Migraine is a common genetically linked neurovascular disorder. Approximately ∼12% of the Caucasian population are affected including 18% of adult women and 6% of adult men ( Migraine is a complex debilitating neurovascular disorder, characterized by recurrent attacks of headache that differ in intensity, frequency and duration. The headache is often associated with an assortment of symptoms which can include nausea, emesis, photophobia, phonophobia, and occasionally, visual sensory disturbances. Migraine is estimated to affect approximately 12% of the Caucasian population  and showMost migraine sufferers probably possess a number of genes that together contribute to susceptibility. Thus far genetic linkage and association studies have implicated a number of susceptibility genes and causative mutations that are of significant clinical relevance in migraine. However not all migraine genes have been uncovered and further research is necessary to determine the definitive molecular genetics of migraine. Numerous theories regarding the causes and underlying mechanisms that result in migraine symptoms have also been proposed.The pathophysiology of this disorder implicates both neurological and vascular mechanisms. Current research suggests that the trigeminovascular system plays a significant role in migraine -6 due toMigraine has a strong inherited component, and a large genetic study  suggestsThe pioneering work of Hayashi in 1954 established the physiological significance of glutamate as an excitatory neurotransmitter . HayashiGlutamate is a nonessential amino acid that does not cross the blood-brain barrier but must be synthesized inside neurons from local precursors . GlutamaThe neuronal/glial cell interface where glutamate cycling occurs contains glutamate receptors, which are responsible for signal input; plasma glutamate transporters, which are responsible for signal termination and vesicular glutamate transporters for signal output through exocytic release . These aGlutamate is the most abundant excitatory neurotransmitter in the brain; it is critical to the communication of nerve cells with one another in practically every circuit in the nervous system. Glutamate communicates in this circuit via two main subtypes of membrane receptors, ionotropic and metabotropic. The family of ionotropic receptors is divided into three groups, referred to as N-methyl-D-aspartate (NMDA) receptors, α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and kainate (KA) receptors on the basis of DNA sequence similarity and their activation by different pharmacologic agonists Table  15). Th. Th15). The difference between ionotropic and metabotropic receptors is that ionotropic receptors are ligand-gated ion channels, while the metabotropic receptors Table  (mGluRs)2+ permeability of the channel . The role of adenosine deaminase RNA editing of glutamate and serotonin receptor transcripts is further exemplified by the disorder, amyotrophic lateral sclerosis (ALS) (MIM 105400). In amyotrophic lateral sclerosis (ALS) editing of mRNA encoding the GluR2 subunit of glutamate AMPA receptors in spinal motor neurons is defective and interferes with normal functioning of the glutamate receptors. Knockout mice in RNA editing genes are lethal implying that this mechanism is essential for survival and dysregulation could potentially affect Glutamatergic function. Genes involved in RNA editing are candidate genes to investigate due to the impact that this mechanism has on the Glutamatergic system.X-ray crystal structures have produced 3D images that have helped understand the structural basis of glutamate receptors revealing which domains are involved in binding to agonists and antagonists and to study the effect of mutations on protein conformation to understand how this relates to neuronal function in neurological disease. So far a number of structures have been described for a membrane-spanning tetrameric glutamate receptor as well as in complex with various agonists, antagonists, and modulators -27. ThesGlutamate concentrations in the extracellular space under normal conditions are kept low and tightly controlled by EAAT transporters present in both nerve endings and surrounding glial cells. Perturbations to this regulatory system can have deleterious effects such as excess release of glutamate, which can induce hyperexcitability in post-synaptic neurons to the point of excitotoxicity and cell death . The traThere are two transport systems: the plasma GluTs, that are responsible for signal termination; and the vesicular GluTs for signal output through exocytic release. Five different ‘high-affinity’ glutamate (excitatory amino acid) transporters have been identified by cloning including Excitatory Amino Acid Transporters located in the plasma membrane, EAAT1, EAAT2, EAAT3, EAAT4 and EAAT5. The vesicular GluTs are crucial for the storage of Glu in synaptic vesicles, three isoforms exist VGLUT1, VGLUT2, VGLUT3 . GeneticObservations by Lucas and Newhouse in 1957  describe2+ ions into the cell from over stimulation with glutamate  such as Multiple sclerosis, Alzheimer’s disease, Amyotrophic lateral sclerosis (ALS), Fibromyalgia, Parkinson’s disease, and Huntington’s disease. The mechanism of this excitotoxicity is thought to be due to the entry of high levels of Calutamate . Ca2+ in and DNA . This isOur understanding of ‘migraine genetics’ is an evolving subject due to its relative novelty in this disease system. There have been some successes such as the recent identification of a functional mutation in the KCNK18 gene. The KCNK18 gene encodes TRESK, a potassium channel that is part of the subfamily K member 2 (K2P) channels, which are expressed throughout the central nervous system, including the trigeminal ganglion neurons . A numbeThe most established molecular knowledge of migraine comes from mutations in the three genes for familial hemiplegic migraine (FHM) – CACNA1A, ATP1A2 and SCN1A -40. FamiIon channel genes play a critical role in normal functioning of the central nervous system where they control important biological functions including the release of neurotransmitters, hormones and muscle contraction . Ion chaet al., 2010 who found a positive association in 3 SNPs in the AMPA receptors GRIA1 and GRIA3 in an Italian population , Huntington Disease, HIV-associated dementia, malignant glioma, and other neurologic disorders . Jen et seizures . The autet al., 2009 in patients with Episodic Ataxia (EA) identified a novel pathogenic mutation C186S in one patient. The mutated EAAT1 protein showed severely reduced uptake of glutamate (Another study of the EAAT1 gene by de Vries lutamate . The sevet al., 2011 evaluated the contribution of polymorphisms in the EAAT2 transporter and found no direct association between this genetic factor and migraine (An association study by Shin migraine . The EAAmigraine -53. A stAlterations in the function or expression of components of this system may be involved in migraine susceptibility. Further research into the Glutamatergic system is necessary to ascertain its role in migraine aetiology.Pharmacological compounds capable of modulating glutamate receptors have helped untangle the functional role of glutamate receptor family members and present promising targets for the treatment of migraine. Considerable scope however remains for the development of novel ligands that will encompass the family of glutamate receptors. Currently the most promising compounds reported in the literature include: topiramate, ketamine and memantine. Several types of drugs, like generic beta blockers, calcium channel blockers, tricyclic antidepressants and anti-epileptic drugs are given to prevent migraines, these are not always effective in all patients. Targeting the glutamatergic system offers a novel approach to treatment in view of the limited efficacy of existing drugs.+ and Ca2+ channels, enhancement of GABA activity, and blockade of ionotropic glutamate receptors (Topiramate is a derivative of the naturally occurring monosaccharide D-fructose that was originally developed as an anticonvulsant and is recognized as an effective medication for migraine prevention . Topirameceptors .A few other compounds Memantine, Ketamine and ADX10059 are drugs that act on glutamate signalling through NMDA receptors , 56. MemMigraine is a disabling costly brain disorder, with hypothesised involvement of neurotransmitters. The major excitatory neurotransmitter of the brain, glutamate and the receptors, upon which it acts, are intimately involved in trigeminovascular nociceptive processing. Given the importance of the Glutamatergic system and its involvement in biological processes involved in the brain, genes of this system remain candidates for further investigation. The role of glutamate antagonists in the treatment of migraine is added evidence of a role for glutamate in migraine. Further research is required to elucidate the mechanism through which GRIA genes may contribute to migraine and to determine if other unknown mutations in components of this system may be contributing to the migraine phenotype.st century. The genetic studies are small and more data is needed to draw any solid conclusion about involvement of Glutamatergic genes in migraine. Nonetheless the genetic evidence is growing with association, linkage and GWAS results bringing to light new variants and genomic regions.Genetic characterization of migraine as a disorder is making steady progress with an increasing number of genomic susceptibility loci now identified. The data and ideas presented above have lent strong support implicating glutamate biology in migraine pathophysiology at the turn of the 21The identification of these migraine specific loci will contribute to more specific pharmacotherapeutics for the patient. Genetic variation greatly affects patient response to treatment and further insight could lead to more individualized treatments leading to better tolerability.


Hypomethylating drugs are useful in the management of Myelodysplastic syndromes, but there are only few reports on chronic myelomonocycitic (CMML) leukemia patients. We describe our experience in 3 CMML patients treated with azacitidine. Two patients obtained partial response after 4 treatment cycles with only minor toxicity and are in continuous partial response, with stable peripheral blood counts, at 29 and 30 cycles from treatment start. Chronic myelomonocytic leukemia (CMML) is a clonal disorder of hematopoietic stem cells often occurring in elderly patients. It was originally classified by the French-American-British (FAB) working group as a myelodysplastic syndrome characterized by monocytosis (> 1000/microl), bone marrow monocyte infiltration, blast cells less than 5% in the peripheral blood and less than 30% in the bone marrow.2CMML is a very hard disease to treat and the prognosis is quite variable, with a median survival of about 20 months. Patients elderly age and comorbidites impact on survival, making bone marrow transplantation only rarely possible. Patients are usually treated with supportive care or mild cytoreductive therapy, like hydroxyurea or low dose cytarabine, until leukemic evolution. In the era of epigenetics, the possibilities offered by the new molecular targeted therapies offer the chance of modifying the natural history of this disease. Among these, the hypomethylating agents Azacitidine and Decitabine have been shown to induce responses in CMML patients. Azacytidine is mostly incorporated into RNA and reaches DNA following reduction by ribonucleotide reductase, while decitabine is directly incorporated into DNA. Both drugs have been shown to produce a direct decrease of DNA methyltransferase activity in vitro and in vivo, reverting aberrant DNA methylation and increasing the expression of silenced genes, leading to cellular differentiation and/or apoptosis.4We treated 3 CMML-2 patients (2 men and 1 woman) of a median age of 63 years (range 54–69), and IPSS intermediate-2/high . Patient6Efficacy of Azacitidine and Decitabine in the treatment of MDS has been confirmed by several national and international clinical trials although a protocol comparing efficacy of the two drugs has not been performed yet. These studies demonstrated efficacy of hypomethylating agents compared to best supportive care.A study by the MD Anderson reported the use of decitabine in 19 elderly CMML patients, with encouraging results, with achievement of 58% complete remission (CR) and 11% hematological improvement (HI), resulting into an overall response rate of 69%.16Our findings confirm highly favorable response rates to azacitidine therapy in high-risk CMML patients, encouraging the application of the treatment in this subset of patients. A correct risk assessment requires the identification of new molecular and clinical features predictive of reponse to hypomethylating agents, to make an effective patient-targeted approach feasible.


