Extensive infiltration of the surrounding healthy brain tissue is a cardinal feature of glioblastomas, highly lethal brain tumors. Deep infiltration by the glioblastoma cells renders complete surgical excision difficult and contemporary adjuvant therapies have had little impact on long-term survival. Thus, deep infiltration and resistance to irradiation and chemotherapy remain a major cause of patient mortality. Modern therapies specifically targeted to this unique aspect of glioblastoma cell biology hold significant promise to substantially improve survival rates for glioblastoma patients. In the present paper, we focus on the role of adhesion signaling molecules and the actin cytoskeleton in the mesenchymal mode of motility that characterizes invading glioblastoma cells. We then review current approaches to targeting these elements of the glioblastoma cell migration machinery and discuss other aspects of cell migration that may improve the treatment of infiltrating glioblastoma. Glioblastoma brain tumors are the most common adult brain cancer and have a very poor prognosis. Despite intensive efforts, there has been little improvement in the ability to successfully treat these tumors, and a major reason for this failure is the unique ability of the glioblastoma cells to extensively spread throughout the surrounding healthy brain tissue. There is increasing realization that the molecular regulators of glioblastoma invasion may be key to the development of new therapeutic approaches . DevelopGliomas are a class of primary tumors that arise in the brain, and the most common and lethal form is the grade IV glioblastomas (previously known as glioblastoma multiformae) . While bThe low survival rates for glioblastoma are, in part, a consequence of the extensive infiltration of healthy brain tissue that is a cardinal feature of these tumors. Diffuse infiltration throughout the brain makes these tumors refractory to successful surgical excision, and tumor recurrence is almost inevitable, with 90% of patients developing new lesions within 2-3 cm of the original site  or at dide novo origin or may develop from a low grade glioma but are histopathologically indistinguishable [Glioblastomas are characterized histopathologically by diffuse infiltration, increased cellular proliferation, increased angiogenesis, nuclear atypia, and necrosis , and tumuishable . Howeveruishable .de novo glioblastoma accounts for approximately 90% of glioblastoma and is chiefly diagnosed in older patients with a mean age of 62 years [Primary or 62 years . Genetic62 years , 16 and 62 years , 18, del62 years , and ina62 years , 21. EGF62 years  and the 62 years , 24. The6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that protects cells by removing alkyl groups from the O6 position of guanine. The activity of MGMT reduces the cytotoxicity of alkylating chemotherapy agents (such as temozolomide) and contributes to drug resistance by reversing the DNA damage induced by such agents [Another group of glioblastomas is known to arise from previously identified gliomas, usually developing as a result of malignant transformation several years after initial diagnosis of a low grade astrocytoma . This tyh agents , and theh agents , 32 and h agents .The brain tumors are one of the few classes of solid tumor observed in both pediatric and adult patient populations. Interestingly, the trend for incidence of the different grades of brain tumor tends to be reversed in the pediatric population with the grade IV glioblastomas being less common . However, the 5-year survival rate for pediatric patients is only 5–15% , and thuMost solid cancers progress to disseminated metastatic disease, evidenced by secondary tumors arising in sites distal to the primary tumor. The rarity of glioblastoma spread outside the brain reflects the important interaction between the glioblastoma cells and the unique brain environment. Thus, the mechanisms of glioblastoma infiltration represent a potentially specific target for treating glioblastoma.in vivo mouse models has established that glioblastoma cells migrate individually with a mesenchymal mode of motility [μm length, termed filopodia  collagen gels, organotypic brain slice cultures, and imaging of fluorescently labeled glioblastoma cells in motility –40. Thismotility . Charactmotility , 42. In motility , and thumotility . Understilopodia . These sThere are intensive research efforts currently underway to try and understand the critical molecular regulators of mesenchymal migration. Although our understanding of this process remains incomplete, it is useful to understand the present state of play and consider how this currently and, may in the future, informs therapeutic approaches to the treatment of glioblastoma. The focal adhesions (FAs) are points of linkage between the ECM, the transmembrane integrin receptors, and the internal actin cytoskeleton. In addition to their role as anchorage points to the matrix, the FAs also transmit information bidirectionally between the cell and the external environment , and theα and a β subunit [α and 8 β subunits, and combinations of these subunits create receptors for specific ECM components including fibronectins, laminins, and collagens [αv, β1, and β3) in cell lines derived from disseminated cancers [αvβ3 is highly expressed on the cell surface across a range of tumor types, including glioblastoma [αvβ3 receptor is an important attachment factor for ECM proteins with the exposed arginine-glycine-aspartate (RGD) motif such as fibronectin [αvβ3 receptor in the angiogenesis that is characteristic of the highly vascularized glioblastomas [αvβ3 in mesenchymal cell migration. This receptor is found in the initial rapidly turned over adhesions  that form at the migrating cells leading edge [αvβ3 receptor is highly expressed in glioblastoma [The integrin receptors are heterodimeric transmembrane complexes consisting of an  subunit . Mammalsollagens –51. Duriollagens . Althoug cancers , there i cancers . Howeverblastoma . The αvβronectin –58. Muchlastomas . Howevering edge  and may ing edge . Given tblastoma –67 and iComponents of the Focal Adhesion Kinase (FAK)/Src tyrosine kinase migration signaling network  are upreIntegrin receptors lack any intrinsic catalytic activity and instead function by recruiting an array of cytoplasmic proteins  which inImportantly, a second FAK subfamily member, Related Adhesion Focal Tyrosine Kinase (RAFTK)/Proline-rich tyrosine kinase (Pyk2), may also play a role in glioblastoma infiltration. These two proteins have overlapping functions and downstream partners, and while FAK is ubiquitously expressed, RAFTK/Pyk2 has a more restricted expression and is predominantly seen in brain and hematopoietic cells. RAFTK/Pyk2 is correlated with increased malignancy in glioblastoma , and oveSrc is similarly activated in numerous tumor types , and theVia this activity of FAK and Src and their target molecules, including those of the Cas family of proteins and paxillin, a signaling network is established that culminates in the activation of GTPase proteins, such as Rac. In turn, this determines the dynamic state of the actin cytoskeleton that is essential to the morphological progression of mesenchymal cell migration , 101. Th+2- and calmodulin-dependent myosin light chain kinase (MLCK) [Cells migrating with a mesenchymal phenotype have a characteristic array of polymerized actin, displaying short, branched filaments at the leading edge and longer tension-bearing filaments in the cytoplasm known as stress fibers . Directee (MLCK) . Desphose (MLCK) , 111.