrial sponsorship, and were published between 2000 to 2004. The trials varied in size , and geographic locations  and Dyer et al. (2000), provided each student with alcohol-free instant hand sanitizer [et al. (2000), Guinan et al. (2002), Morton et al. (2004), and Thompson et al. (2004) provided each class with alcohol-based instant hand-sanitizer dispensers [The trials also varied with respect to the intervention administered. White anitizer ,41, whilspensers ,15,16,31spensers ,41, educspensers , one stuet al. (2001) reported a significant number of dropouts (857 of 1626 students did not complete the study) however no explanations were offered [The quality of reporting of the 5 trials that were examined in detail was low. Only one study was described as being randomized and double-blinded, however, it failed to describe a detailed and appropriate method of randomization and allocation concealment was unclear . Four of offered . Four st offered . Other cet al. (2000), the experimental group had a 20% (95% CI = 19–21%) reduction in absences due to communicable illness, the experimental group in the trial completed by Guinan et al. (2002) had a 49% (95% CI = 42–56%) reduction, White et al. (2001) demonstrated a 33% (95% CI = 17–45%) reduction in the experimental group, Dyer et al. (2000) had a 34 % (95% CI = 10–50%) reduction in absences due to communicable illness in the experimental group in the first phase and a 56 % (95% CI = 31–72%) in the second phase, and Morton et al. (2004) reported a significant odds ratio for McNemar's test .All six studies varied in their definition of communicable illness-related absenteeism, refer to Table et al. 1999, indicated this item to be of great concern for the parent's of school-aged children [Many studies have examined the importance of preventing the transmission of infectious diseases in the school environment, one such studied completed by Cramer children . The moschildren ,8,10,28.children -50. EffeCan the evidence from these six trials reported here be used to promote this type program in elementary schools at the present time? This systematic review of antimicrobial rinse-free hand sanitizers for prevention of illness-related absenteeism in elementary school children is the first review, of the author's awareness, to assess this issue. Although randomized controlled trials are the study design least likely to provide biased estimates of effect, due to the nature of school-based interventions, the inclusion of both randomized and non-randomized cluster controlled trials was required . Of the Four of the six studies used an alcohol-based product, the other two using a benzalkonium chloride based disinfectant. The FDA in the United States has indicated that insufficient data exits to classify the latter compounds as safe and effective to use as antiseptic handwashes. They are also adversely affected in the presence of organic material such as food residues, which may be an issue in schools . Four stSeveral limitations were encountered when completing this review, the major one being the scarcity of high quality studies. Additionally, although content experts, primary authors and industrial companies were contacted, no grey literature was found. The possible existence of unpublished non-significant trials should not be discounted. The validity of performing a quantitative synthesis was considered, however based on a qualitative inspection of heterogeneity and estimates of intervention effectiveness this was not deemed appropriate. Sources of heterogeneity included study designs, population characteristics, intervention characteristics, case definition and primary outcome measure. Thus, sensitivity and subgroup analyses were not performed, and publication bias was not assessed quantitatively. Another limitation was the fact that one reviewer was used to do the broad screen of articles and review the two citations identified between September 2003 and the present time. This may have biased the results; however, it is believed that this reviewer would overestimate the citations to be included.In wake of the recent worldwide emergence of Severe Acute Respiratory Syndrome (SARS), the importance of proper hand hygiene has been brought to the spotlight. Comprehensive hand hygiene programs with occasional reinforcement are an inexpensive intervention, which potentially can work for a broad population, with minimal adverse effects. Future research should concentrate on developing study protocols that are scientifically sound with regards to randomization generation, blinding, allocation concealment and other factors that will minimize or avoid bias. Hand hygiene programs are the most important infection control measure in the school environment and have potentially large public health and economic implications therefore their design, implementation, and analysis should be carried out with the rigour.The author(s) declare that they have no competing interests.EM conceived and designed the study as part of a graduate course in systematic reviews, reviewed trials for inclusions, abstracted data, participated in data analysis, and drafted and revised the manuscript.NLS participated in initial study design, reviewed trials for inclusion, abstracted data, participated in data analysis, and revised the manuscript.Both EM and NLS agreed upon the final revision.Appendix 1 – Syntax for searchesAppendix 2 – List of corresponding authors, content experts and industrial companies contactedAppendix 3 – Data collection formThe pre-publication history for this paper can be accessed here:Additional file 1 - Syntax for searchesClick here for fileAdditional file 2 - List of corresponding authors, content experts and industrialcompanies contactedClick here for fileAdditional file 3 - Data collection formClick here for file


Peromyscus maniculatus) are the principal natural hosts of SNV, in which the virus establishes life-long persistence without conspicuous pathology. Little is known about the mechanisms SNV employs to evade the immune response of deer mice, and experimental examination of this question has been difficult because of a lack of methodologies for examining such responses during infection. One such deficiency is our inability to characterize T cell responses because susceptible syngeneic deer mice are not available.Human infections with Sin Nombre virus (SNV) and related New World hantaviruses often lead to hantavirus cardiopulmonary syndrome (HCPS), a sometimes fatal illness. Lungs of patients who die from HCPS exhibit cytokine-producing mononuclear infiltrates and pronounced pulmonary inflammation. Deer mice (Mus musculus) granulocyte-macrophage colony stimulating factor. These cells are capable of processing and presenting soluble protein to antigen-specific autologous helper T cells in vitro. Inclusion of antigen-specific deer mouse antibody augments T cell stimulation, presumably through Fc receptor-mediated endocytosis.To solve this problem, we have developed an in vitro method of expanding and generating competent antigen presenting cells (APC) from deer mouse bone marrow using commercially-available house mouse (The use of these APC has allowed us to dramatically expand deer mouse helper T cells in culture and should permit extensive characterization of T cell epitopes. Considering the evolutionary divergence between deer mice and house mice, it is probable that this method will be useful to other investigators using unconventional models of rodent-borne diseases. Bunyaviridae) are rodent-borne and can cause hemorrhagic fever with renal syndrome (HFRS4) or hantavirus cardiopulmonary syndrome (HCPS) [Hantaviruses (family e (HCPS) . While He (HCPS) -4. In NoPatients afflicted with HCPS exhibit pronounced pulmonary inflammation due to capillary leak syndrome, with the consequent hypotension often leading to rapid decline and death . Virus iPeromyscus maniculatus) are the principal reservoir host of SNV [Deer mice  [Recent advances in hematopoietic stem cell research have identified an important role for granulocyte-macrophage colony stimulating factor (GM-CSF) in the expansion and maturation of bone marrow cells into competent APC -40. We pusculus) . This leusculus) , it is pWe used RACE to obtain the complete 5' end of GM-CSF. This sequence was translated using the default translation table within MacVector. The polypeptide is predicted to have a 25 residue signal peptide based upon orthologous sequences from other species -45 Figu. The recDeer mouse bone marrow cultures contained mostly cells that appeared dead or dying after 24 hours in culture with GM-CSF. However, at 48 hours clusters of cells were apparent, while control wells without GM-CSF had fewer live cells than at 24 hours. By day 3, adherent stromal cell foci were conspicuous, while semiadherent and nonadherent cells were more evident and these became the prominent cells for the duration of culture. Day 12 bone marrow cells incubated for an additional 48 hours were large, ranging from 12 to 18 μm in diameter, and possessed macropinocytic vesicles and processes Figure . AlthougDay 8 deer mouse bone marrow cells were cultured with various concentrations of house mouse GM-CSF. Two days later, proliferation was assessed Figure  and maxiBM-APC and T cells were examined for the expression of orthologous I-Eβ and TCRβC, respectively, by RT-PCR Figure . For I-EDeer mice were immunized with keyhole limpet hemacyanin (KLH), and 10 days later the lymph nodes, spleens and bone marrow were processed for in vitro expansion of polyclonal T cells (lymph node cells), while the bone marrow cells and splenocytes were frozen. Sera were tested for antibodies to KLH by ELISA and in each deer mouse tested the titer was greater than or equal to 8,000 (data not shown). For recall proliferation, KLH, in vitro-propagated T cells (14 days with IL-2) and BM-APC (14 days with GM-CSF) or freshly thawed splenocytes were cultured together for 72 hours, and proliferation was assessed by MTS assay Figure . For eacDeer mouse antiserum raised against KLH and incubated with antigen for one hour prior to addition of cells increased the sensitivity of T cell proliferation Figure , suggestTo our knowledge, no