In utero haematopoietic stem cell transplantation (IUHSCT) is a non-myeloablative approach for the prenatal treatment of genetic disorders. However, in target disorders, where there is not a selective advantage for donor cells, a useful donor-cell chimerism has not been achieved. There are three possible barriers to engraftment following IUHSCT: limited space in the fetus due to host-cell competition; the large number of donor cells needed, and the immunological asset of recipient. Animal models have shown different levels of resistance to IUHSCT engraftment. In primate, goat, rat and mouse the levels of engraftment that has been achieved were low and not therapeutic. Among 46 cases of IUHSCT reported in humans, successful engraftment was obtained only in cases of X-SCID. Useful levels of chimerism has not been achieved in non-immunodeficiency diseases, and a detectable engrafment, was reported only in one case of ß-thalassemia transplanted at 12 weeks of gestation by fetal liver cells. In one α-thalassemia case, where α-globin-dependent hemoglobin production and anemia are present during fetal period, microchimerism and tolerance were suggested.To overcome the IUHSCT engraftment barriers, it is necessary to develop strategies to improve the competitive capacity of donor cells and to define the gestational age of the possible immunological “window of opportunity” in the human fetus.In utero haematopoietic stem cell transplantation (IUHSCT) is a non-myeloablative promising approach for the prenatal treatment of a variety of genetic disorders and could be an alternative option to therapeutical abortion in some congenital diseases like haematological hereditary syndromes.Observations of “naturally” occurring hematopoietic chimerismAlthough IUHSCT has been clinically successful in severe combined immunodeficiency disease (SCID), a therapeutic donnor-cell chimerism (more than 1%)The reasons for this failure are unclear and reflect a limited understanding of the engraftment barriers involved in clinical IUHSCT.There are three possible barriers to allogenic engraftment following IUHSCT : limited space in the fetus due to host-cell competition from a robust and highly proliferative fetal hematopoietic compartment; the large number of donor cells needed to successfully compete with host cells for these sites; and the immunological asset of recipient that determines the grade of the possible fetal tolerance which is conditional, depending on timing and appropiate presentation of antigenth weeks of gestationThe last barrier has been supported by successful allogenic IUHSCT in sheeps and monkeysThe goal of therapeutically relevant levels of chimerism following a single in utero transplantation has been difficult to achieve. The induction of tolerance with low levels of chimerism to facilitate postnatal cellular transplantations using minimally ablative conditioning regimens is a more realistic goal for clinical application of IUHSCT.Hematopoietic stem cells (HSC) have been isolated from fetal liverAll these cell sources were employed to perform IUHSCT; however, if postnatal cellular therapy is needed, the donor cells must be obtained from a renewable source as adult related bone marrow or mobilized peripheral bloodAn “experiment of nature” first described by Owen in 1945 is the best supporting evidence that IUHSCT might work : dizygotic cattle twins that share cross-placental circulation were born chimeric for their sibilings blood elements, and this state of “chimerism” persisted for life and was associated with specific transplantation toleranceThe first animal model was the sheep. Early gestational transplantation of allogenic, fetal liver–derived HSC into normal sheep fetuses resulted in a multilineage hematopoietic chimerismOther normal animal models have shown different levels of resistance to engraftment after in utero transplantation. In the normal primate, goat, rat and mouse (12–16) the levels of engraftment that has been achieved were low and not therapeutic: successful allogenic IUHSCT in monkeys occurred if donor and recipient fetuses are in a preimmune conditionAlmeida-Porada et al. 2000, used the in utero model of human–sheep HSC transplantation to investigate ways of improving engraftment and differentiation of donor cells after transplantationHayashi et al 2002, obtained the induction of prenatal tolerance in mice where a combined approach of IUHSCTx, performed by intraperitonel injection, followed by postnatal donor lymphocyte infusion has converted low-level mixed hematopoietic chimerism to complete donor chimerismA mouse model described by Perenteau et alHowever, further studies are needed to better characterize the timing of human fetal NK-cell maturation.The reasons for the failure in the correction most diseases and the absence of predicting tolerance in absence of immunosuppression, both in human or animal models, of IUHSCT are still unclear and reflect a limited understanding of the engraftment barriersSince Touraine et al published the first instance of intrauterine transplantation in a human fetus affected by bare lymphocyte syndrome (BLS) in 1989However, successful well-documented engraftment after in utero therapy was obtained only in cases of X-linked SCID by Touraine, Flake and WenglerUseful levels of chimerism has not been achieved in non-immunodeficiency diseases, as metachromatic leukodystrophyParticularly, a detectable engrafment, using IUHSCT, was reported only in one case of thalassemia major transplanted at 12 weeks of gestation by fetal liver cellsOnly in one α-thalassemia case, transplanted at 13 weeks of gestation with paternal CD34+ enriched cells, microchimerism and tolerance were suggestedth and the 18th weeks of gestation using cryopreserved fetal liver haematopietic stem cells and without immunodepression, was also unsuccessfulOur experience, in cases treated between the 13th and the 20th week of gestation, generated an alloimmune response as demonstrated by an high cytotoxic T cell precursor (CTLp) frequency against donor cellsParticularly, a fetus, affected by ß-thalassemia major who underwent a fetus-to-fetus transplantation between the 14We set-up a clinical protocol by which two female fetuses, 16–20 weeks old, were transplanted with circulating hematopoietic paternal stem cells after one week of low dose dexamethasone treatment with the aim to overcome the engraftment barrier by inducing a transient mild fetal immuno suppression and induce a donor-specific tolerance. During these procedures no adverse events were observed both for the mothers and the fetuses. At birth, both fetuses showed the presence of Y chromosome of donor origin but only one, at two months of age, showed the presence of paternal blood group allele A cDNA in peripheral blood and an increase of HbA concentration from 3% to 14.4%, suggesting that erithroid microchimerism has been occurred. At 5 months of age haemoglobin decreased to 6.8 g/dl and the girl started a regular blood transfusional regimenTo overcome the barriers that hinder the success of IUHSCT, it is necessary to develop strategies to improve the competitive capacity of donor cells to achieve significant engraftment, and to define the gestational age of the possible immunological “window of opportunity” in the human fetus.A significant barrier to allogeneic engraftment following IUHSCT is limited space in the fetus due to host-cell competition from a robust and highly proliferative fetal hematopoeitic compartment in circumstances where a competitive or survival advantage does not exist for donor cellsThus, strategies that selectively increase donor HSC homing and engraftment in the fetal recipient may increase both the levels and frequency of engraftment following allogeneic IUHSCT. Dipeptidyl peptidase CD26 inhibition may represent a novel approach to increasing the efficacy and success of HSC/HPC transplantationRecent in vitro studies have demonstrated that increased cleavage of stromal-derived factor 1 (SDF-1) by CD26, a chemokine involved in hematopoietic cell chemotaxis, homing, mobilization and survival, results in a loss of its chemotactic effect on primitive hematopoietic cells. Instead, its inhibition results in SDF1–induced migration In vivo, postnatal studies in a murine bone marrow transplantation (BMT) model found increased short-term bone marrow (BM) homing of enriched HSC and low-density BM donor cells after inhibition or absence of CD26 activity. Reduction of CD26 activity on low-density donor BM cells also resulted in increased long-term engraftment, competitive repopulation, secondary transplantation, and mouse survival following lethal irradiation. Therefore, CD26 inhibition may be an important adjunct to other strategies that are directed toward overcoming the barriers to fetal engraftmentOther manipulations that selectively influence expression of homing receptors and engraftment include cotransplantation with stroma. The use of stromal cotransplantation has increased short- and long-term donor cell expression in the sheep modelTherefore, the recognition of an immune barrier suggests potential immune-based strategies for application to IUHSCT that might prevent loss of engraftment.th week of gestation suggested by the presence of alloreactive T lymphocytes from the 10th week of gestation that could explain the failure of engraftment in fetuses older than this gestationth week of gestation. However, at this so early gestation it is impossible to carry out the infusion of CD34 into the umbilical cord by intravascular route or by their infusion into intraperitoneal cavity. One possible future approach could the infusion of these cells into the coelomic cavity.Some studies in miceCoelocentesis at 6–10 weeks of gestation has been successfully used to obtain fetal DNA for very early prenatal diagnosis of genetic diseasesThe injection of donor haematopoietic stem cells into this cavity could potentially induce tolerance in the fetus making successive booster stem cells infusions possible. This procedure has already been evaluated in pre-immune fetal sheep in which the injection of human stem cells through the coelomic cavity was associated with a significant level of engraftmentThe intrauterine approach was criticized by several authorities in this field who claimed that this approach did not offer any advantage over postnatal transplantation. The main arguments against intrauterine transplantation were that the fetal invasive procedure carried a certain additional risk.Conversely, promoters of intrauterine transplantation claimed that reconstitution of the immune system before birth results in reduced susceptibility to infections in the neonatal period, and to an improved psychosocial situation for the family. Other potential advantages for an intrauterine approach include cost savings and a reduced risk of GVHD in the fetal environment.Thus, a consensus has been difficult to reach and fetal transplantation has not been widely adopted, despite the fact that successful cases treated in utero had outcomes comparable with the best reported with postnatal transplantation