αvβ3 integrin receptor complexes exhibit enhanced actin polymerization activity, thus providing evidence to suggest a direct interaction between actin nucleation and adhesion sites [Given the critical role of FA and actin interaction in cell migration, much research has concentrated on the mechanistic relationship between actin nucleators and FA proteins in the formation of new actin structures . The acton sites . Althougon sites , the facon sites , 120 sugon sites . These don sites , and theThe driving force for cell movement is derived primarily from the coordinated assembly and disassembly of actin filaments and the Rho family GTPases, RhoA, Rac1, and Cdc42 are critical regulators of this process. Together these enzymes regulate the organization of actin filaments and cooperate to control polarity, protrusion and adhesion during cell movement , 122. OtRho proteins can also regulate the actin depolymerizing factor ADF/cofilin, and thus actin nucleation, indirectly through Rho kinase (ROCK) and p21-activated kinase-1 (PAK-1). RhoA activates ROCK while Cdc42/Rac1 activates PAK-1/-2/-3. ROCK and PAK in turn phosphorylate and activate LIM-motif-containing kinase protein (LIMK), which inactivates cofilin and thus the recycling of actin filaments at early lamellipodial extensions , 135–137The history of treating malignant gliomas dates back over a century. The first surgical operation to treat brain cancer was reported in 1884 but barely made an improvement on patient survival . In the In vivo imaging of glioma cell invasion has revealed that the main tumor mass can grow by cooption [The standard treatment for newly diagnosed glioblastomas consists of initial surgery to remove as much of the tumor as possible, followed by radiation therapy and chemotherapy . Surgicacooption  and thatcooption . These fcooption . This suα3β1, αvβ1, αvβ3, and αvβ5 [αvβ3 expression levels can differ across glioma cell lines [αvβ3 and αvβ5 receptors are expressed both in endothelial cells associated with new vasculature and in the glioma tumor cells [αvβ3 has been observed in angiogenic endothelial cells in tumors [αvβ3 receptor plays a vital role in angiogenesis [αvβ3-dependent adhesion [αvβ5 receptor as both of these integrins interact with their ligands via specific RGD motifs [in vitro, including apoptosis and blocking cell adhesion, migration, and invasion [μg/mL while 50 μg/mL can induce significant apoptosis [Integrin receptors reported to be upregulated on glioma cells include and αvβ5  althoughll lines . The αvβor cells , 155–157n tumors  and is ln tumors , 160. Thn tumors , and theogenesis , 162, anadhesion . NotablyD motifs . Importainvasion . In precinvasion . Furtherpoptosis . Cells tpoptosis , dosage-poptosis , and detpoptosis , 168. Phpoptosis , 170, anpoptosis . The drupoptosis , and thepoptosis .in vitro experiments demonstrated increased apoptosis and decreased angiogenesis [TAE226 is a novel small molecule designed to inhibit FAK by blocking phosphorylation of FAK tyrosine 397. The mechanism of action is specific for FAK autophosphorylation as it does not affect total FAK protein expression and has no effect on either EGF-induced EGFR phosphorylation or serum-induced PDGFR phosphorylation . TAE226 ogenesis . In studogenesis  while inogenesis .β, and Kit) [in vitro [Inhibition of Src activity in either two-dimensional (2D) or 3D culture models can successfully block glioblastoma invasion , 179. Foand Kit) . This agand Kit) . Preclinand Kit) , and an and Kit) . Furtherin vitro .Similar to Dasatinib, AZD0530 is also an orally available drug that inhibits Src. Although still in the preclinical stage of testing for glioblastoma treatment, AZD0530 has been found to inhibit both FAK and paxillin phosphorylation and activity. The resulting abrogation of adhesion-dependent signaling pathways consequently inhibits cell migration . In otheThe mammalian target of rapamycin (mTOR) is a protein kinase upstream of PI3K/Akt signaling pathway, and activity of this protein is inhibited by rapamycin. Involved in various signaling pathways implicated in glioblastoma, mTOR plays a role in a number of cellular processes, including cell proliferation and growth, angiogenesis . CriticaThe approaches outlined above highlight the potential—and in some cases, success—of targeting adhesion signaling pathways in the treatment of glioblastoma. The highly unique behavior exhibited by the glioblastoma cells in response to the specialized morphology and molecular structure of the brain suggests that there is considerably more scope to target these pathways and derive specific and efficacious new therapies. Below, we consider some of the adhesion signaling pathways and mechanisms that might be amenable to therapeutic exploitation along with some of the envisaged caveats.β-integrin by antisense β-1 mRNA in a rat model of glioma caused a significant reduction of brain invasion, attributed either to impaired interaction with the ECM in the brain or interference with β-1-dependent signaling pathways [αvβ3 integrins in the brain have revealed that the activation state of integrins rather than expression level controls metastasis and angiogenesis in tumor cells, and this process is highly susceptible to cues from the brain microenvironment [Downregulation of pathways . Similarpathways . Studiesironment . Thus, tironment  highlighIn recent times, it has become clear that invading and migrating cancer cells have a range of motility modes at their disposal that allows the cells to migrate through distinct extracellular environments , 193–195The determining role of the actin cytoskeleton in the morphological and mechanical properties that are indispensable for mesenchymal cell migration suggests that the actin filament system represents a major potential target for antimesenchymal migration strategies. However, the ubiquitous expression and vital contribution of actin to all cells and tissues in the body mean that nonspecific disruption of the actin cytoskeleton would cause unacceptable and life-threatening side effects. A more useful approach would therefore be to target specific actin regulatory molecules that are either restricted in their tissue distribution or are functionally specialized. For example, the tropomyosin family of actin-associating proteins has emerged as critical regulators of cell migration , 198–201γ), slingshot (SSH), LIMK, or chronophin may represent valid targets for anticancer treatment as each of these molecules is mediator of cofilin signaling. Approaches to inhibit or stabilize these pathway intermediates may suppress the pathway activity sufficiently to derive therapeutic benefit in the absence of unacceptably high toxicity levels in nontumor cells.The dynamics of actin filaments are critical in cellular processes ranging from cell division to apoptosis and migration. The dynamics are therefore subject to regulation by a vast repertoire of actin-regulatory proteins. Actin-regulatory proteins that modulate actin function in cell migration include ADF/cofilin . Cofilinde novo actin nucleation at the periphery of the cell, resulting in lamellipodia extensions and membrane ruffling [The Rho GTPases RhoA, Rac1, Cdc42, and Ras indisputably control signal transduction pathways that cooperate to promote cell movement . Given truffling , 210. Inruffling , a findiruffling . Indeed,ruffling . These sruffling , 215 allruffling  overcomeruffling .The PI3K pathway contributes to cytoskeletal remodeling via signaling through the serine/threonine kinase Akt . ImportaGiven the critical role of migration in the progression to metastatic, disseminated cancer, there are increasing numbers of studies taking a global approach to identifying molecules that are essential regulators of cell migration. In one example, investigators used an siRNA screening approach to identify proteins that modulate the migratory response of a breast epithelial cell line . The invFinally, we note that the blood-brain barrier (BBB) constitutes a major hurdle for any systemic chemotherapy delivery to treat glioblastoma. The BBB protects the brain by excluding molecules by size and biochemical properties and generally prevents the translocation of lipophilic molecules larger than 500 Da to the brain –234. HowThere is clearly an urgent need to develop new approaches to treating glioblastoma—the current poor rate of survival means that even small improvements in therapy have the potential to significantly improve life expectancy and/or quality of life for patients diagnosed with this tumor. In the present paper, we have highlighted recent understanding of the cellular mechanisms that regulate mesenchymal cell migration that is characteristic of infiltrating glioblastoma. New targeted therapies focusing on the unique cell biology that facilitates infiltration of the brain parenchyma offer promise to treating patients with glioblastoma. Understanding cell migration is a necessary first step in developing anti-migration pharmaceuticals. It is unlikely that any agent targeted against infiltration will be effective unless used in combination with therapies that target other aspects of tumor biology. Current clinical trials generally measure improvements in time to progression, but overall impact on long-term survival has been modest. The challenge for the future is to translate migration and invasion research into new therapies for use in the clinical setting and to the design of clinical trials including outcomes and markers that report successful inhibition of infiltration. As our understanding of the cell machinery that regulates migration continues to improve apace, the possibility of improved patient outcomes by pursuing novel targeted therapies moves forward towards reality.