previous efforts have been made to develop long-term cultures of T cells from unconventional laboratory rodents. The principal reason for this is that highly inbred strains, required for conventional long-term T cell work, are not available from rodents not routinely used in laboratory work. At least for deer mice, we have developed a method of fulfilling this need by using commercially-available house mouse GM-CSF. This cytokine apparently binds to the GM-CSF receptor on deer mouse cells such that it generates competent APC from the bone marrow. These cells are capable of processing and presenting soluble antigen to autologous antigen-specific helper T cells.Our initial suspicions that house mouse GM-CSF might bind to deer mouse GM-CSF receptor was the result of previous work  in whichWe used a method that has been shown to generate dendritic cells in house mice; however, the cells obtained from deer mouse bone marrow more closely resembled macrophages rather than DC. These cells contained many large macropinocytic vesicles, but conspicuous dendrites typical of DC were not observed. Microscopically, these cells also appeared sensitive to TNF, which decreased macropinocytosis, but it had no effect on the capacity of these cells to present antigen to T cells as has been reported for human DC derived from blood mononuclear cells . TNF can7 bone marrow cells from a deer mouse, which is sufficient for freezing 5 vials at 2 × 106 cells each. Each vial is used to seed a 100 mm bacterial Petri dish, which produces about 107 BM-APC at 14 days of culture. For deer mice, the most significant limitation for cells is from the spleen. Although deer mice are slightly smaller than BALB/c mice, their spleens are disproportionately small (unpublished observations). We routinely recover 7 × 106 splenocytes from a deer mouse, while BALB/c house mice usually provide 10-fold more. Because of this limitation, we have begun to use BM-APC to propagate T cells. This method involves culturing of bone marrow cells with GM-CSF for 10 days, then freezing aliquots of 106 cells. Three days before T cell restimulation, the 10-day BM-APC are thawed and cultured with GM-CSF, then used for restimulation with fresh antigen in one well of a 24-well plate. The T cells are fed fresh IL-2 DMM-5 at two-day intervals for expansion.Since the deer mice are outbred, this method requires the immunization and collection of cells from individual animals Figure . These cWe have used this method to establish nine T cell lines, six specific for KLH and three specific for SNV nucleocapsid antigen (data not shown). Based upon typical cell yields, it should be possible to assay several thousand wells on 96-well plates, which we estimate to be sufficient for many T cell activities, including cloning, peptide epitope mapping, TCR variable gene segment usage, and cytokine profiling.We believe the methods described in this work will allow the characterization of antigen presentation and T cell responses in infected deer mice. Many viruses impair pathways involved in APC and T cell functions so that they can evade a sterilizing immune response. With hantaviruses and their rodent hosts, millions of years of evolution have presumably allowed a coadaptation of the viruses and host immune responses such that pathology does not occur and the virus is not eliminated. It is possible that hantaviruses possess some as yet unidentified mechanism for suppressing an aggressive inflammatory immune response in rodent hosts, which is ineffective in human infections and often leads to inflammatory immunopathology.Because of the substantial evolutionary divergence of deer mice and house mice (about 25–50 million years) , it is lA limitation of this approach is that BM-APC lose their ability to proliferate in the presence of GM-CSF after four to six weeks, similar to what has been observed with house mouse bone marrow-derived cells ,54. SincWe have developed a method for generating large numbers of competent antigen presenting cells from deer mouse bone marrow using house mouse GM-CSF. This method resulted in the production of antigen-specific T cell lines from outbred deer mice. Inclusion of antigen-specific antibody in cultures augments T cell proliferation, suggesting the APC express Fc receptors. This method will allow characterization of APC and T cells in deer mice and may be extended to other rodent species that are important in infectious disease research.The deer mice used in these experiments were from a colony of animals established with deer mice trapped in western Colorado . All proSigmodon hispidus, AAL55394) and human (NP_000749) were aligned using MacVector's  clustal algorithm.The 5' end of the deer mouse GM-CSF cDNA was obtained by using RACE. Briefly, a primer Table  was desiTotal RNA from activated splenocytes was reverse-transcribed using an oligo-dT primer and Superscript II . TCRβC cDNA sequences from house mouse, rat and human were aligned with MacVector. PCR primers Table  were desDeer mice were bilaterally immunized subcutaneously at the base of the tail with 20 μg of KLH  emulsified in CFA (Sigma). Ten days later, draining lymph nodes, spleens and bone marrow were recovered for in vitro experiments. For production of high-titer KLH antiserum, deer mice were immunized i.p. with 20 μg of KLH emulsified in CFA and boosted with 20 μg in IFA one month later. Sera were collected 7 days after boosting.6 cells per vial in 1 ml of 10% DMSO/DMM-5 and stored at -70°C.Immunized deer mice were euthanized by cervical dislocation and the draining lymph nodes, spleens and bone marrow from individual animals were separately collected in Hank's balanced salt solution for processing. The lymph nodes served as a source of antigen-specific T cells, while the splenocytes were treated with ammonium chloride  to lyse RBCs, then frozen in 10% DMSO/5% FBS deer mouse medium  in aliquots for use as autologous APC for additional rounds of in vitro T cell stimulation. The bone marrow cells were collected from tibiae and femurs, washed twice in DMM-5, and aliquotted at 2 × 106) was quick-thawed in a 37°C water bath and cultured without washing in 100 mm bacterial Petri dishes in DMM-10 containing 20 ng/ml recombinant house mouse GM-CSF  at 37°C under 7% CO2. Fresh GM-CSF/DMM-10 was provided on days 3, 6, 8, 10, and 12 for the generation of APC. Cells were collected with a scraper for use in experiments. Cells were processed by cytospin and stained with Wright's stain for morphological examination.Bone marrow-derived APC (BM-APC) were generated with modification of a previously described method for dendritic cells (DC) . One viaDeer mouse splenocytes depleted of RBC by ammonium chloride treatment were incubated with a suboptimal dose of PHA (2 μg/ml) (Sigma) in DMM-5 and recombinant human IL-2 (R&D Systems) for 48 hours. Proliferation was determined by MTS assay . The means and standard deviations of duplicate samples were calculated, with the mean of cells without IL-2 subtracted from sample means.Total RNA was extracted from 14 day T cell and BM-APC cultures  and converted into cDNA. Class II expression of the bone marrow cells was assessed by PCR using a forward primer from exon 2 and a reverse primer that overlaps the boundaries of exons 2 and 3 of deer mouse I-Eβ (Table P. leucopus IgG (H&L)  for 1 hour, then horse anti-goat IgG-HRP conjugate for 1 hour . ABTS substrate (Sigma) was incubated for 15 min, and plates were read at 414 nm. Means were calculated with the background  subtracted.Sera were collected at euthanasia by cardiac puncture. Plates were coated with 5 μg/ml KLH in PBS overnight and washed 5× with wash buffer (PBS-0.1% TWEEN-20). Plates were then blocked with blocking buffer (5% nonfat powdered milk in wash buffer) for 1 hour at room temperature. The sera and remaining reagents were diluted in blocking buffer. Sera were incubated in duplicate for 2 hours at room temperature, followed by goat anti-6 cells per well (24 well plate) with 20 μg/ml KLH in DMM-5. After a 4 day incubation, the lymph node cells were collected and washed twice in DMM-5. The number of recovered lymph node cells varied between animals, but between 2 × 105 and 5 × 105 cells were recovered and plated with fresh antigen and 3 × 106 thawed autologous splenocytes in DMM-5 in 24 well plates in 1 ml of DMM-5. At 2 day intervals, cultures were fed by removing 750 μl of media and replacing it with DMM-5 containing 20 U/ml of recombinant human IL-2. When cultures were greater than 80% confluent, cells were passaged 1:2 into additional wells or into T25 tissue culture flasks. This process was continued for 14 days to expand T cells. Cultures were also restimulated at two week intervals with fresh mitomycin-C treated autologous splenocytes as above, or 2 × 106 BM-APC to continue propagation of T cell lines.In vitro stimulation of helper T cells was performed essentially as described elsewhere ,57. Lymp4) or splenocytes (2 × 105) were used to stimulate 105 T cells in the presence of KLH in 96-well plates. In other experiments, the effects of house mouse tumor necrosis factor (20 ng/ml) were evaluated on APC morphology and capacity to stimulate T cell proliferation. Lastly, antisera to KLH were produced in deer mice and used to assess the capacity of the BM-APC to capture and process antigen for presentation to T cells. In these experiments, antiserum or normal deer mouse serum were diluted to 1:2,000, a saturating dilution in ELISA as described above, with KLH and incubated for 1 hour at room temperature. BM-APC and T cells were then added and the cultures incubated for 72 hours prior to determining proliferative responses by MTS. Means and standard deviations were calculated from duplicate samples.BM-APC were examined for functional capacity to process and present KLH to sensitized autologous deer mouse T cells expanded in culture. For these experiments, mitomycin-C treated BM-APC -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliumBD conducted bone marrow cell culture work. DGW and TAC performed RT-PCR experiments. JP cloned and sequenced the TCRβ cDNA. RMF cloned and sequenced the MHC class II cDNA. TS immunized deer mice and generated T cell lines.