Epidemiological studies have provided overwhelming evidence for a causal role of chronic HBV infection in the development of hepatocellular carcinoma (HCC), but the molecular mechanisms underlying virally-induced tumorigenesis remain largely debated. In the absence of a dominant oncogene encoded by the HBV genome, indirect roles have been proposed, including insertional activation of cellular oncogenes by HBV DNA integration, induction of genetic instability by viral integration or by the regulatory protein HBx, and long term effects of viral proteins in enhancing immune-mediated liver disease. In this chapter, we discuss different models of HBV-mediated liver cell transformation based on animal systems of hepadnavirus infection as well as functional studies in hepatocyte and hepatoma cell lines. These studies might help identifying the cellular effectors connecting HBV infection and liver cell transformation. Thus, for HBV and HCC, as well as for hepatitis C virus (HCV) and HCC, etiological relation might be viewed as indirect, being caused by persistent viral replication and chronic tissue injury, rather than by inherent oncogenic features of the virus [A number of viruses including the hepatitis B virus (HBV) have been recognized as human oncogenic agents ,2. The che virus . The rolhe virus ,11). Nothe virus ).In this article, we summarize some of the findings that support these notions and discuss the possible implications for our understanding of HBV-related tumorigenesis.2.The human hepatitis B virus is the prototype of the hepadnavirus family, which includes closely related viruses infecting a limited number of primates, mammals and birds . MammaliWHV-infected woodchucks develop chronic hepatitis and HCCs that are in many points similar to those associated with HBV infection in humans, although the liver of this rodent species is not susceptible to cirrhosis. In experimental inoculations shortly after birth, virtually all woodchucks that become chronic WHV carriers develop HCCs with a median tumor-free survival of 24 months and a median life expectancy of 30–32 months . Moreovemyc family genes. The patterns of WHV DNA insertion in c-myc in these tumors share common aspects with those of Moloney murine leukemia virus (MLV) in murine T-cell lymphomas [Search for oncogenes at integration sites in woodchuck HCCs has led us to demonstrate that WHV acts mainly as an insertional mutagen of ymphomas .myc genes, particularly in the intronless retroposon N-myc2 and in two nearby loci on the X chromosome called b3n and win [myc2 gene have been found preferentially in large, advanced stage tumors, highlighting the strong oncogenic activity of N-myc2 [myc or N-myc oncogenes, evidence for a direct role of Myc activation in woodchuck tumorigenesis came from the finding that virtually all transgenic mice carrying WHV insertion sites in c-myc and N-myc2 developed HCC [The highest frequency of viral integrations was found in the woodchuck N- and win –28  gene and resulted in the fusion of 29 amino-terminal residues of the HBV pre-S1 gene to the DNA-binding and hormone-binding domains of RARß [In human HCCs, HBV DNA integration sites have been mapped to multiple regions on virtually all chromosomes, suggesting that viral integrations are distributed randomly throughout the host genome ,43. Thes of RARß ,45. Expr of RARß . In a se of RARß ,48. Such of RARß . In thesMore recently, the notion that HBV integrates randomly into human chromosomes has been challenged by large-scale analysis of HBV DNA integration sites using the Alu-PCR approach. These studies have revealed that targeting of cellular genes by HBV integration is a more frequent event than suspected previously ,50. As pBesides acting by cis–or trans-activation, HBV insertions have been associated with major genetic alterations within the cell genome, including large deletions, duplications and chromosomal translocations –60. The 4.Among the viral gene products, the HBx protein encoded by the HBV X gene has been termed “viral oncoprotein” by many authors, and it has been involved in liver cell transformation because of its pleiotropic activities on cell cycle regulation, signaling pathways and DNA repair (reviewed in –71) Fig. HoweverThe oncogenic potential of HBx has also been addressed in transgenic mice, with again divergent results that probably reflect the use of different murine genetic backgrounds and various promoters yielding different expression levels of the viral protein. Development of HCC associated with high-level expression of HBx in the liver was essentially described for a transgenic mouse line generated in the outbred CD-1 background . In otheWAF1/Cip1 is a master effector of tumor suppressor pathways that promote cell cycle arrest in response to various stimuli, and its genetic ablation might cooperate with HBx in cell transformation. Recently, it has been shown that constitutive liver expression of HBx and insulin receptor substrate-1 (IRS-1) contribute cooperatively to the development of dysplasia and eventually HCC [Transgenic expression of the viral protein cooperates with chemical carcinogens such as diethyl nitrosamine (DEN) in accelerating hepatocarcinogenesis . By crosally HCC . The mulThe mechanisms implicating HBx in the tumoral process have been investigated in HBx transgenic models. It has been shown that hepatic expression of HBx is associated with a significant increase in S-phase hepatocytes in young animals but not in adult mice , while o4.Recent studies have led to a consensus agreement on the fact that HBx interferes with the normal control of cell cycle progression. This function may be attributed to the ability of HBx to bind different cellular partners and to activate transcription as well as various signaling cascades –99. The Interestingly, in some studies these deleterious effects of HBx have been observed in the context of viral replication, and they were detectable in dividing, but not in quiescent hepatoma cells . DiffereOther studies have shown that HBx effects on centrosome dynamics and mitotic spindle formation are associated to the binding of HBx with different cellular partners implicated in centrosome formation. It has been reported that HBx interacts with HBXIP, a major regulator of centrosome duplication, required for bipolar spindle formation and cytokinesis. This interaction appears to be responsible for the formation of defective spindles and subsequent aberrant chromosome segregation . In otheAlthough the precise HBx activities that interfere with the mitotic cell cycle remain to be determined, these data provide insight into possible mechanisms leading to deregulation of the cellular mitotic machinery by a viral protein. They also provide a strong link between HBx expression and chromosomal instability in HBV-related carcinogenesis.cis- or trans-activation of cancer-related genes. The large variety of pathological properties attributed to the viral protein HBx indicates that multiple cooperative mechanisms may operate in the development of liver cancer. Future studies identifying more precisely the cellular effectors that mediate the tumorigenic effects of HBV are poised to advance the field of liver cancer biology and to develop adapted therapeutic strategies.Cancer arises from stepwise accumulation of genetic changes that confer unlimited, self-sufficient growth and resistance to normal homeostatic mechanisms. The long latency of HCC development after the primary HBV infection may be interpreted as a sign of an indirect action of the virus. It is generally admitted that host immune responses against infected hepatocytes triggers continuous necrosis and cell regeneration, leading to chronic inflammatory disease that in turn favors the accumulation of genetic alterations. In this view, long-term toxic effect of viral gene products, notably the HBx protein, as well as exogenous carcinogens such as aflatoxins and alcohol, potentiate the action of chronic liver inflammation. However, tumor onset might also depend on the occurrence of a decisive HBV integration event that would promote genetic instability or lead to


Abundant herbivores can damage plants and so cause conflict with conservation, agricultural, and fisheries interests. Management of herbivore populations is a potential tool to alleviate such conflicts but may raise concerns about the economic and ethical costs of implementation, especially if the herbivores are ‘charismatic’ and popular with the public. Thus it is critical to evaluate the probability of achieving the desired ecological outcomes before proceeding to a field trial. Here we assessed the potential for population control to resolve a conflict of non-breeding swans grazing in river catchments. We used a mathematical model to evaluate the consequences of three population management strategies; (a) reductions in reproductive success, (b) removal of individuals, and (c) reduced reproductive success and removal of individuals combined. This model gave accurate projections of historical changes in population size for the two rivers for which data were available. Our model projected that the River Frome swan population would increase by 54%, from 257 to 397 individuals, over 17 years in the absence of population control. Removal of ≥60% of non-breeding individuals each year was projected to reduce the catchment population below the level for which grazing conflicts have been previously reported. Reducing reproductive success, even to 0 eggs per nest, failed to achieve the population reduction required. High adult and juvenile survival probabilities (>0.7) and immigration from outside of the catchment limited the effects of management on population size. Given the high, sustained effort required, population control does not represent an effective management option for preventing the grazing conflicts in river catchments. Our study highlights the need to evaluate the effects of different management techniques, both alone and in combination, prior to field trials. Population models, such as the one presented here, can provide a cost-effective and ethical means of such evaluations. How to manage species and environments for the benefits of conservation, economic, and human well-being objectives is a central challenge facing ecologists Population control measures on vertebrate herbivores typically focuses on reducing population size by (i) reducing reproductive success to reduce recruitment into the population, or (ii) removing individuals to reduce population size directly Cygnus olor Gmelin, 1789) are charismatic herbivores known to reduce plant abundances in both aquatic and terrestrial habitats Large vertebrate herbivores can damage vegetation of ecological and socioeconomic value through consumption, trampling and altered nutrient concentrations Cygnus atratus Latham, 1790) populations in the United States and New Zealand respectively faced widespread public opposition, legal challenges, and damaged relations between different stakeholder groups, in particular government wildlife officials, landowners and animal welfare groups Mute swans are protected under the EU Wild Birds Directive (2009/147/EEC), implemented in the UK through the Wildlife and Countryside Act (1981), making it illegal to capture, kill or injure swans, or to disturb or damage swan nests or eggs Mathematical models allow the projection and evaluation of the consequences of management decisions on species abundances and distributions In this study we first tested the ability of an age structured population model, incorporating territory availability, to generate accurate projections sensu  of histo−1 of river. This estimate seems sensible as the two chalk river systems for which grazing conflicts are known have densities which exceed this; the River Wylye density is 5.5 ind. km−1 and the current River Frome density is 3.8 ind. km−1The River Frome  is a shallow (<1.5 m depth), mesotrophic chalk river that flows through a catchment dominated by mixed pastoral and arable agriculture 1), juveniles (N2), non-breeding adults (N3) and breeding adults (N4). Adult swans were further subdivided into breeding and non-breeding adults based on territory availability; adults were defined as breeding if they were randomly allocated one of the fixed number of territories (T).We constructed an age-structured population model with a twelve month time step All model parameters were derived from data presented in studies of chalk river swan populations Table 1.i.e. relative numbers of adults, juveniles and cygnets) on the mean (± SD) September counts given in i.e. until they have become juveniles i.e. a longer section of river should receive more