HNF1B gene to exhibit reciprocal associations with prostate cancer risk. We aimed to identify whether these variants may have an effect on cancer risk in general versus a specific effect on prostate cancer only.Genome-wide association studies have found type 2 diabetes-associated variants in the HNF1B, rs4430796 and rs7501939, which are in linkage disequilibrium . Overall, the analysis included 16 datasets on rs4430796 with 19,640 cancer cases and 21,929 controls; and 21 datasets on rs7501939 with 26,923 cases and 49,085 controls. Malignancies other than prostate cancer included colorectal, breast, lung and pancreatic cancers, and melanoma. Meta-analysis showed large between-dataset heterogeneity that was driven by different effects in prostate cancer and other cancers. The per-T2D-risk-allele odds ratios  for rs4430796 were 0.79 ] per G allele for prostate cancer (p<10−15 for both); and 1.03  for all other cancers. Similarly for rs7501939 the per-T2D-risk-allele odds ratios  were 0.80  per T allele for prostate cancer (p<10−15 for both); and 1.00  for all other cancers. No malignancy other than prostate cancer had a nominally statistically significant association.In a collaborative analysis, we collected data from GWAS of cancer phenotypes for the frequently reported variants of HNF1B variants have a highly specific effect on prostate cancer risk with no apparent association with any of the other studied cancer types.The examined  HNF1B gene (formerly TCF2) have been demonstrated to be associated both with the risk of prostate cancerA large number of epidemiological studies have suggested correlations between type 2 diabetes (T2D) and various cancersHNF1B was previously known to be mutated in individuals with maturity-onset diabetes of the young type 5 (MODY 5)HNF1B variants modulate also the risk of other malignancies, or show specificity for prostate cancer, requires large sample sizes. Here we present the results of a large collaborative meta-analysis of HNF1B, rs4430796 and rs7501939, which have the most consistent associations with both prostate cancer and T2D. Relevant data were collected on the two variants from GWAS on cancer phenotypes in Caucasian populations in order to examine whether they have an effect on cancer risk in general, on few specific cancers, or only on prostate cancer.A definitive answer on whether All the originally contacted investigators of cancer-related GWA studies agreed to participate in this collaborative analysis, with the exception of the investigators of 3 GWA studies The contributing teams and datasets are shown in Overall, the collaborative analysis included data on rs4430796 for 19,640 cancer cases and 21,929 controls; for prostate cancer there were 11,145 cases and 9,650 controls, while for all other cancers there were 8,495 cases and 12,279 controls. The collected data on rs7501939 included 26,923 cases and 49,085 controls; for prostate cancer there were 12,898 cases and 40,371 controls, while for the other cancers there were 14,025 cases and 43,893 controls. Malignancies other than prostate cancer in these datasets included colorectal, breast, lung and pancreatic cancers, and melanoma . deCODE −10) and rs7501939  according to fixed effects calculations, while by random effects calculations there was nominal significance . The reason for this diversity is that there was very large between-study heterogeneity in the effect sizes , and this makes the fixed effects calculations less reliable. Results were qualitatively similar when we increased the variance in deCODE, FHS, and IARC estimates to account for the overlapping control group (not shown).The meta-analysis of all datasets  showed a−15 by fixed effects and 0.79  p = 10−13 by random effects), and rs7501939  0.80  p<10−15 by fixed effects and 0.79 , p = 2×10−11 by random effects) , although the heterogeneity pertained only to the exact magnitude of the genetic effects and a nominally statistically significant association was seen in each of the datasets except for the Framingham study where the number of prostate cancer cases was more limited.The heterogeneity was largely driven by the diversity in the effect sizes between prostate cancer and all other cancers. A meta-analysis limited to prostate cancer datasets gave consistent associations with both rs4430796  0.79 , p<10effects) . There wConversely, the results for all other cancers suggested no significant association and results were consistent across studies. The summary OR was 1.03 and 1.00 for the two polymorphisms respectively (p = 0.14 and 0.81 by fixed effects) and the 95% CIs excluded ORs deviating more than 7% from the null (OR = 1.00) for rs4430796 and more than 4% from the null for rs7501939 . The Q-tThere was also no convincing evidence for an association between either of the two polymorphisms and any of the other cancers (besides prostate cancer), when each cancer type was evaluated separately. Point estimates were in the opposite direction (odds ratio 1.03–1.05) for pancreatic and lung cancer, but were not nominally statistically significant . The difThe current collaborative analysis documents that both rs4430796 and rs7501939 have robust support for association with prostate cancer, while we did not observe any convincing evidence for an association of any of the other cancers examined with either polymorphism. When data from all other cancers, excluding prostate cancer, were combined the summary effects had 95% CIs that excluded even subtle associations. Apart from prostate cancer, when other datasets for each individual cancer type was combined, the 95% CIs consistently excluded associations with modest effects. This would suggest that the effects mediated by these polymorphisms are specific to T2D and prostate cancer and they do not involve any other cancer types.HNF1B gene encodes a transcription factor and it was initially identified as a MODY geneThe HNF1B variants for T2D, prostate cancer, and other cancers, also in non-Caucasian populations. Preliminary data suggest that both of the examined variants had consistent associations with T2D in Caucasian, Asian (Hong Kong), and West African ancestry participantsHNF1B variants and prostate cancer. This may be due to different associations in different sub-phenotypes. For example, some data suggest that the rs4430796 A allele may primarily increase the risk for early-onset (<50 years) prostate cancer rather than later-onset diseaseThe two variants that we assessed are not necessarily the functional culprits. GWA studies typically derive markers of phenotypes that are probably linked with the functional genetic variationHNF1B variants conclusively have pleiotropic effects on both T2D and prostate cancer, the pleiotropy apparently does not extend to other cancer types. Genetic effects may offer a way to dissect comorbidity between specific cancers and metabolic phenotypes. Besides HNF1B, other gene loci have started appearing where variants are identified that modulate susceptibility to both T2D and some malignancy, e.g. prostate cancer for the JAZF1 locus gene CDKN2A locus In conclusion, while the two examined We used the NHGRI catalogue of published GWA studiesHNF1B variants, rs4430796 and rs7501939. The risk alleles for prostate cancer are A and C for rs4430796 and rs7501939 respectively. The risk alleles for T2D are G and T for rs4430796 and rs7501939 respectively. The two SNPs have modestly high LD in Caucasians, but low LD in Africans . Investigators were requested to provide all GWA data that they had obtained for evaluation of any cancer phenotype, including any additional unpublished datasets. Additional genotyping for the two specific variants was encouraged, when a GWA platform had been used that did not directly genotype these polymorphisms (e.g. Affymetrix or Perlegen rather than Illumina). When a study had data on more than one cancer type, data were requested to be provided separately for each cancer type. Investigators were asked to provide also information and clarifications about the design of their studies, and to ensure that population stratification and cryptic relatedness had been appropriately addressed and appropriate quality controls were available for the genotyping. All GWAS investigations that contributed data on these SNPs used stringent QC standards  and the two SNPs fulfilled these standards. Approval from local institutional review boards and steering committees was obtained, as deemed necessary for each study by its investigators. The contributed data were checked for completeness and with logical queries and any missing or unclear information was clarified through communication with the contributing investigators.We communicated with the corresponding and principal investigators of all of these studies to request their participation in the collaborative meta-analysis. The investigators of these studies were asked to contribute relevant data on genotype frequencies in cancer cases and non-cancer controls for the For each SNP, we performed meta-analyses including the data from all eligible cancer studies (regardless of the specific cancer phenotype addressed) and also subgroup meta-analyses, with each subgroup limited to studies addressing a specific cancer phenotype. A separate analysis compared the results of the association for prostate cancer versus the association for all other cancers combined.2 metricAll analyses followed the per allele (log-additive) model of inheritance with effect sizes expressed in the odds ratio (OR) scale using both fixed and random effects modelsBased on the accumulated total sample size and given the minor allele frequencies of these two polymorphisms in HapMap CEU , the meta-analysis had 95% or higher power to detect an association of OR = 1.10 at alpha = 0.05 with each of the two polymorphisms for overall cancer risk, prostate cancer risk, or other cancer risk. Reported p-values are two-tailed. Analyses were performed in STATA 10.0 .