Esophagectomy is considered the gold standard for the treatment of high-grade dysplasia in Barrett's esophagus (BE) and for noninvasive adenocarcinoma (ACA) of the distal esophagus. If all of the metaplastic epithelium is removed, the patient is considered "cured". Despite this, BE has been reported in patients who have previously undergone esophagectomy. It is often debated whether this is "new" BE or the result of an esophagectomy that did not include a sufficiently proximal margin. Our aim was to determine if BE recurred in esophagectomy patients where the entire segment of BE had been removed.Records were searched for patients who had undergone esophagectomy for cure at our institution. Records were reviewed for surgical, endoscopic, and histopathologic findings. The patients in whom we have endoscopic follow-up are the subjects of this report.Since 1995, 45 patients have undergone esophagectomy for cure for Barrett's dysplasia or localized ACA. Thirty-six of these 45 patients underwent endoscopy after surgery including 8/45 patients (18%) with recurrent Barrett's metaplasia or neoplasia after curative resection.Recurrent Barrett's esophagus or adenocarcinoma after esophagectomy was common in our patients who underwent at least one endoscopy after surgery. This appears to represent the development of metachronous disease after complete resection of esophageal disease. Half of these patients have required subsequent treatment thus far, either repeat surgery or photodynamic therapy. These results support the use of endoscopic surveillance in patients who have undergone "curative" esophagectomy for Barrett's dysplasia or localized cancer. The incidence of esophageal adenocarcinoma has increased more rapidly than any other form of cancer since the 1970s and now represents the majority of esophageal neoplasms in the West . Other rDetection of dysplastic Barrett's esophagus or mucosal adenocarcinoma is important because it allows the opportunity to intervene prior to the development of invasive neoplasia. Unfortunately, no medical or surgical GERD treatment has been consistently and convincingly demonstrated to prevent the development of adenocarcinoma . TraditiAfter approval by the Mayo Foundation's Institutional Review Board for Research, the electronic medical records of Mayo Clinic patients in Jacksonville, Florida, were searched to find all patients who had undergone esophagectomy for cure at the Mayo Clinic surgical facility, St. Luke's Hospital, Jacksonville, Florida, since 1995. This time period was chosen to coincide with the routine availability and clinical use of pre-operative staging with endosonography in our institution. The records of these patients were reviewed for pre-operative and post-operative staging results including computed tomography and endosonography studies. In addition, endoscopic, surgical studies and histopathological studies were studied. Specifically, the surgical specimens were reviewed to ensure that the esophagectomy specimen, including lymph node sampling, was adequate and the proximal margin was completely free of Barrett's metaplasia, dysplasia or carcinoma. The patients, in whom we have at least one follow-up endoscopy study, with biopsies obtained for histologic confirmation of mucosal disease, are the subjects of this report. Esophageal disease was staged according to the Tumor-Lymph node-Metastasis (TNM) criteria .Since 1995, 45 patients have undergone esophagectomy for Barrett's dysplasia or localized adenocarcinoma with curative intent in our institution. At operation, none of these patients were found to have extension of malignant disease to paraesophageal lymph nodes and all esophageal glandular mucosa was resected with only normal squamous mucosa remaining at the proximal surgical margin. Subsequently, 36 of these patients (80%) have undergone endoscopy after surgery including 8/45 patients (18%) who were found to have recurrent Barrett's glandular mucosa after curative resection and are described in the table.Open transthoracic esophagectomy (Ivor Lewis procedure) with pyloroplasty was performed in most patients (39/45) including the patients diagnosed with recurrent Barrett's disease. Five different surgeons performed these operations. Most patients had evidence of gastric stasis (retained food) at endoscopy . Anastomotic dilation was performed at endoscopy in 16/36 patients . It is possible that patients with anastomotic strictures may be at increased risk of recurrent Barrett's esophagus because of worse reflux although their swallowing symptoms may, alternatively, be related to other factors such as anastomotic ischemia or surgical sutures. Patients frequently used aspirin (42%) or COX-2 specific non-steroidal anti-inflammatory agents (25%). Twice-daily proton pump inhibitors were routinely prescribed for these patients although patient compliance is difficult to assess because of high drug costs and limited symptomatic improvement. While the small number of patients limits our analysis, these factors were found to occur in a proportional number of patients with Barrett's disease and no clear trends could be identified.2N0M0 in a 78-year-old man. This metaplastic glandular epithelium was detected at a follow up of 90 months and 17 months, respectively. In the other 3 patients, short segment Barrett's low-grade dysplasia has been found in lengths between 10–25 mm after complete resection of the esophagus for Barrett's high-grade dysplasia (1 patient) and Barrett's T3N0M0 carcinoma (2 patients). This recurrent Barrett's glandular dysplasia was detected at a follow up of 42–47 months. Erosive esophagitis was also noted in 4 of 5 patients indicating uncontrolled reflux disease. Subsequently all patients have been treated with high doses of proton pump inhibitors (such as esomeprazole 80 mg twice a day or 40 mg three or four times per day) in an attempt to maximally control reflux of acid and digestive juices from the stomach into the cervical esophagus.Five of these patients have been diagnosed with Barrett's metaplasia or low-grade dysplasia have been followed for more than 12 months in surveillance endoscopy programs monitoring the stability of the glandular epithelium. Two of these 5 patients have been found to have short segment Barrett's metaplasia with lengths of 10 mm and 15 mm, after complete esophageal resection for Barrett's high-grade dysplasia in a 72-year-old man and Barrett's adenocarcinoma T2 or T3N0M0 adenocarcinoma. These patients varied in age from 58–80 years of age. These patients were found to have more severe erosive esophagitis suggesting worse acid reflux and mucosal injury compared to the non-carcinoma recurrent Barrett's patients. Recurrent Barrett's multi-focal high-grade dysplasia, over a 10 mm segment length was detected 88 months after esophagectomy in one patient and was successfully ablated with porfimer sodium photodynamic therapy using the methods described elsewhere [1N0M0 was confirmed at repeat esophagectomy. Finally, a diminutive polypoid mass proximal to the surgical anastomosis was found in a 58-year-old woman who had 7 months previously undergone esophagectomy for Barrett's mucosal adenocarcinoma. Computed tomography with contrast enhancement noted esophageal wall thickening and suspicious lymphadenopathy. Repeat resection confirmed the tumor histologic grade of T2N1M0 adenocarcinoma.Three other patients developed recurrent Barrett's disease after curative resection of esophageal Tlsewhere . High-grOver the past four decades, the incidence of esophageal adenocarcinoma has risen dramatically, particularly in older white men . PreviouAfter undergoing esophageal resection, the native squamous mucosa of the cervical esophagus will be brought into contact with the acid-secreting mucosa of the gastric body. This reconstruction allows acid and duodenal juice to reflux from the gastric conduit to the remaining cervical esophagus. Reflux of gastric and duodenal content is an important factor in the pathogenesis of Barrett's metaplasia, dysplasia and esophageal adenocarcinoma -20. It iIn the study of Oberg et al, despite the use of potent acid-suppressing medications, severe esophageal acid exposure was noted in most patients. Patients with recurrent Barrett's epithelium were found to have significantly more severe acid exposure that occurred predominantly in the supine position . There wis or T1) were detected at surveillance endoscopy and were treated with endoscopic laser ablation, mucosal resection or surgical resection. While squamous cell carcinomas are not related to gastroesophageal reflux, this paper also suggests that esophageal cancer patients (squamous or adenocarcinoma) are predisposed to the development of metachronous carcinomas in the remnant cervical esophagus. This is consistent with DeMeester's experimental model of Barrett's dysplasia and adenocarcinoma occurring after complete gastrectomy with esophago-jejunostomy and reflux of bile and digestive enzymes into the cervical esophagus [Murata and colleagues recently reported the diagnosis of metachronous squamous cell carcinomas in five of 253 patients (2%) who had undergone esophagectomy for thoracic esophageal squamous cell carcinoma more than two years