immigrants as it has a larger surrounding area to supply such immigrants. The mean annual numbers of juvenile and adult immigrants to a 16.9 km length of chalk river have been estimated as 1.66±0.73 SD and 10.58±4.44 SD respectively We estimated T as 38±9.24, after the mean densities of breeding pairs per length of chalk river reported by Even deterministic models with relatively simple structures can yield complicated population dynamics To test the accuracy of the projections of our model, we compared model projections with historical population data for the swan populations of a 34.8 km section of the River Wylye We tested three management strategies; (a) the effects of manipulating reproductive success, (b) the effects of removing a given percentage of the non-breeding subpopulation, and (c) the combined effects of (a) and (b), on the projected swan population size. The non-breeding subpopulation was targeted as grazing conflicts have been reported for these, but not breeding, individuals and thus removal of breeding birds is likely to prove unacceptable to stakeholders We assessed the sensitivity of the projected population size to changes in the mean value of each parameter using the one-at-a-time method of local sensitivity analysis The population sizes projected by our model, as a mean percentage of the observed population sizes, were 91% (range 72–126%) for the River Wylye population (i.e. a population size which did not vary thereafter (The model projected a 54% increase in population size, from 257 to 397 individuals, within 17 years if no management action were taken Figure 3As expected, reducing the number of eggs per nest below the current mean value of 4.4 resulted in lower population sizes; greater reductions in clutch size resulted in lower population sizes (Our model projected that the sustained removal of ≥60% of non-breeding birds each year would achieve a stable population below the target threshold of 155 swans (A range of different combinations of annual egg and non-breeder removals were projected to reduce population size below our target threshold of 155 swans Figure 4Varying parameters by ±5% indicated that the stable population size projected by our model was affected most strongly by changes in the survival rate of non-breeding adults, which comprised the majority of the non-breeding population, with some, albeit lower, effects of the survival rates of breeding adults, juveniles and cygnets Figure 5In this study we constructed and tested a mathematical model of mute swan populations causing conflicts with agricultural, fisheries and conservation interests. The model built and improved on previous swan population models by assessing management at the catchment-scale and by incorporating territoriality, which is a key factor regulating mute swan population dynamics In common with a range of other studies of animal populations, our model suggested that reductions in populations could be achieved through reduced productivity or the removal of individuals Several mechanisms were likely to have prevented clutch reductions and ≤60% removals of non-breeders from reducing population size below 155 individuals. Firstly, the high apparent survival rates of juveniles, non-breeding adults and breeding adults meant that these individuals persisted within the population. Similar studies have also concluded that adult survival rates have the greatest effect on model projections for long-lived herbivores The annual removals of ≥60% of non-breeding individuals would require sustained, high-levels of management intervention in the swan population. Such removals could be achieved through licenced translocation or culling of individuals. Translocations would likely be the more ethically acceptable option, especially with the public, as it is non-lethal Due to the substantial ecological, practical and ethical obstacles to effective population control highlighted in our study, alternative management strategies for grazing conflicts such as habitat alterations or feeding deterrents could be explored. Currently, there are few published data on the efficacy of different habitat management strategies and other ethical interventions on alleviating the grazing conflicts associated with charismatic vertebrate herbivores. Further research should test whether changes in habitat management could alleviate grazing conflicts associated with herbivores. Economically or ecologically valuable plant stands and associated biota could be protected by the establishment of sacrificial feeding areas near to areas of overgrazing, as have been suggested for grazing conflicts between geese and agriculture Our results highlight the importance of considering the metapopulation dynamics of the target species. Management which focuses exclusively on a single population, or a limited spatial area containing only part of a population, may be confounded by immigration from other populations within the landscape. Given the limitations of population control, it is questionable whether such control should be attempted for well-established species subject to immigration and high survival


Critical to advancing the systems-level evaluation of complex biological processes is the development of comprehensive networks and computational methods to apply to the analysis of systems biology data . Ideally, these networks will be specifically designed to capture the normal, non-diseased biology of the tissue or cell types under investigation, and can be used with experimentally generated systems biology data to assess the biological impact of perturbations like xenobiotics and other cellular stresses. Lung cell proliferation is a key biological process to capture in such a network model, given the pivotal role that proliferation plays in lung diseases including cancer, chronic obstructive pulmonary disease (COPD), and fibrosis. Unfortunately, no such network has been available prior to this work.To further a systems-level assessment of the biological impact of perturbations on non-diseased mammalian lung cells, we constructed a lung-focused network for cell proliferation. The network encompasses diverse biological areas that lead to the regulation of normal lung cell proliferation , and contains a total of 848 nodes  and 1597 edges . The network was verified using four published gene expression profiling data sets associated with measured cell proliferation endpoints in lung and lung-related cell types. Predicted changes in the activity of core machinery involved in cell cycle regulation  are statistically supported across multiple data sets, underscoring the general applicability of this approach for a network-wide biological impact assessment using systems biology data.To the best of our knowledge, this lung-focused Cell Proliferation Network provides the most comprehensive connectivity map in existence of the molecular mechanisms regulating cell proliferation in the lung. The network is based on fully referenced causal relationships obtained from extensive evaluation of the literature. The computable structure of the network enables its application to the qualitative and quantitative evaluation of cell proliferation using systems biology data sets. The network is available for public use. The immediate goal of this work was to construct a computable network model for cell proliferation in non-diseased lung. Lung epithelial cells are stimulated to proliferate upon injury as a mechanism for renewal . AlteratComprehensive and detailed pathway or network models of the processes that contribute to lung disease pathology are needed to effectively interpret modern "omics" data and to qualitatively and quantitatively compare signaling across diverse data sets. The ultimate goal of this work is to evaluate the biological impact of xenobiotics and environmental toxins on experimental systems such as lung cell cultures or whole rodent lung. Network models representing key biological processes as they occur in non-diseased cells are crucial for this effort. Tumor cell lines and other cell contexts representing advanced disease states have genetic changes and altered signaling networks that may not be present in normal, non-diseased cells. Thus, the network model described in this report is focused on biological signaling pathways expected to be functional and to regulate cell proliferation in non-diseased lung.Many different approaches can be taken to develop biological models. Biological pathways such as those captured by KEGG (Kyoto Encyclopedia of Genes and Genomes)  are manuReverse Causal Reasoning (RCR) is a systems biology methodology that evaluates the statistical merit that a biological entity is active in a given system, based on automated reasoning to extrapolate back from observed biological data to plausible explanations for its cause. RCR requires an extensive Knowledgebase of biological cause and effect relationships as a substrate. RCR has been successfully applied to identify and evaluate molecular mechanisms involved in diverse biological processes, including hypoxia-induced hemangiosarcoma, Sirtuin 1-induced keratinocyte differentiation, and tumor sensitivity to AKT inhibition -9. TheseThe Cell Proliferation Network reported here provides a detailed description of molecular processes leading to cell proliferation in the lung based on causal relationships obtained from extensive evaluation of the literature. This novel pathway model is comprehensive and integrates core cell cycle machinery with other signaling pathways which control cell proliferation in the lung, including EGF signaling, circadian clock, and Hedgehog. This pathway model is computable, and can be used for the qualitative systems-level evaluation of the complex biological processes contributing to cell proliferation pathway signaling from experimental gene expression profiling data. Construction of additional pathway models for key lung disease processes such as inflammatory signaling and response to oxidative stress is planned in order to build a comprehensive network of pathway models of lung biology relevant to lung disease. Scoring algorithms are under development to enable application of this Cell Proliferation Network and other pathway models to the quantitative evaluation of biological impact across data sets for different lung diseases, time points, or environmental perturbations.The construction of the Cell Proliferation Network was an iterative process, summarized in Figure in vitro and in vivo. This lung-focused, fully referenced Cell Proliferation Network provides the most comprehensive publicly available connectivity map of the molecular mechanisms regulating proliferative processes in the lung.The Cell Proliferation Network represents a broad collection of biological mechanisms that regulate cell proliferation in the lung, and was built using a framework that is amenable to computational analyses. The Cell Proliferation Network  were prioritized. However, in many cases, the results of the relevant detailed experiments have not been published. Thus, as a second priority, relationships derived from cell types that are found in the normal lung , but not explicitly from lung were used. The network was focused on relationships derived from experiments done in human systems, though relationships from mouse and rat were also included. Canonical mechanisms, such as the regulation of E2F transcription factor family members by the retinoblastoma protein RB1, were included in the network even if literature support explicitly demonstrating the presence of the mechanism in lung-related cells was not identified. It was assumed that the individual relationships within canonical mechanisms (for example CDKN1A inhibiting the kinase activity of CDK2) can occur in the lung. However, if canonical relationships with specific lung contexts were found in the literature, they were used. If needed for