The use of herbal medicines  has recently gained popularity in Europe and the United States. Nevertheless the exact mechanism of the preventive effects of these products is still far to be clearly established, being its knowledge necessary to successfully apply these therapies to avoid stone formation.The effect of oral lemon juice administration on calcium oxalate urolithiasis was studied in male Wistar rats. Rats were rendered nephrolithic by providing drinking water containing 0.75% ethylene glycol [v/v] (EG) and 2% ammonium chloride [w/v] (AC) for 10 days. In addition to EG/AC treatment, three groups of rats were also gavage-administered solutions containing 100%, 75% or 50% lemon juice [v/v] (6 μl solution/g body weight). Positive control rats were treated with EG/AC but not lemon juice. Negative control rats were provided with normal drinking water, and were administered normal water by gavage. Each group contained 6 rats. After 10 days, serum samples were collected for analysis, the left kidney was removed and assessed for calcium levels using flame spectroscopy, and the right kidney was sectioned for histopathological analysis using light microscopy.Analysis showed that the rats treated with EG/AC alone had higher amounts of calcium in the kidneys compared to negative control rats. This EG/AC-induced increase in kidney calcium levels was inhibited by the administration of lemon juice. Histology showed that rats treated with EG/AC alone had large deposits of calcium oxalate crystals in all parts of the kidney, and that such deposits were not present in rats also treated with either 100% or 75% lemon juice.These data suggest that lemon juice has a protective activity against urolithiasis. Kidney stone formation or urolithiasis is a complex process that is a consequence of an imbalance between promoters and inhibitors in the kidneys . The recRenal calculi can be broadly classified in two large groups: tissue attached and unattached . AttacheTo further investigate the potential of lemon juice as a therapy for lithiasis, the present study examined the effect of lemon juice on experimentally EG-induced calcium oxalate (CaOx) nephrolithiasis in rats.ad libitum access to standard chow and tap water, and were kept under a controlled 12 h light/dark cycle at 22 ± 2°C.Thirty male Wistar rats weighing approximately 280 g were acclimated for 3 days in cages before experiments commenced. Experiments were conducted in accordance with internationally accepted standard guidelines for the use of animals. Rats had ad libitum access to regular food and drinking water, and administered 6 μl distilled water per 1 g of body weight by gavage (intra-gastric administration). Groups 2, 3, 4 and 5: ad libitum access to regular food, and ad libitum access to drinking water containing 0.75% [v/v] ethylene glycol (EG) and 2% [w/v] ammonium chloride (AC) in order to promote hyperoxaluria and CaOx deposition in the kidneys. Groups 2, 3 and 4 were also administered 6 μl lemon juice solution/g body weight by gavage at the following concentrations: Group 2, 100% lemon juice; Group 3, 75% [v/v] and Group 4, 50%. Group 5 rats were administered 6 μl distilled water/g body weight by gavage (positive control). All rats were weighed daily.The thirty rats were divided into five groups comprising six animals per group. Each group underwent a different treatment protocol for 10 days. Group 1: negative control, After the 10-day experimental period, rats were anaesthetized and blood was collected from the retro-orbital region, centrifuged at 10,000 × g for 10 min , and thet-test was used to assess differences between means. Conventional Windows software was used for statistical computations. A P value < 0.05 was considered to indicate a significant difference.Results are presented as mean ± standard error (S.E.). A one-way ANOVA was used to determine the significance of differences among groups. Student's Serum analysis showed that urea and creatinine levels were higher in Groups 2, 3, 4 and 5 compared to Group 1  weighed less than the negative control rats (Group 1) at the completion of the experiment  resulted in increased kidney calcium levels compared to the negative control rats, while the administration of 100% lemon juice reduced this calcium accumulation (Group 2) Fig. .Examination of kidney paraffin sections showed that Group 5 rats  had the greatest amount of CaOx deposition, and this was present in all parts of all three major areas of the kidney. Intratubular and interstitial crystals were observed on the cortex Figs.  and 4e. Urinary lithiasis is generally the result of an imbalance between inhibitors and promoters in the kidneys. Human kidney stones are usually composed of CaOx , and sevMany in vivo models have been developed to investigate the mechanisms involved in the formation of urinary stones, and to ascertain the effect of various therapeutic agents on the development and progression of the disease -33. RatsThe present study examined the effect of various lemon juice concentrations on the deposition of CaOx crystals within the rat kidney. Previous studies concluded that medicinal plants had little effect on the urinary chemistry of urolithiasis ,35. The The association of crystals with renal tubular cells is considered a potential factor in the process of renal stone formation. Indeed, calculations considering the rate of crystal growth even at its maximum speed and tubular fluid flow suggest that a single crystal would not become large enough to be retained and occlude the lumen during its normal transit through the nephron . FurtherLemon juice has a high antioxidant capacity due to the presence of citrate, vitamin C, vitamin E and flavonoids such as eriocitrin, hesperetin ,38 and lIn urolithiasis, the glomerular filtration rate (GFR) decreases due to stones in the urinary system obstructing urine outflow. This leads to the accumulation of waste products in the blood, particularly nitrogenous substances such as urea, creatinine and uric acid. In addition, increased lipid peroxidation and decreased levels of antioxidant potential have been reported in the kidneys of rats supplemented with a calculi-producing diet . In thisThe present study found that the administration of lemon juice effectively prevented the development of urolithiasis in rats. These findings support the use of lemon juice as an alternative medicine to prevent urolithiasis. Further research is necessary to clarify the mechanism underlying this preventative effect of lemon juice.The author(s) declare that they have no competing interests.MT participated in this study by gavage of rats, measurement of body weight and analysis of kidney calcium levels. AL performed the statistical analysis. KE participated in the animal experiments. FL participated in laboratory management. IZ examined the histological samples. YE participated in analytical determinations. AO performed image processing. FG participated in the evaluation and discussion of the obtained results. AC participated in coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Campylobacter jejuni isolates have been demonstrated very recently. Prior work with Campylobacter temperate bacteriophages, as well as evidence from prophages in other enteric bacteria, suggests these prophages might have a role in the biology and virulence of the organism. However, very little is known about the genetic variability of Campylobacter prophages which, if present, could lead to differential phenotypes in isolates carrying the phages versus those that do not. As a first step in the characterization of C. jejuni prophages, we investigated the distribution of prophage DNA within a C. jejuni population