previously. These superficial carcinomas (Tsophagus . Also, iKonishi et al reported finding an adenocarcinoma in Barrett's esophagus following a total resection of the gastric remnant in a 52-year-old man who had undergone distal gastrectomy for gastric cancer nearly twenty years previously . The BarThese studies have demonstrated that the cervical esophagus is exposed to high amounts of acid and refluxate despite the use of proton inhibitor medications and often in the absence of severe reflux symptoms. Although our group of patients has been observed for only a median of 2 years after esophagectomy, our study confirms that the development of metaplastic columnar mucosa in the cervical esophagus is a common complication related to reflux associated injury to the squamous epithelium. Further, our findings suggest that this recurrent glandular mucosa is unstable and predisposed to the development of dysplasia and invasive carcinoma, as has already developed in most of patients.The early detection of this recurrent disease remains vitally important to preserve all possible treatment options including surveillance endoscopy follow-up, endoscopic ablation with porfimer sodium photodynamic therapy, and if necessary repeat esophagus resection surgery. Our specific recommendations include surveillance endoscopy every 6–12 months for patients who have undergone "curative" esophagectomy for Barrett's dysplasia or adenocarcinoma. In addition, we also routinely recommend indefinite use of proton pump inhibitors, regardless of symptom status, starting at twice daily dosing and increasing as necessary to control reflux symptoms and mucosal damage due to acid, bile and digestive enzymes. Whether these drug doses should be titrated based on ambulatory pH and impedance test results remains to be determined. We have generally been disappointed by prokinetic agents such as metoclopromide in improving reflux symptoms in these patients. For esophagectomy patients who develop recurrent Barrett's metaplasia we recommend the use of COX-2 inhibitors or aspirin chemoprevention to protect against the development of metachronous Barrett's carcinoma. ,30.None declared.All authors participated in the study design and coordination as well as case collection and review of histopathologic and endoscopic results. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


P. aeruginosa is a pathogen that causes acute and chronic lung infections by interacting with the pulmonary epithelial cells. We use this example for examining the ways of triggering the response of the eukaryotic cell(s), leading us to a better understanding of the details of the inflammatory process in general.Binding of a bacteria to a eukaryotic cell triggers a complex network of interactions in and between both cells. Considering a set of genes co-expressed during the antibacterial response of human lung epithelial cells, we constructed a promoter model for the search of additional target genes potentially involved in the same cell response. The model construction is based on the consideration of pair-wise combinations of transcription factor binding sites (TFBS).It has been shown that the antibacterial response of human epithelial cells is triggered by at least two distinct pathways. We therefore supposed that there are two subsets of promoters activated by each of them. Optimally, they should be "complementary" in the sense of appearing in complementary subsets of the (+)-training set. We developed the concept of complementary pairs, i.e., two mutually exclusive pairs of TFBS, each of which should be found in one of the two complementary subsets.We suggest a simple, but exhaustive method for searching for TFBS pairs which characterize the whole (+)-training set, as well as for complementary pairs. Applying this method, we came up with a promoter model of antibacterial response genes that consists of one TFBS pair which should be found in the whole training set and four complementary pairs.We applied this model to screening of 13,000 upstream regions of human genes and identified 430 new target genes which are potentially involved in antibacterial defense mechanisms. In spite of numerous sophisticated approaches devoted to this subject . We staWe suggest a simple, but exhaustive method for searching for TFBS pairs which characterize the whole training set, and combinations of mutually exclusive pairs (complementary pairs). The idea of starting the analysis with a "seed" of sequences allows a very biology-driven way of initial filtering of information.To enhance the statistical reliability and to get additional evidence in TFBS combination search, we applied the principal idea of phylogenetic footprinting (using orthologous mouse promoters), yet proposing a different view on applicability of this approach.Finally we came up with a promoter model which we applied to screening of 13,000 upstream regions human genes. We identified 430 new target genes which are potentially involved in antibacterial defense mechanisms.In every step of our investigations we tried to combine purely computational approaches with the preexisting experiment-based knowledge, as it is represented in corresponding databases and literature, and with our own biological expertise. To develop a promoter model, the first task is to select those transcription factors, the binding sites of which shall consitute the model. The overwhelming majority of methods and tools estimating the relevance of predicted TF binding sites in promoter regions are based on their over- and underrepresentation in a positive (+) training set in comparison with some negative (-) training set. If, however, a binding site is ubiquitous, or very degenerate, so that it can be found frequently in any sequence, the comparison with basically any (-)-training would not reveal any significance for its occurrence. That tells nothing about their functionality in any specific case, which may be dependent on some additional factors and/or other conditions. Therefore, basing the decision about the relevance of a transcription factor for a certain cellular response solely on whether its predicted binding sites are overrepresented in the responding promoters may lead to a loss of important information. Thus, we did not rely on this kind of evidence but rather chose the candidate transcription factors according to available experimental data. We found 5 factors reported in literature as taking part in anti-bacterial or similar responses and selected them as candidate TFs ,15,18-29On the other hand, some of the factors, which have also been mentioned in literature as potentially relevant  or mighFinally, we constructed our promoter model of binding sites of 5 TFs , considering their pairwise combinations and some combinations of higher order .predictive model, since it is also important to have minimal occurrence of a discriminating feature in the (-)-training set. In the further work we considered all pairs with p < 0.005, but as this did not reasonably restrict the list of considered pairs, we had to apply an additional filtering approach. For this purpose we used a simple characteristic such as the percentage of sequences in (+)- and (-)-training sets. By operating directly with percentages we could easily filter out those pairs which would identify too many false positive sequences, thus getting rid of a substantial part of useless information. This procedure allows to estimate immediately the applicability of the model to identify further candidate genes that may be involved in the cellular response under consideration (see Methods).In several steps of the model construction we had to estimate overrepresentation of a feature in the (+)-training set compared with the (-)-training set. We operated with the number of sequences that possess the considered feature, in our case a pair of TFBS, at least once. Otherwise, mere enrichment of a feature in the (+)-training set may be due to strong clustering in a few members of that set which would not lead to a useful prediction model. At the first step the T-test has been performed ), but it appeared to be a weak filter: for example, we could find several pairs which showed, if estimated with T-test, a remarkable overrepresentation (p < 0.001), but with a difference of 97% in the (+)-training set versus 85% in the (-)-training set, which is of no practical use to construct a The main problem of promoter model construction are the numerous false positives. Developing our approaches we applied some anti-false-positives measures :• distance assumptions• identification of "seed" sequences• phylogenetic conservation• subclassification into complementary sequence sets.In the following, we will comment on each item in more details.The commonly accepted view that functionally cooperating transcription factors may physically interact with each other triggered us to introduce certain assumptions concerning the distances between the considered TFBS. Transcription factors can interact either immediately with each other or through some  mediator proteins (co-factors). Principally there can be many ways of taking this into account, since our knowledge about the mechanisms of interaction is limited. In this work we used two different approaches to consider distances in the promoter model development.at a certain distance, but within a certain distance range, we considered the pairs occurring in segments of a certain length.In the first case we based our assumptions on the structure of known composite elements. We assumed that the binding sites of interacting TFs should occur in a distance of not more than 150 bp to each other  Directly interacting factors should have the binding sites at a close distance.