completing critical mechanisms within the network, relationships with other tissue contexts were used, provided they reflected proliferative processes that can occur in the normal lung. Causal relationships derived from embryonic tissue contexts were included, as the embryonic lung represents a model for non-diseased lung cell proliferation ,11. As aThe Cell Proliferation Network represents the biological mechanisms leading to cell proliferation in a specific organ, the lung. Thus, biological boundaries were designed to focus the network on the cellular processes and signaling pathways with a described role in regulating lung cell proliferation, with a particular emphasis on the proximal connections to core cell cycle machinery. Following an exhaustive search of the literature, a set of pathways were selected for inclusion, while other pathways with less direct relevance for proliferation were excluded, creating the mechanistic biological boundaries of the network. These biological mechanism boundaries were used to ensure that the Cell Proliferation Network represented the most relevant proliferative mechanisms that occur in the non-diseased lung.Cell proliferation can be directly or indirectly influenced by a wide range of factors, including external biological stimuli (e.g. growth factors) and internal metabolic alterations (e.g. ATP homeostasis). The broad range of factors that can influence cell proliferation, coupled with the observation that many proteins involved in regulating cell proliferation have varying degrees of biological promiscuity , necThe Cell Proliferation Network was constructed in a modular fashion using a "building block" framework in which a core Cell Cycle building block is connected to additional biological pathways that contribute to cell proliferation in the lung Figure . These sIncludes canonical elements of the core machinery regulating entry and exit from the mammalian cell cycle, including but not limited to cyclin, CDK, and E2F family members.Includes common extracellular growth factors involved in regulating lung cell proliferation, namely EGF, TGF-beta, VEGF, and FGF family members. The EGF family members EGF and TGF-alpha play critical roles in regulating the proliferation of airway epithelial cells through EGF receptor activation ,15. FGF7This block contains diverse elements of the common intra- and extracellular pathways involved in mediating lung cell proliferation, including the Hedgehog, Wnt, and Notch signaling pathways. Hedgehog signaling regulates cell proliferation and branching morphogenesis in the developing mammalian lung ,20. SimiIncludes the signal transduction pathways leading to cell proliferation that originate from the interactions of common cell adhesion molecules (including ITGB1 complexes with ITGA1-3 chains) and extracellular matrix components .Includes the main known epigenetic modulators of lung cell proliferation including the histone deacetylase (HDAC) family and DNA methyltransferase (DMT) family member DNMT1. For this block, connections from these epigenetic mediators to the core cell cycle components  were prioritized.In order to verify the content of the network, we used publicly available data from experiments in which cell proliferation was modulated in the lung or lung relevant cell types. Specifically, we analyzed transcriptomic data sets using Reverse Causal Reasoning (RCR), which identifies upstream controllers ("hypotheses") that can explain the significant mRNA State Changes in a given transcriptomic data set. Upon completing the literature model, a search was initiated for transcriptomic data sets to verify and expand the model using public data repositories such as GEO (Gene Expression Omnibus) and ArrayExpress. The ideal data set would have been collected from either whole lung or a specific untransformed lung cell type, involves a simple perturbation affecting cell proliferation , have cell proliferation phenotypic endpoint data , and have raw data available with at least three biological replicates for each sample group to clearly identify statistically significant changes in gene expression. Although this ideal data set was not found, these criteria were used to identify four "next best" data sets for these purposes Table . The EIF® Technology Platform heatmap key for Figure We performed RCR analysis on each of these four cell proliferation transcriptomic data sets and evaluated the resulting hypotheses. Foremost, we assessed whether nodes in the cell proliferation literature model were predicted as hypotheses in directions consistent with their biological roles  correctly present in the literature model and 2) detectable using this computational approach. However, equally important were the predictions for nodes in the peripheral building blocks, which 1) identify additional mechanistic detail for the proliferative pathways modulated and 2) can be used together with the hypothesis predictions in the core Cell Cycle block to assess the coverage of the literature model by all four data sets . For the purposes of highlighting the peripheral mechanisms involved in lung cell proliferation, hypotheses within the growth factors building block were especially well represented, including predicted increases in PDGF, FGFs 1, 2 and 7, HGF, and EGF and its receptors  appears to be very well-suited for the evaluation of mechanisms guiding lung cell proliferation using gene expression microarray data sets.In addition to verifying the cell proliferation literature model, RCR on the four cell proliferation data sets was used to identify other mechanisms impacting cell proliferation in the lung. The prediction of a hypothesis in a cell proliferation data set may suggest involvement in proliferation; however, they may also reflect other biological processes that are affected by the experimental perturbations in these data sets. Therefore, each of the hypotheses predicted by RCR in these four data sets that were not already included in the model was investigated to determine its role in lung proliferation. Hypotheses that were determined to play a role in lung proliferation based on surveys of the literature were then further examined to determine how they could best be integrated into the existing literature model. These nodes  were then added to the model, creating a more robust and comprehensive network of lung proliferation. The literature model supplemented with these data set-derived nodes is referred to in this paper as the integrated Cell Proliferation Network, as it takes into account not only known proliferative mechanisms operating in the lung from the literature, but also additional mechanisms determined to play a role in lung cell proliferation identified by RCR on cell proliferation data sets. For example, the transcriptional activity of Zbtb17 (MIZ-1), was predicted to be increased in the CTNNB1 data set Figure . MIZ-1 iThe content of the Knowledgebase (the substrate used to build the proliferation network) used in this study is constantly updated with the latest scientific information. As such, the proliferation model itself is dynamic, and has the flexibility to represent a contemporary view of lung cell proliferation as scientific knowledge advances. RCR prediction of a given node using gene expression data sets requires a minimum of four observed RNA expression changes that are consistent with the predicted change in node activity in the Knowledgebase. Thus, one reason that a network node may not be predicted as a hypothesis using RCR on the cell proliferation data sets is that the Knowledgebase contains too few causal connections from the node to downstream RNA expressions. To address this, we took advantage of the dynamic property of the Knowledgebase to perform targeted knowledge curation around specific nodes in order to increase the likelihood of detecting them as hypotheses using RCR. The extent of these curation efforts was limited to a subset of nodes in the proliferation network, however the structural framework used in the construction of this network allows for additional knowledge to be incorporated in the future.In order to evaluate the content of the Cell Proliferation Network we assessed the coverage of network nodes predicted by RCR (on the four cell proliferation data sets) as a percentage of total network nodes that were capable of being predicted. In all, 229 of the 848 nodes in the Cell Proliferation Network met the minimum criteria to be predicted changed by RCR (i.e. there were four or more observed RNA expression changes consistent with the predicted change contained in the Knowledgebase) and are termed the "possible nodes". Of these 229 "possible nodes", RCR predicted changes in 102 (45%) in at least one of the four cell proliferation data sets. Seventy one (31%) were predicted based on the RhoA data set alone, while 31 (14%), 19 (8%) and 47 (21%) were predicted based on the CTNNB1, NR3C1, and EIF4G1 data sets, respectively. Notably, many of the nodes for which a prediction was not possible exert their influences on proliferation via non-transcriptional events, such as phosphorylation, degradation, etc., or have limited published information regarding their influences on gene expression. As such, these nodes would be more likely predicted to increase or decrease when using a combination of systems biology data types (e.g. gene expression and phosphoproteomic arrays). These results further verify the Cell Proliferation Network, as well as the method of using RCR to predict proliferative mechanisms using systems biology data.As noted in the "Network verification and expansion" section, the ideal publicly available data set for verifying the network would have adhered to collection of quality control criteria including 1) non-diseased lung tissue focus, 2) simple perturbation of primarily cell proliferation , 3) relevant endpoint data, and 4) statistical soundness. The data sets used for evaluating the model were chosen because they all met criteria 2-4 detailed above, and were also done in lung cell relevant contexts. In fact, two of the data sets were derived from experiments done in embryonic lung, and two were done in cell types that best approximated the biology occurring in lung cells.The network described here is the first step in the larger objective of creating an integrated network of lung biology. The Cell Proliferation Network portrays the signaling pathways involved in normal lung cell proliferation with expanded coverage relative to existing representations. However, it relates only a subset of the processes involved in many complex lung diseases. For example, lung cancer is a disease of uncontrolled cell proliferation, but also involves response to DNA damage and apoptosis evasion components, among others ,49. SimiCell proliferation is a complex biological process with relevance to several common lung diseases. Modern systems biology data, such as transcriptomics, are useful in unraveling the detail embedded in complex processes like cell proliferation, but require the appropriate tools. The publicly available lung focused Cell Proliferation Network described here represents the most comprehensive and fully referenced mechanistic representation of the signaling pathways that regulate normal lung cell proliferation in existence, and is compatible with analysis using systems biology data. The adaptable and computable structure of the network makes it a useful tool for a wide variety of research investigators across broad scientific disciplines.The nodes and edges comprising the Cell Proliferation Network were added to the model from the Selventa Knowledgebase, a comprehensive repository containing over 1.5 million nodes  and over 7.5 million edges (connections between nodes). The Selventa Knowledgebase is derived from peer-reviewed scientific literature as well as other public and proprietary databases. In addition to containing