assessed the DNA and protein sequence variability within a subset of the putative prophages found.Prophages integrated within the chromosomes of C. jejuni DNA using probes from genes within the three putative prophages of the C. jejuni sequenced strain RM 1221 demonstrated the presence of at least one prophage gene in a large proportion (27/35) of isolates tested. Of these, 15 were positive for 5 or more of the 7 Campylobacter Mu-like phage 1  genes tested. Twelve of these putative prophages were chosen for further analysis. DNA sequencing of a 9,000 to 11,000 nucleotide region of each prophage demonstrated a close homology with CMLP 1 in both gene order and nucleotide sequence. Structural and sequence variability, including short insertions, deletions, and allele replacements, were found within the prophage genomes, some of which would alter the protein products of the ORFs involved. No insertions of novel genes were detected within the sequenced regions. The 12 prophages and RM 1221 had a % G+C very similar to C. jejuni sequenced strains, as well as promoter regions characteristic of C. jejuni. None of the putative prophages were successfully induced and propagated, so it is not known if they were functional or if they represented remnant prophage DNA in the bacterial chromosomes.Southern blotting of Campylobacter. There was evidence for recombination among groups of prophages, suggesting that the prophages had a mosaic structure. In many of these properties, the Mu-like CMLP 1 homologs characterized in this study resemble temperate bacteriophages of enteric bacteria that are responsible for contributions to virulence and host adaptation.These putative prophages form a family of phages with conserved sequences, and appear to be adapted to  Campylobacter spp., especially C. jejuni, have been recognized as the most frequent cause of bacterial enteric infection in many countries [Campylobacter genomes have now been fully or partially sequenced [Campylobacter isolates of interest it will be necessary to either sequence more genomes or explore the roles of mobile genetic elements such as transposons, plasmids, and bacteriophages.Though ountries -3, thereequenced ,5 and a equenced -8. HowevCampylobacter fetus (at the time known as Vibrio fetus) in 1968 after induction with mitomycin C, induction in aging cultures, or induction using co-cultivation methods [Campylobacter temperate bacteriophages are capable of horizontal DNA transfer. Using co-cultivation techniques, Bryner and colleagues [Vibrio fetus (Campylobacter fetus). Four groups of bacteriophage from lysogenic strains were defined on the basis of differential lysis of a panel of test isolates [C. jejuni in enteric disease of children demonstrated the presence of temperate bacteriophages that mediated resistance to typing phages and were capable of lysing a stock strain of C. jejuni [Campylobacter isolates [Lysogenic, or temperate, bacteriophages were first recovered from  methods , indicatlleagues  induced,isolates , suggest. jejuni . These pisolates .Autoagglutinated bacteria appeared to be "leaky", and phage tail-sheaths were associated with bacterial cells.Campylobacter spp. Several investigators attempted unsuccessfully to isolate and propagate temperate bacteriophages from C. jejuni [C. hyoilei [C. jejuni [Campylobacter jejuni integrated elements (CJIEs) [Campylobacter Mu-like phage 1 (CMLP 1). Elements similar to these CJIEs were found quite frequently when a large panel of isolates was tested using a DNA microarray, and CMLP 1 appeared to integrate essentially randomly in the genome [C. jejuni genome, producing changes in pulsed-field gel electrophoresis (PFGE) patterns [C. jejuni were found to result from inter-genomic inversions between Mu-like prophages; activation of dormant Mu-like prophages subsequent to predation by virulent bacteriophage was also noted [After this initial work there was a period in which temperate or lysogenic bacteriophages were not demonstrated in . jejuni ,16. Howe hyoilei . The ver. jejuni . At leaspatterns . Genome so noted . It appeC. jejuni of prophages homologous to CJIE 1, 2, and 4 of RM 1221. One of the questions we wanted to answer was whether these Campylobacter Mu-like prophages were similar in their heterogeneity to lambdoid prophages found, for instance, in many of the Enterobacteraceae. In this study we have therefore investigated the sequence diversity in approximately 9 – 11 kb regions of twelve bacteriophages homologous to CMLP 1 of C. jejuni strain RM 1221. Sequence variability due to apparent insertions and deletions was detected, and results supported the concept that these bacteriophages are modular in nature, with mosaic genomes. There was no evidence for the presence of lambdoid prophages similar to those found in the Enterobacteriaceae.At the time this study was begun, there were no data available on the distribution or variability within Campylobacter population tested  was detected in only 3 isolates. No sequences with homology to λ phage were found in any of the isolates tested in Southern blotting experiments.All CMLP 1 gene homologs used for Southern blot experiments were found in two isolates from bovine stools, but were completely absent from other bovine isolates. The single isolate from a chicken that carried a CMLP 1 homolog was either missing some of the genes found in the RM 1221 prophage or had genes that were divergent in at least two regions of its sequence. Both ovine isolates did not appear to carry the phage, though one had a single phage gene homolog.On the basis of the results of the Southern blot experiments described above, 12 isolates were chosen for sequence analysis of putative prophages homologous to CMLP 1. Our intent was to determine whether there was any sequence variability among these prophages and characterize the nature and extent of the changes present. If warranted, a subset of the prophages would then be completely sequenced at a later date. Since virulence genes are often inserted among the late genes of bacteriophage λ -23, we dApproximately 10 – 11 kb (~30%) of each prophage, equivalent to a region from about cje0215 to cje0232 of the RM 1221 genome, was sequenced by PCR amplification of approximately 1 – 3 kb fragments of the integrated phage genomes followed by chromosomal walking to fill in the complete sequence. In the case of isolate NC 13266 an amplicon was not obtained for the region adjacent to the cje0220 homolog, so that the sequenced region is shorter than those of the other CMLP 1 homologs. Different primer sets were used to obtain PCR amplicons from this region for the two main groups of isolates. Primer sets phs5 and phs6 proved capable of amplifying DNA from the group containing NC 13255, NC 13265, and other isolates, while the pcc1 primer set was used to amplify product from members of the group containing the Walkerton outbreak strain 1 isolates. The % G+C of the sequenced regions of the 12 prophages and the homologous region of RM 1221 CMLP 1 ranged from 30.66 to 31.93.The phylogenetic relationships among the CMLP 1 homologs are shown in Figure Campylobacter chromosome as a whole. Isolates 00–2425, 00–2538, and 00–2544 had identical sequences in this insert. Other isolates had slightly less identity for this 372 bp insert: 98% in 00–3477, NC 13256, and NC 13266; 97% identity in 99–7046, 00–6470, and NC13255; and 96% identity in isolates 00–0949 and 00–2828. There were no other sequences closely related to this insert sequence found in BLAST (blastn) searches of the nr/nt database. The next most closely related oligonucleotide was a 58 bp sequence from the Mus musculis chromosome (accession number gb/AC163020.9/) that had 