(b) The factors interacting through some co-factor may have binding sites on some medium distance, depending on the size and other properties of the co-factor (and the factors themselves).(c) We can also expect direct interaction of another type, when the two factors are not located in the nearest neighborhood, but their interaction requires the DNA to bend or even to loop. This means that the distance is no longer a close one, although we cannot estimate the distance range for this case; thus, we allowed different ranges of distances, excluding only the closest ones.We searched for pairs in three distance ranges, roughly called "close", "middle" and "far", all with adjustable borders, so that moving them we could get the best proportion of percentages in (+)- and (-)-training sets. We used the search in the distance ranges as a starting point, but some of the found pairs required optimization of the borders, so that they finally did not fit into any of the predefined ranges. The initial "close" range was taken as 5–20 bp, to exclude the overlapping of the sites, but to allow close interaction; however, the border had to be shifted in many cases up to 50 bp. The initial "middle" range was chosen from 21 to 140 bp (the number of nucleotides wrapping around the core particle of the nucleosome); the "long" range had its upper border at 250 bp.Initially the idea of "seed" sequences was exploited because of the desire to make use of preexisting biological knowledge about the expressed genes and also because of doubts in the reliability of the available data set. Different experimental approaches differ in their reliability. The microarray analysis is not absolutely reliable ,34-36, sTherefore, we started our analysis with a group of "seed" sequences, which we considered for distinct reasons more reliable and preferable. Choosing a seed group, we took into consideration two kinds of evidence; the first was the source of information, i. e. the methods with which the gene has been shown to participate in the response. We took the promoter sequences of those genes which have been reported by other methods but microarray analysis ,38-47,47The second kind of evidence was whether we could find any additional biological reasoning for the gene to participate in this kind of reply. For instance, a well-known participant of the NF-κB-activating pathway such as IκBα, or participants of different pathways which are likely to be triggered here as well, like c-Jun or PKC, were estimated as the first candidates for the "seed" group.Methods) -training set was reduced by at least two orders of magnitude: depending on the "seed" the number of considered pairs varied from 50 to 400. In the next steps this number was reduced by another order of magnitude Table .Each "seed" is characterized by its own set of pairs. To ensure the robustness of the obtained results, we undertook the "leave-one-out" test, removing consecutively one sequence of the "seed" set (for the combined "seed" sets which included human and mouse orthologs we excluded simultaneously both orthologous sequences). This has been repeated for each sequence (or ortholog pair). Only the robust pairs have been taken into further consideration.Evolutionary conservation of a  TFBS is generally accepted as an additional criterion for a predicted site to be functional . HowThe observed fact is that functional features are not necessarily bound to conserved regions, as long as we speak about primary sequence conservation. Dealing with such degenerate objects as TF binding sites, one should not expect an absolute conservation of their binding sequences. From the functional point of view, it seems to be more reasonable to expect that not the sequences, but the mere occurrence of binding sites and/or their combinations as well as (perhaps) their spatial arrangement would be preserved among evolutionarily related genomes. That is the approach that we use in the present work, completely refraining from sequence alignments. We search for those pairs of TFBS which can be found in human and corresponding mouse orthologous promoter regions, considering the promoter as a metastring of TFBS. We took a feature (the pair of TFBS) into account only if we could identify it in both orthologous promoters, not taking into consideration in what region of the promoter it appeared; we also did not try to align metastrings of TFBS symbols, since they may be interrupted by many additional predicted TFBS . While this work was in progress, we found a very similar approach in the work of Eisen and coworkers ,55, who The idea that combinations or clusters of regulatory sites in upstream regions provide specific transcriptional control is not new ,8,56. NeIn the following, we will formalize our approach and describe the logics of our investigation.All procedures are described for the example of pairwise combinations, but principally all of them can be applied to combinations of higher orders. We restricted our attempt to pairs for sake of computational feasibility.Methods. A pair is taken into account if it has been found in a sequence at least once.We consider all possible pairwise combinations of TFBS in each sequence, as described in m and n located in a distance range from r1 to r2 (where r1 ≤ r2) on either strand of DNA (+ or -). We can denote the sets of sequences containing pairs in different relative orientation as, .Let us consider two TFBS To allow inversions of DNA segments containing pairs, we consider three classes of combinations Fig. :i = 1,...3  represents the set of sequences with a pair of i-th class m, ni) .In more general form for  be a fraction of the sequences  in the (+)-training set, and  the fraction of sequences  in the (-)-training (control) set.Let  by choosing appropriate values for m, n, i and r1, r2. Also, we are interested only in pairs, which are present in at least a minimum fraction of (+)training sequences (C1) and in a defined maximum fraction of (-)-training sequences (C2). They can be filtered in advance.We have to solve now the optimization problem to maximize the difference  for whichThus, we search for such C1,2 ≤ 1 are adjustable parameters.where 0 ≤ C1 = 0.8 and C2 = 0.4. We could not find pairs which would satisfy more stringent parameters, i. e. either higher C1 or lower C2; on the other hand, requirement (1) was found to be satisfied by a lot of different combinations which gave rise to the same Pt and Pc.For single pairs we chose r1, r2) from the expression  ). Each possible type of pair is determined by values of m, n and i. We can list all types of pairs and assign a number j to each pair in this list. Then each type of pair is characterized by mj, nj, ij:To make the analysis more specific, we can consider combinations of pairs instead of single pairs. For sake of simplicity, we will omit furtheron  we can identify  and , which appear in the (+)training set simultaneously:For two different A triple or a combination of a higher order can be represented in the same way.The antibacterial response of the cell is triggered by at least two distinct pathways, and it may be therefore supposed that there are subsets of promoters activated by each of them. Optimally, they should be "complementary" in the sense of appearing in complementary subsets of the (+)-training set Fig .Seed sequences; Table Complementary pairs were searched first in a "seed" subset of the (+)-training set of sequences Fig , step 1.C1 is therefore always set to 1, );(a) they together cover the whole subset , with an allowed overlap (defined by the parameter C5).