a vast collective of causal relationships derived from healthy tissues, the Knowledgebase is particularly enriched in disease areas such as inflammation, metabolic diseases, cardiovascular injury, liver injury and cancer. Knowledge Assembly Models (KAMs) are subsets of the global Selventa Knowledgebase designed to facilitate reasoning and computation Figure .The human KAM is the set of causal assertions from human sources that has been augmented with orthologous causal assertions derived from either mouse or rat sources, and is competent for RCR : Automated Hypothesis Generation). Similarly, the mouse KAM is the set of causal assertions derived from mouse sources that has been augmented with orthologous causal assertions derived from either human or rat sources. Each KAM contains approximately 90,000 total nodes and 400,000 total edges, incorporating information from over 35,000 distinct citations. An example causal assertion is increased transcriptional activity of EGR1 (early growth response 1) causing an increase in the expression of CCND1 (cyclin D1). Each such causal assertion has a specific scientific citation, and the assembled collection of these causal assertions is referred to as either the human or mouse KAM in this paper. The Selventa Knowledgebase and KAMs provide a framework for developing computable, qualitative models of specific areas of biology.When analyzing public gene expression data sets for the construction and verification of the network, the full human KAM was used as the substrate for RCR; however the Cell Proliferation Network itself  reflects a subset of all the causal assertions in the human KAM.Reverse causal reasoning (RCR) was used to verify and expand the Cell Proliferation Network using cell proliferation experiments with publicly available transcriptomic profiling data. RCR interrogates a species-specific KAM to identify upstream controllers of the RNA State Changes  observed in the data set. These upstream controllers are called "hypotheses", as they are statistically significant potential explanations for the observed RNA State Changes. Hypothesis generation is performed automatically by a computer program that utilizes the KAM to identify hypotheses that explain the input RNA State Changes, prioritized by multiple statistical criteria. The substrate for analysis of RNA State Changes observed in the cell proliferation data sets is a species-specific KAM, which is derived from the global Selventa Knowledgebase. For the EIF4G1 data set, the human KAM was used, while the mouse KAM was used for the RhoA, CTNNB1, and NR3C1 data sets.Each hypothesis is scored according to two probabilistic scoring metrics, richness and concordance, which examine distinct aspects of the probability of a hypothetical cause explaining a given number of RNA State Changes (see next section). Richness is the probability that the number of observed RNA State Changes connected to a given hypothesis could have occurred by chance alone. Concordance is the probability that the number of observed RNA State Changes that match the directionality of the hypothesis  could have occurred by chance alone. A scored hypothesis is considered to be statistically  significant if it meets richness and concordance p-value cutoffs of 0.1. Following automated hypothesis generation , each scored hypothesis meeting the minimum statistical cutoffs for richness and concordance is evaluated and prioritized by a group of scientists based on its biological plausibility and relevance to the experimental perturbation used to generate the data. Evaluation and prioritization was based on multiple criteria, including the mechanistic proximity of the hypothesis to non-diseased lung biology and evidence that the hypothesis is present or has activity in normal lung or lung-related cells. When constructing this network, each hypothesis was collaboratively evaluated by teams of scientists from both Philip Morris International (PMI) and Selventa. For a more comprehensive and detailed explanation on hypothesis scoring and evaluation, please refer to .Many hypotheses identified using RCR on the cell proliferation data sets were already represented in the literature model; those that were not represented in the literature model were investigated by evaluation of their biological relevance to the lung context and whether they are causally linked to phenotypes and processes relevant to cell proliferation in the literature. Hypotheses meeting the above criteria were then added to the literature model as data set-driven nodes, creating the integrated network model. Thus, RCR allowed for verification, testing, and expansion of the Cell Proliferation Network using publicly available proliferation data sets.http://www.ncbi.nlm.nih.gov/gds, while data for E-MEXP-861 was downloaded from ArrayExpress http://www.ebi.ac.uk/microarray-as/ae/. The data from PMID15186480 was obtained from a link within the online version of the paper http://jbiol.com/content/3/3/11. Raw RNA expression data for each data set were analyzed using the "affy" and "limma" packages of the Bioconductor suite of microarray analysis tools available for the R statistical environment [p-values for each probe set on the expression array [Four previously published cell proliferation data sets, GSE11011 (EIF4G1), GSE5913 (RhoA), PMID15186480 (CTNNB1), and E-MEXP-861 (NR3C1), were used for the verification and expansion of the Cell Proliferation Network Table . These dironment -56. Robuon array . The Benp-value of 0.05 was obtained and they had an absolute fold change greater than 1.3. An additional expression abundance filter was applied to three of the data sets; probe set differences were considered significant only if the average expression intensity was above 250 in either the control or treated group for the EIF4G1 and RhoA data sets, and above 10 for the NR3C1 data set. No abundance threshold was applied to the CTNNB1 data set. These criteria were applied to optimize State Change numbers for RCR. NetAffx version na30 feature annotation files, available from Affymetrix http://www.Affymetrix.com, were used for mapping of probe sets to genes. Genes represented by multiple probe sets were considered to have changed if at least one probe set was observed to change. Gene expression changes that met these criteria are called "State Changes" and have the directional qualities of "increased" or "decreased", i.e., they were upregulated or downregulated, respectively in response to the experimental perturbation. The number of State Changes for each data set is listed in Table Probe sets were considered to have changed qualitatively in a specific comparison if an adjusted AKT: v-akt murine thymoma viral oncogene homolog; ATP: adenosine-5'-triphosphate; BEL: Biological Expression Language; CCND1: cyclin D1; CDK: cyclin-dependent kinase; CDKN: cyclin-dependent kinase inhibitor; COPD: chronic obstructive pulmonary disease; CTNNB1: catenin, beta 1; DMT: DNA methyltransferase; DNA: deoxyribonucleic acid; E2F: E2 transcription factor; EGF: epidermal growth factor; EGR1: early growth response 1; EIF4G1: eukaryotic translation initiation factor 4 gamma, 1; FDR: false discovery rate; FGF: fibroblast growth factor; FGFR2: fibroblast growth factor receptor 2; FN1: fibronectin 1; GEO: Gene Expression Omnibus; GLI: glioma-associated oncogene family zinc finger; HDAC: histone deacetylase; HOX: homeobox family; HGF: hepatocyte growth factor; HIF1A: hypoxia inducible factor 1, alpha subunit; HRAS: v-Ha-ras Harvey rat sarcoma viral oncogene homolog; IC/EC: intracellular and extracellular; IGF2: insulin-like growth factor 2; IL1B: interleukin 1, beta; ITG: integrin; JAK: Janus kinase; KAM: Knowledge Assembly Model; KEGG: Kyoto Encyclopedia of Genes and Genomes; LEF1: lymphoid enhancer-binding factor 1; MAPK: mitogen-activated protein kinase; MCF10A: Michigan Cancer Foundation cell line 10A; MEK: mitogen-activated protein kinase kinase; MIR: micro RNA; mRNA: messenger ribonucleic acid; mTOR: mammalian target of rapamycin; MYC: v-myc myelocytomatosis viral oncogene homolog; MYCN: v-myc myelocytomatosis viral related oncogene, neuroblastoma derived; NIH3T3: National Institutes of Health cell line 3T3; NR3C1: nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor); p53: tumor protein p53; PDGF: platelet derived growth factor; PGE2: prostaglandin E2; PMID: PubMed identifier; RB1: retinoblastoma 1; RCR: Reverse Causal Reasoning; RhoA: ras homolog gene family, member A; RMA: Robust Microarray Analysis; RNA: ribonucleic acid; SBML: Systems Biology Markup Language; SRC: v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog; STAT: signal transducer and activator of transcription; TGF: transforming growth factor; VEGF: vascular endothelial growth factor; WNT: wingless-type MMTV integration site family; ZBTB17: zinc finger and BTB domain containing 17 (MIZ-1).The authors declare that they have no competing interests.JW contributed to the network design, biological content, interpretation of results, and manuscript revision. WS contributed to the network design, biological content, interpretation of results, manuscript revision, and project co-ordination. BF, SG, NC, WH, AH, CM, RL, CP, MT, EV contributed to the network design, biological content, interpretation of results, and manuscript preparation. SE contributed to the network design, biological content, and interpretation of results. AVH, BW contributed to the biological content and interpretation of results. MM contributed to project co-ordination and manuscript preparation. AM, MP, JH contributed to system concept and supervised the project. RK contributed to system concept, network design, interpretation of results, manuscript preparation and supervised the project. All authors read and approved the final manuscript.The Cell Proliferation Network Model nodes-edges-evidence. this excel file contains the complete list of evidences associated to all nodes and edges present in the Cell Proliferation Network.Click here for fileThe Cell Proliferation Network Model. this file contains the Cell Proliferation Network Model in "OWL Web Ontology Language" format.Click here for file