88% identity with the Campylobacter sequence.The major structural features of the CMLP 1 homologs from all 12 isolates are compared with CMLP 1 in Figure Wolbachia endosymbiont of Drosophila melanogaster (accession number gb/AF420275.1/AF420275).The group of sequences that included the three Walkerton outbreak strain 1 isolates had a common 1068 nucleotide region of divergent sequence within cje0222 (tail tape measure protein). The % G+C of the insertion in isolate 00–2425 was 34.93, higher than the 31.66 % G+C of the entire sequenced part of this putative prophage. This insertion had an identical sequence over its entire length in isolates 00–2525, 00–2544, 00–2538, and NC 13256, and was 99% identical over its entire length with isolate 00–3477. These isolates were the only ones that had such a high degree of homology in BLAST (blastn) searches of the nr/nt database; the next most closely related sequence was a 58 nt fragment from a 2043 bp phage-related tail gene of a Isolate NC 13266 had a different region of divergent sequence (648 nucleotides) with a % G+C of 25.89 that incorporated one end of cje0222 and adjacent genes. While the first 335 bp of this showed moderate homology with the homologous region in the other prophages, including RM 1221, the final 310 bp had no homology to any sequences in the nr/nt database and had a 22.58% G+C. Finally, all sequences in the group containing Walkerton outbreak strain 1 isolates, plus NC 13255, had a common divergent sequence of 43 nucleotides after the ORF homologous to cje0231. This short sequence had no homology to the consensus sequence of the other strains and only low homology to any other sequences in the nr/nt database.Insertions of different sizes and at different locations were detected in NC 13266 (48 nucleotides), in NC13255 (43 nucleotides in cje0228), and 00–2818 (24 nucleotides near cje0229). Deletions were also found. A common deletion of 98 nucleotides was found in CMLP 1 homologs from 00–2425, 00–2538, 00–2544, 00–3477, NC 13255 and NC 13266. Isolate 00–2818 had a 120 nucleotide deletion within cje0220 and NC 13255 had an 88 nucleotide deletion near cje0229. In addition to the 98 nucleotide deletion described above, the CMLP 1 homolog of isolate NC 13266 had a 39 nucleotide deletion at the end of cje0230. It should be noted that, in addition to the insertions and deletions, all CMLP 1 homolog sequences contained single nucleotide changes not apparent on the schematic diagram shown in Figure Consistent with these major structural features, split decomposition analysis of the 12 partial prophage genomes exhibited a reticulate structure suggestive of extensive recombination among this population (data not shown).Campylobacter promoter elements.CMLP 1 from RM 1221 appeared to produce functional lytic phage particles  suggestiC. jejuni [Most of the ORFs encoding genes with putative bacteriophage functions were preceded by the consensus ribosome binding sequence (AAGGA) for . jejuni  in all iFor the ORFs encoding the putative cje0220 homolog the situation was a little more complicated. In isolates 99–7046, 00–0949, 00–2818, 00–6470, NC 132355, and NC 13265 this ORF had four possible alternative start sites with the start codons TTG (beginning with amino acids LHSKEWSG...), GTG (beginning with amino acids VAFSDAT...), and ATG . Only the GTG and latter ATG start codons were preceded by the consensus ribosome binding sequence. In the cje0220 homolog of isolates 00–2425, 00–2538, 00–2544, 00–3477, NC13256, and NC 13266 there were two predicted start sites; the first producing a peptide beginning with "MARK..." and the second producing the RM 1221-like peptide beginning with "MKNNT...". Both start sites were preceded by appropriate ribosome binding sequences, though their placement relative to the start codon differed.For some other ORFs the consensus ribosome binding sequence was not present adjacent to the putative start codon, and possible ribosome binding sites containing a suitable combination of "A"s and "G"s – preferably containing a GG dinucleotide – were sought. The cje0229 homolog in each putative prophage was preceded by a sequence consisting of GGGAGAG, while each cje0225 homolog was preceded by GGGCA, cje0224 by AGAGG, cje0222 by AAAGGG in all isolates except NC 13266, and cje0221 by ATGGA. Only the promoter regions of cje0218 homologs were devoid of a sequence resembling the consensus ribosome binding site or one of the potential ribosome binding sites proposed here.Campylobacter promoters are quite heterogeneous in both sequence and placement [C. jejuni sodB -10 sequence, TAATATT. A 2A12, proA-like -10 sequence was found preceding cje0226, cje0222, and only the ATG start codon encoding the "MKNNT..." start site of cje0220. The other two potential start sites for this ORF were not preceded by any of the known -10 promoter sequences.The -10, -16, and -35 regions of lacement . For thisodB (TAATATT), ileS (TAGAATT), and glyA (TATTGTT). All cje0225 ORFs were preceded by the lysS -10 oligonucleotide (TTTAAAC), while cje0221 was preceded by the 23ES-like sequence (TACAATT) and cje0217 by the 1G9-like sequence (TATATTA). A large proportion of the ORFs identified may therefore be expressed though, once again, there is no evidence yet that these putative prophages are capable of being induced. ORFs corresponding to cje0229, cje0228, cje0227, cje0224, cje0219, cje0218, and cje0216 did not have any of the experimentally determined -10 promoter sequences upstream of the start codon. It is possible that many of these ORFs carry alternative -10 sequences.cje0230 from all isolates except NC 13265 carried the orf1-like -10 promoter sequence, TATCTTT. Isolate NC 13265 had three different -10 sequences in its promoter region; these were identical to the sequences determined for Translations were obtained using DNA sequences for each ORF corresponding to a CMLP 1 gene, and the resulting peptides were compared. Results of phylogenetic analysis of these proteins are shown in Figure Most of the available prophage sequence was occupied with open reading frames corresponding to the CMLP 1 genes identified previously . As noteDifferences noted in the DNA sequence were apparent as differences in the translated protein products. The largest possible open reading frame present in cje0216 of isolate 00–2818 was 28 amino acids longer at the N-terminus than any of the cje0216 proteins from other isolates.C. jejuni and in Mu phages [Interesting variations were found in cje0220, which produces a protein with homology to a DNA adenine methylase. The DNA insertion within this gene in NC 13266 produced a region of peptide homology with cje0220 of strain 260.94, which was quite different from all other cje0220 peptides. Only a partial protein sequence was obtained for the cje0220 homolog in NC 13266. The 120-nucleotide deletion within cje0220 of 00–2818 resulted in an in-frame deletion within the C-terminal third of the protein. Four possible start sites were evident in cje0220 proteins from isolates 00–0949, 00–2818, 00–3477, NC 132356, and NC 13265, especially when the alternative start codons (UUG/TTG and GUG/GTG), known to be functional in both u phages ,25, wereVariability was also evident in the protein sequences of cje0222, which was homologous to phage tail tape measure proteins. The C-terminal third of the protein was highly conserved in all sequences. In isolate NC 13266 the remaining sequence was very similar to that of strain 260.94 , 00–3477, NC 13255, and strain RM 1221 replaced the consensus C terminal peptide of KYKKM with NTKVKK. Interestingly, the 43 nucleotide insertion into cje0228 of NC 13255 translated into a C-terminal peptide extension of