(b) each of them can be found in not more and not less than a certain number of sequences -training set of 33 sequences, looking only for the combinations found in the second step  Fig. , step 4.The percentage of the pair occurrence in the (-)-training set has been counted on the first step with the subsequent filtering of pairs.A rather large number of combinations satisfied the requirements described in the previous section. However, when we selected those that were robust in a "leave-one-out" test for the "seed" sets, the final list of potential model constituents was shortened down to only 2 ubiquitous and 12 complementary pairs.We found one satisfactory pair which should be found in all promoters of target genes:AP - 1, NF - κB(1)AP-1, NF - κB, class 1, distance from 10 to 93 bp; see Fig. -training set 52%) -training set revealed the presence of all pairs under consideration). But the simultaneous usage of all the pairs could overfit the model, so we did not apply them all, sacrificing a bit of specificity for sake of a higher sensitivity.Finally, we came up with 4 complementary pairs Fig.  composedC1, C2  for common pairs; for complementary pairs we considered the case with C3 = 0.3 C4 = 0.7 C5 = 0.2. The probability to find by chance a "seed" of 12 sequences which would produce at least one pair common for the random selection of 33 sequences (including the "seed") depends on the chosen C1, C2 and is found to vary between p < 0.0005  and p = 0.02 . We failed to find any complementary pairs after 1000 iterations of the permutation test with the parameters used for the "real" (not permuted) model construction. These results suggest that the success of the model construction based on the search for combinations of TFBS is strictly dependent on the selected training set  and that the significance of the findings, depending on the correct choice of the adjustable parameters, is high enough to claim their non-randomness. Thus, we can say that in the described case the pairs found in the given (+)-training set with the given parameters are the real characteristics of this set.In order to avoid the overfitting of the model and to demonstrate the significance of our results, we performed a permutation test. For that, we conducted 2000 iterations of random permutation of (+) and (-) labels in the training sets and tried to rebuild the model using the procedure described above. The rate of correct classification on this random selection was estimated. The cases of common and complementary pairs were considered separately. The analysis was made for different The model consists of two kinds of combinations of pairs: ubiquitous pairs , and complementary pairs. We can divide the model into two modules, one for each kind of combination.M1 and M2 be modules comprising ubiquitous pairs and complementary pairs, respectively.Let M1 comprises the pair AP-1, NF-κB(1).Module M2 comprises all complementary combinations listed in the Fig. m) in M2.Module S(M) the set of sequences which possess the whole model M; then we can also consider S(M1) and S(M2) , and S(m) – the set with a submodule m.To apply the model means to search for sequences containing all these combinations. Let us call Thenm); in this case we consider four submodules, so the sequences containing M2 can be found as:Module M2 consists of submodules (S(M2) = S(m1) ∩ S(m2) ∩ S(m3) ∩ S(m4),where the set with each submodule we must consider as a union of sequence sets containing the complementary pairs:The final result of application of the model M can be presented asS(M) = S(M1) ∩ S(M2)The model gives 3.4% of false positives and re-identifies 52% of the whole (+)-training set, but these 52% comprise all most reliable sequences of the set .Applying our promoter model to screening of 13000 upstream regions from a collection of human 5'-flanking sequences , we idenP. aeruginosa binding. One of the results of our work is a list of potential target genes, enriched with different regulatory proteins, including transcription factors and known participants of the ascending pathways. This theoretical result must have two practical consequences: first, it allows to restrict further experimental research to a manageable number of candidate genes; second, it enables to understand or to clarify some uncertain details concerning the triggering pathways, and thus to make some new predictions based on this information. There is a number of published tools for searching for regulatory modules  [7,5nd Noble ,59-63. Tnd Noble . Any of nd Noble , and theIn the present work we tried to address the listed problems. We could not, of course, improve the situation with the paucity of experimental data, only endeavored to make our data searches as accurate and exhaustive as possible. In principle we developed our approaches basing them, whenever possible, on biological reasoning. We find it extremely important to use as much experimental evidence as it is available at the moment. In our approach we alternated two different kinds of steps – expanding the data and restricting it: exhaustive data search – "seed" and distance constraints – exhaustive enumeration of all possible pairs – complementary pair constraints.To avoid the problem of low confidence in the (+)-training set (which may occur not only in our specific case), we developed the approach of "seed" sequences. The difference from the "seeds" used in cluster analysis is that in our approach the choice of the "seed" is biologically based. Although the "seed" approach is, obviously, a restrictive measure, moreover, a pre-process restriction, which may result in missing potentially relevant additional sequence features, we find it useful and appropriate when the choice of the "seed" is made on a solid biological basis. After having applied the restrictive "seed" technique and distance assumptions, we undertake an exhaustive, complete enumeration of all possible pairs of potential TF binding sites that can be found in the (+)-training set, which in turn reveals a large number of combinations. This list of all found pairs is processed under a new kind of constraints imposed by the search for complementary pairs.The search for complementary pairs is a completely new approach, which supplies us with a new kind of information. It enables to identify subsets of the (+)-training set which possess different regulatory modules, thus suggesting their triggering by different regulatory pathways. This kind of information becomes extremely important in two cases: (i) when two or more pathways are presupposed to be triggered in the cellular response, like in the case considered in this work; (ii) when the (+)-training set consists of not really co-regulated, but of co-expressed genes, without precise information about which of them are regulated by the same mechanism. The identification of complementary pairs and, consequently, groups of sequences enables to better define the co-regulated genes thus providing a partial, although only predicted, confirmation of the co-regulation, and at the same time to better understand the ascending pathways.P. aeruginosa this pathway is not the only one [The final result of our search supported the idea of complementary pairs. There is a lot of evidence in literature that interleukin 8, β-defensin, monocyte chemoattractant protein and different mucins are regulated through LPS-triggered pathway(s) ,15,38. Oonly one , but we Our approach, as any other, has its limits. It has been shown for the genuine composite elements of certain types   that oneThe next source of limitations we see in the preselection of factors according to published data. Obviously, we can not expect that the experimental data is exhaustive; some of the transcription factors may be not reported just because their participance in a certain process has not yet been investigated. On the other hand, statistical overrepresentation, as it has already been mentioned before, can not be taken by itself as proof of biological functionality or its lack; some TFBS cannot be overrepresented due to their degenerate nature. We had no other idea of how to take into account those TFBS which are not overrepresented, but to rely on published experimental data. We find that the usual methods based on statistical overrepresentaion are even more restrictive, but maybe the best solution could be found in merging both approaches – i.e., using the experimental evidence along with statistical ones, for instance using Bayesian techniques.P. aeruginosa binding, and further development of the methodological approaches, making them more flexible and applicable to any similar task. The list of predicted target genes has to be evaluated experimentally, but may have its value for further research already on the present step. The future work on reconstructing the intracellular pathways triggering the genetic program of the antibacterial cell response will be well supported with the information picked up from this list. It may give some hints for the next steps of experimental research, for instance providing information about the first candidates to be checked. The information about the complementary subsets of regulated genes helps to better understand the triggering pathways, and the complementarity of their function is a subject for further consideration.We see the perspectives of this work in two different fields: further investigation of regulatory networks triggered by The methodological approaches presented in this paper can be, of course, applied to other objects. In this work we focused on the experimentally proven basis for the initial choice of transcription factors. This kind of evidence is stronger than any prediction, but it can work only when this information is available, which may be not the case for some other sets of genes or cellular situations. In the next step of development we would like to allow also an exhaustive computational search through the whole list of known TFs for potential constituents of the models. The usage of Bayesian techniques, as mentioned in the previous paragraph, would be also appropriate for this kind of predictions.We suggest a methodology for promoter model construction based on the search of TFBS pairs and show how it works in the particular case of antibacterial response of human lung epithelial cells. We show that the method allows to identify and predict subsets of target