High field 1H magnetic resonance in vivo, showed increased hypothalamic myo-inositol concentrations as compared to other cerebral structures. 1H and 13C high resolution magic angle spinning (HRMAS) revealed increased neuroglial oxidative and glycolytic metabolism, as well as increased hypothalamic glutamatergic and GABAergic neurotransmissions under orexigenic stimulation. We propose here an integrative interpretation of all these findings suggesting that the neuroendocrine regulation of appetite is supported by important ionic and metabolic transcellular fluxes which begin at the tripartite orexigenic clefts and become extended spatially in the hypothalamus through astrocytic networks becoming eventually MRI and MRS detectable.We review the role of neuroglial compartmentation and transcellular neurotransmitter cycling during hypothalamic appetite regulation as detected by Magnetic Resonance Imaging (MRI) and Spectroscopy (MRS) methods. We address first the neurochemical basis of neuroendocrine regulation in the hypothalamus and the orexigenic and anorexigenic feed-back loops that control appetite. Then we examine the main MRI and MRS strategies that have been used to investigate appetite regulation. Manganese-enhanced magnetic resonance imaging (MEMRI), Blood oxygenation level-dependent contrast (BOLD), and Diffusion-weighted magnetic resonance imaging (DWI) have revealed Mn The hypothalamus is a small cerebral structure responsible for the integral homeostasis of vital systemic functions including global energy metabolism, appetite, thirst and osmoregulation, thermoregulation, circadian rhythms, and some fundamental survival responses such as aggressiveness  and Spectroscopy (MRS) approaches are known to be well endowed to observe hypothalamic morphology and function. Briefly, Manganese-enhanced MRI (MEMRI) techniques allow to monitor neuronal activation through the accumulation of Mnin vivo.In this review, we examine the information gained thus far with these approaches and provide an integrative interpretation that highlights the vital role of hypothalamic neuroglial compartmentation in the cerebral control of global energy homeostasis Recent years have witnessed an important development in the understanding of the hypothalamic mechanisms involved in appetite control and global energy homeostasis  and oxyhemoglobin (diamagnetic) changes, and by studying this ratio, BOLD  provides information on the diffusion behavior of water molecules in biological tissues, and can be used to probe and define tissue structures at microscopic scales  of a representative mouse in the fed (left) or overnight-fasted (right) state, in the hypothalamus, ARC, VMN, and DMN nuclei, respectively. Bar graphs in panels 5C,E,G, and I show mean values of the same parameter in six mice. The significant increase of Dslow with fasting in all nuclei may be interpreted as the consequence of activation-induced astrocytic swelling.An example on the use of DWI imaging in the study of hypothalamic activation is illustrated in Figure 1H MRS in vivo and ex vivo1H and 13C HRMAS. These methods have been shown to overcome the need to use large voxel volumes, a limitation precluding earlier the applications of MRS in vivo to hypothalamic physiology.The use of imaging methods may be complemented by several advanced spectroscopy strategies including mainly 1H MRS (14.1 Tesla) has been shown to be able to obtain high quality metabolic profiles from the mouse hypothalamus in vivo either unilaterally (2.2 μL voxels) or bilaterally (4.4 μL voxels)   and the reduced sensitivity of the method, imposed the use of relatively large voxel sizes in vivo, exceeding significantly the dimensions of the hypothalamus. To overcome this, a novel collection of High Resolution Magic Angle Spinning (HRMAS) 13C methods ex vivo were implemented recently. By acquiring NMR spectra of biopsy samples, inclined 54.7° with respect to the static magnetic field, the dipolar couplings that broaden the resonances in vivo are removed and high resolution spectra similar to those obtained in solution, can be obtained from samples as small as 5–10 mg, a size comparable with that of the rodent brain hypothalamus. Using this technology, authors investigated  glucose into hypothalamic metabolites  GABA and (3-13C) lactate, while the infusion of the orexigenic peptide ghrelin did not affect 13C labeling in these metabolites. These results revealed that overnight fasting appears to increase GABAergic neurotrasmission and glycolysis, but additional factors other than ghrelin were required to elicit this complex hypothalamic response.d Figure  the effe1H and 13C HRMAS and infusions of (1-13C) glucose, a primarily neuronal substrate or (2-13C) acetate, a predominantly glial substrate  glucose, but not from 13C acetate. Together, this evidence showed for the first time that the increased MEMRI effect associated to neuronal activation of the orexigenic pathways in the obese mice was accompanied by increased oxidative metabolism and glutamate-glutamine cycling. Together with earlier 13C HRMAS evidences on increased GABAergic performance after overnight fasting, the picture emerges that orexigenic stimulation results in increased glutamatergic and GABAergic neurotransmission, implying augmented transcellular cycling of glutamate-glutamine and GABA between neurons and astrocytes. Present results support the view that the synaptic transmission events supporting neuroendocrine signaling in the hypothalamus follow similar neuroglial compartmentation mechanisms to other types of cerebral sensorial or motor activations.The neuroglial mechanisms underlying leptin signaling in the hypothalamus were recently investigated in control and ob/ob mice, combining MEMRI with 1H and 13C NMR spectroscopy.In summary, the evidence accumulated recently by MRI and MRS methodologies has contributed importantly to investigate the role of metabolic compartmentation during neuroendocrine regulation in the hypothalamus. Figure + and Ca2+ channels. Mn2+ substitutes Ca2+, accumulating in excited neurons during depolarization  glucose as reflected by 13C HRMAS. This reveals that hypothalamic activation by fasting involves the excitation of both glutamatergic (activatory) and GABAergic (inhibitory) terminals, as expected for an hypothalamic feed-back loop mechanism (Figure Briefly, the firing of orexigenic neurons involves voltage dependent Nae Glynn, . The astm Figure .+ ions accumulated in the extracellular space during the orexigenic action potentials, may enter surrounding astrocytes through stimulation of the Na+/K+/2Cl− cotransporter (Hertz et al., + concentrations are known to be tightly coupled to neuronal activation, and have been detected using metallographic microscopic imaging approaches (Goldschmidt et al., ARs has been recently proposed to be involved with cell volume regulation processes and water exchange in the brain (Cesetti et al., 2+ accumulations.The volume changes inferred in the hypothalamus during orexigenic activation merit further attention. The Kmyo-inositol levels detected by high field 1H MRS (Lei et al., 1H MRS in vivo, indicate that volume regulation processes in the hypothalamus may play an important role in hypothalamic function.The volume changes inferred by DWI find an adequate support when considering the increases in hypothalamic 1H and 13C MRS have revealed osmolite accumulation and increased glutamatergic and GABAergic neurotransmissions. Taken together, these results suggest an important role for hypothalamic metabolic compartmentation during appetite regulation, as in other cerebral activations. Naturally, these interpretations do not exclude additional contributions to MRI or MRS from other potential mechanisms.In summary, the above sections indicate that MRI and MRS methodologies have provided important insight into the hypothalamic mechanisms underlying appetite regulation. Briefly, MEMRI approaches reveal neuroglial manganese accumulation, BOLD shows concomitant oxygen consumption and associated hemodynamic responses and DWI discloses water diffusion changes compatible with glial swelling. in vivo.Finally, the approaches reviewed here may provide a valuable tool to further investigate glutamatergic or GABAergic neurotransmissions in the hypothalamic control of global energy balance and in the development of improved treatments against feeding disorders, obesity, and diabetes. In addition, this arsenal of new methodologies may be easily extended to explore other hypothalamic functions, opening a new avenue in the research of hypothalamic performance The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The vitamin folate is recognized as beneficial health-wise in the prevention of neural tube defects, anemia, cardiovascular diseases, poor cognitive performance, and some forms of cancer. However, suboptimal dietary folate intake has been reported in a number of countries. Several national health authorities have therefore introduced mandatory food fortification with synthetic folic acid, which is considered a convenient fortificant, being cost-efficient in production, more stable than natural food folate, and superior in terms of bioavailability and bioefficacy. Other countries have decided against fortification due to the ambiguous role of synthetic folic acid regarding promotion of subclinical cancers and other adverse health effects. This paper reviews recent studies on folate bioavailability after intervention with folate from food. Our conclusions were that limited folate bioavailability data are available for vegetables, fruits, cereal products, and fortified foods, and that it is difficult to evaluate the bioavailability of food folate or whether intervention with food folate improves folate status. We recommend revising the classical approach of using folic acid as a reference dose for estimating the plasma kinetics and relative bioavailability of food folate. The preventive effect of high folic acid intake against neural tube defects (NTD) is considered one of the most important nutritional discoveries in the period 1976-2006  in varioThe chemically most stable folate form is synthetic folic acid , which iIt is commonly assumed that food folates, which mainly exist in their polyglutamyl form, are absorbed in the jejunum as monoglutamyl folates after removal of the polyglutamyl chain by intestinal γ-glutamyl hydrolase , and the4-folate) is the substrate for both nucleotide synthesis and the methylation cycle (3-H4folate) donates a methyl group to vitamin B12, which transfers it to homocysteine, avoiding accumulation of homocysteine in the cell and maintaining synthesis of amino acids. The first signs of deficiency appear in tissues with rapid cell turnover and during life stages with increased cell division, such as pregnancy or growth -labeled folate from an oral dose corresponds to that of a differently labeled reference dose of [2H]-folic acid from analogous intravenous administration. This dual-label stable isotope protocol, when used to determine relative folate bioavailability of food folates and added fortificants, produces values of 37-153% ,80,81,82Studies using the oral-fecal balance technique for folate are commonly carried out using ileostomy subjects, who do not possess a colon microflora affecting fecal folate content, in combination with the AUC technique. Absorbed folate is inversely correlated to folate content in stomal effluent. However, not all folate, found in ileostomy effluent or feces, originates from non-absorbed orally ingested folate , and thein vitro that high exposure to folic acid reduces cellular uptake into Caco-2 and kidney cells by down-regulation of genes coding for folate absorption and transport 5-methyltetrahydrofolate or [13C5]folic acid from bread or a breakfast meal (S)-[13C5]5-methyltetrahydrofolate was twice that for food labeled with folic acid, whereas the excretion of labeled folate into ileostomy effluent did not differ significantly and was as low as 10% of the dose, indicating high bioavailability providing that no severe oxidative degradation of non-absorbed labeled folate occurred in frequently sampled ileostomy effluent. For supplemental (6S)-[13C5]5-methyltetrahydrofolate too, a greater median plasma AUC was observed compared with [13C5]folic acid [A recent study uses a combined approach of various short-term techniques in the form of a stable isotope AUC and ileostomy model ,47 to deast meal . The plalic acid  -5-methyltetrahydrofolate (Metafolin®) will be commonly used for fortification purposes, synthetic folic acid reference doses are appropriate for evaluation of the effectiveness of folic acid-fortified foods at population level.However, the data highlight differences in the metabolic handling of folic acid and reduced folates in humans, as evidenced by differing acute post-absorptive plasma kinetics and different effects on fasting plasma folate concentrations after long-term intervention. It is still unclear whether these findings have implications for human health, and they need to be considered in recommendations regarding folic acid fortification of food. As it is not expected that the more expensive and less stable bioactive diastereoisomer (6An immediate consequence is our recommendation to revise the classical approach of using folic acid as the reference dose in estimating the plasma kinetics and relative bioavailability of food folate. The design of future studies on relative folate bioavailability faces the challenge of accounting for the bias from metabolic handling of folate compounds, and existing findings have to be interpreted with caution.