QKIQKIQKIQKIQKIQKM, a change one might expect would have some effect on protein function. Cje0229 of NC 13255 also had a C-terminal extension different from all the other proteins, which were quite conserved overall. The cje0230 peptide sequence was conserved except for the 13 amino acid deletion in NC 13266 corresponding to the 39 nucleotide in-frame deletion in the DNA of this isolate. The C-terminal third of cje0231 from RM 1221 and NC 13265 was different from the corresponding protein in all other isolates, which were very similar.Homologs of proteins cje0217, cje0218, cje0219, cje0223, cje0224, cje0226, and cje0227 were remarkably well conserved overall, suggesting that the complete peptide sequence was required for the proper function of at least some of these proteins.Phylogenetic comparisons for each protein Figures , 5, 6 suIt should be noted that a number of additional potential ORFs producing translation products between 3,000 and 10,000 Daltons were detected, especially when alternative start codons were included in the analyses. For the most part these overlapped the previously characterized proteins on the opposite strand and lacked clearly identifiable ribosome binding sites, and so are not discussed in detail here. Examples include two ORFs that were found in most or all prophage sequences, encoded in the 00–2425 prophage by nucleotides 1680 to1979 and by nucleotides 2487 to 2729. If expressed, these ORFs would produce proteins with molecular masses of 7,653 and 8,369, respectively, that have no homology with any known peptide sequences. In contrast, an ORF encoded by nucleotides 2928 to 3167 of isolate 00–2425 appeared to be present only in 00–2425, 00–2538, 00–2544, 00–3477, and NC 13256. This ORF, if expressed and functional, could contribute to differences in biology among the two groups of isolates.Campylobacter isolates confirms the observation of Parker et al. [The finding of CJIEs 1, 2, and 4 in a number of r et al.  that ther et al. .Prophages homologous to CMLP 1 from strain RM 1221 were found in 4/6 isolates of Walkerton outbreak strain 1, suggesting that the rate of loss or gain of this bacteriophage may be quite high, as previously suggested . The facC. jejuni and C. coli share environmental niches, including the human intestinal environment, with Salmonella and E. coli isolates carrying lambdoid prophages partially responsible for the virulence of these organisms. The complete absence of lambdoid prophages from Campylobacter, if proven true in the long term, could indicate that these bacteria lack receptors for infection by lambdoid phages, that there are effective barriers to lysogeny by these phages, or that the genes carried by these phages do not provide a sufficient selective advantage to the organism to be stably maintained in the population.It was interesting that no lysogenic phage DNA homologous to phage λ was found in the genome of any isolate, as this phage family plays the predominant role in the pathogenesis of many members of the Enterobacteraceae. If one assumes that the presence of genes from the beginning, middle, and end of CJIE 4 indicates the presence of the whole element, it can be seen that this element is present in a number of isolates from which CMLP 1 (CJIE 1) is absent. This in turn indicates that these elements can be inherited independently, though the data do not allow a distinction between the existence of CJIE 4 as a mobile element versus the differential carriage of the two elements through gain or loss of CJIE 1. Results from Southern blots using probes to CJIE 2 are somewhat more difficult to interpret due to the fact that the two genes tested are present in different frequencies in the population. Future work will concentrate on this element alone.C. jejuni found by MLST [C. jejuni populations and that the phage may have been acquired early in the evolution of this organism. It was considered somewhat surprising, therefore, that only three major groups of CMLP 1 homologs were found when DNA sequences were analyzed phylogenetically. Sequence comparisons indicated that the CMLP 1 homolog carried by isolate NC 13266 showed evidence of recombination with the other two groups of phage as well as the acquisition and incorporation of novel DNA sequences. This was supported by our inability to obtain sequence further upstream of the cje0220 gene with primers that produced PCR amplicons from other isolates, a finding that suggests that this region contains unique DNA sequences in NC 13266. The NC 13266 CMLP 1 homolog appeared to be highly related to strain 260.94 in all proteins for which sequence was available from GenBank. Future work will involve cloning and sequencing further regions of NC 13266 using strain 260.94 sequences for developing PCR primers.Phages homologous to CMLP 1 were found in 4 of 13 reference isolates from the UK that represent much of the variability within  by MLST . The isoC. jejuni genome as a whole, suggesting that these sequences were acquired from an organism other than C. jejuni. Recombination appeared to have a major role in the diversification of the prophages under study. Because the 372 bp replacement sequence was found in all prophages except NC 13265 and RM 1221, we would speculate that this event may have occurred first in the evolution of the prophages studied here. The 43 bp replacement was present in only a subset of these prophages, and would appear to have been acquired later. Finally, the 1068 bp replacement was found in only a small subgroup containing the three Walkerton outbreak isolates, suggesting that it was acquired last. Most of the insertions and deletions appear to be strain-specific, and suggest fairly frequent changes in the prophage DNA sequence. The finding of the 98 bp deletion in both NC 13266 (in the absence of the 43 bp replacement) and in the group containing the Walkerton isolates  suggests that at least some of the prophage sequence changes could be modified by additional recombination events. This makes the construction of a tidy evolutionary tree difficult for these integrated bacteriophages.CMLP 1 homologs from isolates 00–2425, 00–2538, 00–2544, 00–3477, and NC 13256 had similar structural features, including a 98 bp deletion and a 1068 bp sequence quite distinct from the analogous sequence in CMLP 1. This 1068 bp sequence, as well as a 372 bp sequence replacement common to most prophages analyzed in this study, had a somewhat higher % G+C than the overall prophage sequence and the Campylobacter temperate phages with similar sequences. It is also possible that there are more families of sequence variants yet to be discovered.The 100% sequence identity of isolates 00–2425, 00–2538, and 00–2544 supports the classification of these isolates as clones of the Walkerton outbreak strain 1 ,27. The Campylobacter. Since CMLP 1 was previously induced from RM 1221 [Campylobacter and that Campylobacter may be the preferred or unique host of these phages, an hypothesis supported by the fact that the % G + C of the sequenced part of the prophages is very close to that of previously sequenced C. jejuni genomes . At thiThe effect of variations in phage DNA sequence on the resulting protein sequence was readily apparent. Some proteins had amino acid sequences quite different from the corresponding RM 1221 sequence, so much so that one would expect the function of the protein to be highly modified or abrogated. Most insertions and deletions were in-frame, so that a full-length protein was made. In these cases, it is possible that the expressed proteins might have modified function. Several proteins appeared to have potential N-terminal extensions compared with the RM 1221 homolog, though