genes potentially triggered by different regulatory pathways and thus possessing different regulatory modules. The methodology is easily applicable to any similar task and does not depend on the number of included TFs and/or number of investigated sequences, which only should not be too low for statistical reasons., release 77-1.Eukaryotic Promoter Database , release 3.0DBTSS, the database of transcription start sites ® Professional release 7.1 TRANSCompel® Professional release 7.1 TRANSFAC® Professional release 4.1 TRANSPATHThe positive (+) training set comprises:P. aeruginosa by means of:1. Promoters of human genes shown to be expressed in epithelial cells after interaction with a. microarray analysis ,b. other methods ,28,37,382. Orthologous mouse promoters., or from DBTSS, the database of proven transcription start sites . The length of the sequences was 600 bp (-500/+100). This region comprises most of then known upstream elements and corresponds to the upstream region used by Davuluri et al. as "proximal promoters" for promoter recognition [The sequences were derived either from Eukaryotic Promoter Database ognition , plus a ognition .The "seed" set is a subset of the positive training set selected for highest experimental reliability (see Table The negative (-) training set was composed of randomly chosen 5'-upstream sequences derived from the TRANSGENOME information resource of annotated human genome features . The setP. aeruginosa binding or in the pathways triggered during similar responses. The search revealed 5 candidate factors: NF-κB [We based our selection of TFs on experimental evidence. For that we undertook an extended literature search, looking for the TFs which have been shown to take part either directly in the response of epithelial cells to s: NF-κB ,23,24,26s: NF-κB ,24,25,27s: NF-κB ,24 and Ss: NF-κB ,29,48.® database [Including C/EBP and Sp1 in the list was additionally reasoned by the fact that these factors are known to be second constituents in the most frequent NF-κB-containing composite elements as they are compiled in the TRANSCompeldatabase . Moreove® [®); (ii) at least one hit for every searched transcription factor could be found in every sequence of the (+)-training set. The lower border for the thresholds was predefined as 0.80/0.79 (core similarity/ matrix similarity).We made this search with the weight matrix approach using the Match™ tool ; the mat® . For theWe considered all the coordinates (with strand information) of all potential TF binding sites found by Match™ for each transcription factor. Further on, we examined all possible combinations of the coordinates, thus revealing all possible pairs in the sequence.Formalization of the approach, choosing the most promising results achieved with either of them. We considered all pairs of TFs within these segments. All the pairs of one type found within one distance range were merged. We considered a pair only if it appeared in the sequence at least once (within a certain distance), not taking in account the number of pairs in each sequence.We worked under two different kinds of distance assumptions as described in ES developed the methodological approaches as well as statistical analysis and conducted the data analysis. EW conceived the study and participated in its design and coordination. Both authors drafted the manuscript. Both authors read and approved the manuscript.The question is, if we choose by chance a subset of sequences, will our algorithm be able to define a model, specific to such a random subset? In other words, will this algorithm allow to make a model of anything, without dependence on the preselection of the sets ((+)-training set and/or the "seed" set)? We tried to prove the validity of the algorithm theoretically.k sequences, 100% of which possess the required common feature: a pair, a combination of pairs or complementary pairs, just by chance. Note that this estimation is written not for the whole model construction process, but only for the first step of it, where we consider only the "seed" sequences.Our algorithm is based on the definition of biologically relevant "seed" sets, in which we search for the candidate pairs . Therefore, in order to answer the question, it is reasonable to estimate the probability to come across a "seed" set of f) of the TFs included in the model and the frequencies of all possible pairs (F), constructed of these sites. If the frequencies of single sites and the pairs of them satisfy the equationLet us consider the frequencies of predicted single sites Fij as the probabilities of independent events, which is a prerequisite for the following formalism.we can interpret Methods). We did not take into consideration distances and orientations; the probability estimated for the general case will decrease further with the addition of new constraints.We measured the frequencies of predicted single sites and the frequencies of all possible pairs in the (-)-training set -training set, mi is the number of sequences possessing the i-th site, and Mij is the number of sequences possessing pairs of the i-th and j-th sites. Fij was then calculated as (1) and compared with the measured value.where For all cases investigated in this work, the difference between the calculated and measured values did not exceed standard deviation (σ), only in one case getting to 1,5 σ (data not shown). This confirms the correctness of using pair frequencies as probabilities in this case.Ppair to find a set of k sequences in N with any (at least one) pair, same in all k. We can enumerate all possible pairs of sites of the considered TFs, considering only the cases of the independent sites (i<j). Let U be the number of all possible pairs, then we can callLet us estimate the probability Fij = Fu,u ∈ {1,..., U}Ppair can be calculated as:It is easy to show, that the probability Ppairs 2to find k sequences with any common pairwise combination of pairs (pair of pairs). The pairs of pairs may consist either of 3 (when one site is shared) or of 4 different sites ; their probabilities therefore are:Let us estimate the probability andfi, fj, fl, fo are the frequencies of the single sites of the considered TFs, i<j<l<o.where We can enumerate all possible pairs of pairs (notating them as Q):V be the number of all possible pairs of pairs, V = t + s.Let k sequences each possessing a pair of pairs of one type, is:Analogously to (2), the probability to find v ∈ {1,..., V}.where k sequences with any complementary pair (complementary combinations). We consider pairs as complementary, if two of them are found in the seed set in not more than 60% of the sequences and not less than 40 %, the allowed overlap being 20%. The two complementary pairs together must cover the whole seed set. In the case studied here, comprising the 12 sequences of the seed set, we fixed that each of the pairs should be present in at least 5, but not more than 7 sequences, and they are allowed to co-occur in 0–2 sequences.Let us estimate the probability to find The probability that we choose 12 sequences, possessing any one pair of complementary pairs in accordance with these requirements can be calculated as:u, w ∈ {1,..., U}, and  are the binomial coefficients (note that this formula implies that Pcompl reaches the maximum when the frequencies of both pairs are 0.5).where Methods). The results are:All the probabilities were calculated for the (-)training set of 2040 5'-upstream sequences and for the set of 5 selected transcription factors . In this case it can be seen that the simultaneous occurrence of 1 or 2 pairs in 12 randomly chosen sequences has a rather high probability, and thus we can not base our model construction on the search of only these features.  describes the probability to find any combination of 3 or 4 sites, therefore, up to 6 pairs;obviously, the simultaneous search of more than 6 pairs will definitely overfit the model, so we do not consider this case). The probability to find 12 sequences sharing complementary pairs is much lower, so the consideration of a complementary combination makes the model much more specific, and the probability of finding a model with a complementary pair "by chance" is sufficiently low for us to claim that the proposed algorithm is valid. Note that this is a very rough estimation, considering only the upper borders; we would like to emphasize once more, that the probabilities were calculated without considering orientation and distance constraints, and that this is the estimation made for only the very first step of analysis: choosing of a seed set with needed properties. Obviously, this value depends on the number of the sequences in the "seed". Note that when we spread our requirements for simultaneous search on the whole (+)-training set (which is the next step of the model construction) the probability of constructing a model "by chance" will drop dramatically.The whole list of genes found with the promoter model when applying it to the collection of 13000 human 5'-upstream sequences. This list is not cleaned from hypothetical genes.Click here for fileThe list of genes (found with the promoter model when applying it to the collection of 13000 human 5'-upstream sequences) cleaned from hypothetical genes.Click here for file