Innervated adult fast and slow skeletal myofibers show Panx1 reactivity in close proximity to dihydropyridine receptors in the sarcolemma of T-tubules. These Panx1 channels are activated by electrical stimulation and extracellular ATP. Panx1 channels play a relevant role in potentiation of muscle contraction because they allow release of ATP and uptake of glucose, two molecules required for this response. In support of this notion, the absence of Panx1 abrogates the potentiation of muscle contraction elicited by repetitive electrical stimulation, which is reversed by exogenously applied ATP. Phosphorylation of Panx1 Thr and Ser residues might be involved in Panx1 channel activation since it is enhanced during potentiation of muscle contraction. Under denervation, Panx1 levels are upregulated and this partially explains the reduction in electrochemical gradient, however its absence does not prevent denervation-induced atrophy but prevents the higher oxidative state. Panx1 also forms functional channels at the cell surface of myotubes and their functional state has been associated with intracellular Ca2+ signals and regulation of myotube plasticity evoked by electrical stimulation. We proposed that Panx1 channels participate as ATP channels and help to keep a normal oxidative state in skeletal muscles.Normal myotubes and adult innervated skeletal myofibers express the glycoprotein pannexin1 (Panx1). Six of them form a “gap junction hemichannel-like” structure that connects the cytoplasm with the extracellular space; here they will be called Panx1 channels. These are poorly selective channels permeable to ions, small metabolic substrate, and signaling molecules. So far little is known about the role of Panx1 channels in muscles but skeletal muscles of Panx1 Also, they share some permeability properties with Cx hemichannels, since they are permeable to small positively  and negatively  charged organic molecules and inorganic ions . In addition, Panx1 forms gap junction channels in exogenous expression systems including Xenopus laevis oocytes and some mammalian cells lines  have revealed that the TM1 region and domains of the first extracellular loop (E1) are exposed to the channel lumen. Also, C-terminal amino acids substitution and reagent perturbation suggest the contribution of this segment to the permeation pathway  mediated by conformational changes of voltage activated dihydropyridine receptors induced by depolarizing membrane potentials. In support of this view it is possible to say that electrical stimulation induces ATP release and uptake of EtdSkeletal muscles develop through a process partially coordinated by extracellular signaling. The coordinate response of cell groups includes the myogenic commitment of mesodermal pluripotent cells, myoblast alignment and fusion. In mice, this process requires the presence of Cx43 expression and functional gap junction channels  and Panx1 channels  does not exhibit potentiation of muscle contraction induced by electrical stimulation. Therefore, it was suggested that this response is due to the absence of ATP release   that reduced the calcium transients and the ATP release (Buvinic et al., 2+ release events that differentially activate Ca2+-dependent signaling pathways, which determine the expression of genes responsible for the slow or fast muscle phenotype (Tavi and Westerblad, 2+/calmodulin-dependent kinases II and IV- and protein kinase C-dependent pathways (Tavi and Westerblad, 2+ signals lead to muscular plasticity by modulating the expression of several genes including IL-6 and c-fos, and the switch between troponin isoform from fast to slow fiber (Buvinic et al., The skeletal muscle activity induces remodeling of structure and functional performance of myofibers, changing the muscular force output, endurance and contractile velocity with respect to a functional demand (Tavi and Westerblad, Panx1 channels are involved in several relevant physiological skeletal muscle processes, such as potentiation of skeletal muscle contraction since they do not occur in the absence of these channels. Currently, Panx1 channels have been clearly involved in potentiation of contraction of skeletal muscles. In addition, Panx1 channels appear to mediate the release of ATP involved in muscle remodeling. People who are active in sports should be cautious with food and drink additives that could reduce the Panx1 channel activity and consequently the potentiation response of muscle contraction. Moreover, to further understand the possible role of Panx1 channels in pathologic responses it may be needed to develop highly selective Panx1 channels inhibitors and/or acute down regulation of Panx1 expression using inducible KO animals or transfections with siRNA or morpholines. Finally, Panx1 channels act as ATP channels and the absence prevent the induction of reactive oxygen species; measured as levels of TBARS in this work Figure , suggestThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Burkitt lymphoma has many forms of clinical presentations and, in children, it is usually discovered due to the presence of an abdominal mass. This rapidly growing tumor is highly malignant, aggressive, and may cause either indirect symptoms, due to pressure phenomena or direct involvement of the bowel lumen, leading to either intestinal obstruction or intussusception. We describe the case of a 4-year-old girl who exhibited an unusual presentation of ileocolic intussusception on a Burkitt lymphoma lesion of the ileum. Burkitt lymphoma represents 8-10% of all tumors in children under 15 years of age [5].  This type of lymphoma has three clinical forms. The endemic form, common in the African continent, develops due to a chromosomal translocation between chromosomes 8 and 14 and often presents as a rapidly growing tumor of the facial bones. The non-endemic form, observed most often in the United States, typically presents with an abdominal mass or ascites. This non-endemic form results from a chromosome 8 translocation involving the c-myc oncogene. The third form is specific to immunocompromised patients and frequently presents as diffuse lymphadenopathy [6]. Burkitt lymphoma has a high proliferation rate and the histological appearance of a “starry sky" because of the high rate of mitosis and spontaneous cell death [3]. Many pediatric patients with Burkitt lymphoma present with intussusception as a first clinical sign, a presentation that potentially leads to the disease detection at an earlier stage [7]. Intussusception caused by Burkitt lymphoma as a cause of acute abdomen is rare, with often misleading symptoms that make the diagnosis more difficult [8]. Children with Burkitt lymphoma have an excellent prognosis with contemporary treatment regardless of disease stage .Intussusception is one of the most common emergencies in children that require the attention of a pediatric surgeon. Especially in older children, in 10% of the cases, there is a pathological lesion  at the lead point of the intussusceptum [11]. Estimated incidence is 2-3 cases per 1000 live births with boys/girls sex ratio 3:2 [11]. Although 90% of intussusceptions occur in children between 3 months and 3 years of age, with a peak incidence between the fifth and the ninth month of life, intussusception has also been reported in utero, in neonates and in adults [9]. The classic clinical triad described for intussusception is made up of abdominal colic, “red currant jelly" stools and a palpable abdominal mass; in children older than 2-3 years the presentation of intussusception is subtler and the classic triad of symptoms may not be present [9], and combined with clinical observation, is usually sufficient to diagnose the intussusceptions [11]. Barium enema, CT and colonoscopy can also be helpful in diagnosis [12]. Ultrasonography is the most efficient examination for the diagnosis of intestinal intussusceptions with a reported accuracy of up to 100% [14]. Non-Hodgkin’s Lymphoma (NHL) represents the third most frequent cancer in childhood and usually is subdivided into 3 histological subtypes: (1) 65% B-cell NHL including both Burkitt lymphoma and diffuse large B-cell lymphoma; (2) 20% lymphoblastic and (3) 15% anaplastic large cell lymphoma [16]. Burkitt lymphoma represents 40%-50% of all NHL cases in childhood, and is more common in males than females [14]. Predisposing factors for lymphoma of the small intestine includes prior malabsorbtion syndromes, inflammatory bowel disease and immunodeficiency states [14]. All these risk factors were absent in our patient who were previously healthy.Primary gastrointestinal lymphoma represents 1%-4% of all gastrointestinal malignancies. Most cases in children involve the distal ileum or ileocecal region [18]. The rapidity of volumetric doubling of this neoplasm frequently leads to an acute abdomen presentation that may mimic other diseases  [12]. In the case we are presenting, the onset was slow, with the patient losing weight and, prior to admission, showing the following two of the three classic signs for intussusception: diffuse abdominal pain and bloody stools without any palpable abdominal mass. The clinical presentation of undiagnosed Burkitt lymphoma is non-specific, which makes this a difficult condition to diagnose. Abdominal pain is present in 80% of the cases of intussusception with Burkitt lymphoma, along with nausea, vomiting, constipation, diarrhea, fatigue or malaise [15]. Along with the assignment of specific treatment protocols of relatively short duration, event-free survival rates for children with B-cell NHL have dramatically increased within the last 20 years [15]. Surgical procedures are now needed for complete resection in limited disease, diagnostic biopsies, management of life-threatening local tumor effects and second-look operations [15]. Surgery may provide important prognostic information through definitive staging offers a chance for cure with or without adjuvant therapy and may prevent complications such as haemorrhage, obstruction or perforation [13]. In patients with proven localized abdominal disease total resection may be performed if is not mutilating (as hemicolectomy performed in the past). The open surgical approach is still the gold standard in most paediatric surgical facilities [11]. Patients with complete resection had a significantly better prognosis compared with patients with incomplete resection or biopsy [15]. The ideal treatment of gastrointestinal lymphoma must be individualized based on the type of disease and its location so that a multidisciplinary approach with surgery and chemotherapy can optimize the chance of cure [19], therapy courses include the following drugs: cyclophosphamide, methotrexate, cytarabine, iphosphamide, etoposide, vincristine, vindesine, adriamycin, doxorubicin, dexamethasone [15]. Burkitt lymphoma patients who present with intussusception require shorter-duration and less intense chemotherapy than patients diagnosed in other ways. In our patient, we performed a segmental resection of the terminal ileum with total resection of the tumour, 2 courses of chemotherapy with excellent outcome at 18 month after surgery.  Burkitt lymphoma is very sensible to chemotherapy [Burkitt lymphoma is a diagnostic entity that the pediatric surgeon should consider when faced with a child with an unspecific clinical presentation, who complains of diffuse abdominal pain, weight loss, and bloody stool (with a “red currant jelly" appearance). The suspicion of intestinal intussusception with a pathological lesion at the lead point of the intussusceptum necessitates surgery. Laparotomy is the gold standard in both diagnosis and treatment, ensuring the excision of the entire tumor with free margins. A multidisciplinary team with an oncologist assures efficient therapeutic management. Short- and long- term outcomes are generally favorable, as shown by data from a review of the literature and confirmed by our patients’ outcome.