Gene expression patterns from peripheral blood cells may be useful as biomarkers for monitoring MS progression and response to therapy, argue Kaminski and Achiron Despite the significant progress in increasing our understanding of the immune mechanisms of multiple sclerosis (MS), in improving clinical classification and brain imaging, and in developing new treatments, the factors that determine the course of the disease are mostly unknown . CurrentThe most commonly used disease-modifying therapies are interferon β (IFNβ)  and glatThe diagnosis and management of disease could be transformed thanks to the completion of the human genome project, the availability of sequence information for nearly every gene, and the advent of novel high throughput technologies  a biological state.Glatiramer acetate: A synthetic protein made of four amino acids found in myelin. It is used as an immunomodulator drug in treating MS.IFNβ: A cytokine that is secreted from fibroblasts in response to stimulation by a live or inactivated virus or by double-stranded RNA. It is used as an immunomodulator drug in treating MS.Microarray: A technology that allows the simultaneous profiling of the expression of thousands of genes (even whole genomes). Multiple gene detectors (oligonucleotides or cDNAs) are deposited on a slide that is hybridized with fluorescently labeled samples.PCR (polymerase chain reaction): The exponential amplification of a DNA fragment using repeated activation of a heat-stable DNA polymerase.Real-time PCR : A method in which the quantitation of the products of PCR is made by measuring fluorescent emission. It is used for accurate quantitation of mRNA.RT-PCR (reverse transcription–polymerase chain reaction): PCR that is performed on cDNA generated from RNA. It is used for mRNA detection and quantitation.Supervised classification: A process in which classifiers are learned from user-defined groups (classes).Unsupervised classification: A process in which classifiers are learned without user-defined groups (classes), i.e., without a predefined training set.In diseases that do not require tissue resection for diagnosis or therapy, it is rare to obtain tissues for analysis. This problem is even more pronounced in diseases like MS, in which the target organ is the very inaccessible brain and spinal cord. Despite these limitations, several groups used microarrays to analyze brain tissues obtained posthumously from patients who had MS and identified genes that characterized either acute or chronic lesions ,10,11. HIn MS, looking for markers of disease activity in the much more accessible peripheral blood does not require a significant leap of faith. MS is an autoimmune disease, and it is possible that some of the cells involved in the pathogenesis of the disease will be found in the bloodstream. Abnormal T cell populations have repeatedly been observed in the peripheral blood of patients with MS ,13,14. WFortunately, recent observations suggest that these doubts are unfounded. Bomprezzi et al.  determinWeinstock-Guttman et al.  analyzedPLoS Biology, Baranzini et al. [And now, in a new study published in last month's i et al.  provide Interestingly, individual and pairs of genes did not perform that well, and all three genes in a triplet were required for the highest accuracy (about 80%–90%). The minimal combinatorial number of genes that contains the most predictive information is not available since combinations of more than three genes were not performed. Although the results were not tested on an independent dataset, as is frequently requested , the autWhat could Baranzini and colleagues' findings mean? Clearly, the most obvious conclusion is that the lack of response did not result from the deactivation of IFNβ. The effect of IFNβ on MX1, IFNAr1, and STAT2 was observed for two years in all patients, suggesting that the response did not depend on IFNβ bioavailability. Considering that PBMCs represent an admixture of multiple cell types, the most plausible explanation is a simple lack of shift in subcellular populations.However, the importance of Baranzini and colleagues' study lies not in its mechanistic insights, but in its clinical relevance. The careful design of the experiment, the use of reproducible real-time PCR instead of microarrays, the meticulous analysis, and the previous observations ,17,19,20


Malignant small bowel tumors are very rare and leiomyosarcoma accounts for less than 15% of the cases. Management of these tumors is challenging in view of nonspecific symptoms, unusual presentation and high incidence of metastasis. In this case report, an unusual presentation of jejunal sarcoma and management of liver metastasis with radiofrequency ablation (RFA) is discussed.A 45-year-old male presented with anemia and features of small bowel obstruction. Operative findings revealed a mass lesion in jejunum with intussusception of proximal loop. Resection of bowel mass was performed. Histopathological findings were suggestive of leiomyosarcoma. After 3-years of follow-up, the patient developed recurrence in infracolic omentum and a liver metastasis. The omental mass was resected and liver lesion was managed with radiofrequency ablation.Jejunal leiomyosarcoma is a rare variety of malignant small bowel tumor and a clinical presentation with intussusception is unusual. We suggest that an aggressive management approach using a combination of surgery and a newer technique like RFA can be attempted in patients with limited metastatic spread to liver to prolong the long-term survival in a subset of patients. Malignant tumors of the small bowel are rare and accounts for the <2 % of total gastrointestinal (GI) malignancy ,2. The aA 45-year-old male was referred to our center with the diagnosis of suspected non-Hodgkin's lymphoma (NHL) of the bowel in December 1999. He had generalized weakness for 2-years along with recurrent vomiting, occasional constipation and melaena for last 2 months. The diagnosis was considered after an ultrasound (USG) guided fine needle aspiration cytology (FNAC) from the intra-abdominal mass done elsewhere and showed features suspicious of NHL.At presentation, patient's general condition was poor and he was dehydrated and pale. There was no peripheral lymphaedenopathy. Abdominal examination revealed an ill-defined, mobile, nontendor lump in left paraumblical region extending up to left lumber region. There was no hepato-splenomegaly. Examination of chest and cardiovascular system was normal. After initial resuscitation with crystalloids and blood transfusion, patient was further investigated.At presentation his hemoglobin was low (6.4 gm%) but rest of the routine haematological investigations were within normal limit. The chest X-ray was normal. Abdominal ultrasound (USG) showed a left upper abdominal mass lesion suggestive of bowel mass. The upper GI endoscopy was normal. The barium meal follow through examination was suggestive of intussusception of proximal jejunum and a suspected mass lesion at the leading edge of intussusceptum. An USG guided core needle biopsy of mass was performed because of earlier suspicion of NHL, which showed smooth muscle bundles with areas of necrosis. Based on the above findings and biopsy report, patient was taken up for exploratory laparotomy 3 weeks after initial presentation.Operative findings revealed an intussuscepted proximal jejunum loop 12 cm distal to duodeno-jejunal flexure and a vascular polypoidal growth measuring 6.5 × 5 × 3.5 cms on serosal surface of jejunum. Liver and spleen were normal. There was no evidence of mesenteric or retroperitoneal lymphaedenopathy, ascites or peritoneal disease. The resection of involved segment of jejunum with 5 cm margins and an end-to-end anastomosis was performed. The postoperative course was uneventful.Pathological examination of the specimen revealed two mass lesions measuring 4.5 cm & 3 cm in continuity  located on mucosal and serosal surface of jejunum respectively. Microscopically it showed spindle cell tumor with a mitotic rate of 5/10 high power fields (HPF). The tumor was negative for desmin, S-100, CD 34 and c-kit (CD117) but focally positive for actin on immunohistochemistry (IHC). The resection margins were free. The final diagnosis of malignant spindle cell tumor – leiomyosarcoma of jejunum was made. Due to negative margins and no evidence of disease elsewhere, no adjuvant therapy was planned and patient was kept on regular follow up. He was assessed clinically at three months interval and an USG of abdomen was performed six monthly. Patient remained disease free for 39 months. After that a follow-up USG showed a space-occupying lesion (SOL) in segment VII of liver and a mass lesion in left upper abdomen. The patient was asymptomatic and abdominal examination revealed no abnormality. A computerized tomographic (CT) scan of abdomen was performed which showed a 2 × 2 cms, brightly enhancing SOL in segment VII of liver  declare that they have no competing interests.SVSD, NKS: Surgical management and review of manuscript.AS, SH, SK: Review of literature and preparation of manuscript.ST, DKP: Radiofrequency ablation.All authors have read and approved the contents of manuscript.