it is not clear whether these would be included in the final protein product. Alternate start codons were responsible for the possible presence of some of these N-terminal peptide extensions. These data confirm observations previously submitted by others to GenBank for isolate CF93-6. Further proteomics and functional studies will be necessary to answer some of the questions raised by the sequence analysis presented here and elsewhere.Escherichia coli, while similar Gifsy and Fels phages encode a number of virulence factors in Salmonella enterica serovar Typhimurium [S. Typhimurium proteins are effectors translocated into eucaryotic cells by type three secretion systems encoded at other locations in the chromosome [Campylobacter prophages exhibit at least some of the properties, including a modular or mosaic structure, in common with prophages from other enteric bacteria, thereby supporting the possibility that they may also have similar functions in virulence and the biology of the bacteria. No morons that could be novel virulence genes were found during the partial sequencing of the CMLP 1 prophage homologs in C. jejuni isolates. To address whether additional novel genes might be present in other parts of the prophage, future work will involve completion of the DNA sequences of a subset of the prophages discussed here. It would also be of interest to determine, using expression DNA microarrays, quantitative RT-PCR, and 2D-DIGE, whether prophage genes are expressed and whether the presence of integrated Mu-like prophages affects the expression of Campylobacter chromosomal genes. Initial work can be done using a naturally occurring strain pair with and without the prophage [Campylobacter.Bacteriophages have critically important roles in genome diversification and the evolution of virulence and host adaptation of other enteric bacteria ,23,28,29himurium . Some ofromosome . In addiromosome ,32. Whilprophage , though Campylobacter jejuni population. Phages in this family appear to have undergone some differentiation, and may be continuing to evolve. Future studies are required to understand how these bacteriophages interact with their host bacteria, and what implications this has for the pathogenesis, ecology, survival, and growth of these bacteria.CMLP 1 and its homologs appear to represent a temperate bacteriophage family that is widely distributed and frequently carried within the C. jejuni and C. coli isolates were chosen from among isolates previously characterized as part of investigations into a large Canadian water-borne outbreak [C. jejuni MLST reference isolates representing most of the major clonal complexes of this organism [2, 5% O2, and 85% N2).Table outbreak . C. jejuorganism  were purorganism . All iso2, 0.5 μM of each primer, 0.2 mM dNTP mix, 200 to 1000 ng of template DNA, and 5 U FastStart DNA polymerase . PCR was performed using a Perkin Elmer 2400 thermocycler. Denaturation and annealing times were 1 min each, while extension times were 1 min for amplicons less than 1 kb and 3 min for products larger than that. Products were electrophoresed on 1.5 % agarose gels then purified using the QIAquick PCR purification kit  or Montage™ PCR Centrifugal Filter Devices  according to the manufacturers' instructions. Sequencing reactions were run using Big Dye Terminator 3.1 Cycle Sequencing kits  according to the manufacturer's instructions, and sequencing was performed using an ABI 3100 or 3730 DNA Analyzer (Applied Biosystems).Template DNA was prepared from bacteria using the PureGene™ Genomic DNA purification kit  according to the manufacturer's recommendations. DNA from strain RM 1221 was used as a positive control in all reactions and strains NCTC 11168 and 81–176 were used as negative controls. A control in which water was substituted for DNA template was included in all runs. 100 μl PCR reaction mixtures consisted of 1× PCR buffer, 2 mM MgClCampylobacter prophages and keeping them in a form suitable for transducing recipient strains [Some investigators have had difficulties inducing  strains ,16. We t2, 5% O2, and 85% N2). Bacteria were embedded in 1.2% Seakem Gold agarose  plugs and these plugs were digested to lyse bacterial cells using the standardized CDC protocol [Pst I and 1.0 μl of a 0.5 mg/ml RNase solution  in a total volume of 100 μl followed by an incubation period of 4 h at 37°C. Finally, the intact plugs containing digested DNA were equilibrated with 0.5 ml of 0.5X TBE  for 15 min at room temperature and placed into a 1% agarose gel. Samples were electrophoresed for 18 h at 60 volts in 0.5 × TBE. Gels were stained with ethidium bromide, photographed, and blotted using a Vacugene XL blotting apparatus . Blotting was performed according to protocol No.1 of the manufacturer's instructions. Blots were cross-linked using UV and probed with 500 ng of labelled probe and 100 ng of labelled λ DNA.Southern blotting was done using bacteria embedded in plugs essentially according to the methods for manual ribotyping of Clark et al. . Bacteriprotocol . Three-qC. jejuni strains RM 1221 or isolate 00–2544 using the primers and conditions listed in Table NC_003912), amplified by PCR, and purified as summarized above. Probes (amplification products) were sent to the DNA Core Facility at the National Microbiology Laboratory for DNA sequence analysis to confirm the identity of the product prior to use in hybridizations. Probe preparation, hybridization and detection were performed using the AlkPhos direct labelling and CDP-Star detection system  under low stringency conditions according to the manufacturer's instructions. Strains RM 1221, NCTC 11168, and 81–176 were included on blots as positive and negative controls; RM 1221 was included on each blot analyzed, while NCTC 11168 and 81–176 were present only on one set of blots. The size standard used was DNA Molecular Weight Marker II . All blots were also developed using only λ DNA as a probe. This provided a test for the presence of λ prophages and further served to ensure that no bands due to potentially lysogenic lambda phages were seen when blots were developed with probes for RM 1221 phage genes.Probes were generated by PCR amplification of bacteriophage genes from either Sequence assembly and editing were done using Seqman™ and EditSeq™ in the Lasergene DNASTAR™ version 5.06 software . The sequences obtained had at least two strands in opposite directions, while some regions had four or five overlapping contigs.Sequences were saved as FASTA (.fas) files. After entry into MEGA™ 3.1  the sequCampylobacter [Prophages were searched for the presence of known ribosome binding sites and -10 promoter sequences . Additiolobacter  and Mu [lobacter  were incEF694684 – EF694695.Sequences have been deposited in GenBank and are numbered CGC was responsible for conception of the study, experimental design, data collection, and analysis. L-KN participated in data analysis and preparation of the manuscript.


Tamoxifen, a selective oestrogen receptor (ER) modulator, increases bone mineral density (BMD) in postmenopausal women and decreases BMD in premenopausal women. We hypothesised that inherited variants in candidate genes involved in oestrogen signalling and tamoxifen metabolism might be associated with tamoxifen effects in bone.ESR1, ESR2, and CYP2D6 were tested for associations in the context of menopausal status and previous chemotherapy, with a mean percentage change in BMD over 12 months.A total of 297 women who were initiating tamoxifen therapy were enrolled in a prospective multicentre clinical trial. Lumbar spine and total hip BMD values were measured using dual-energy X-ray absorptiometry (DXA) at baseline and after 12 months of tamoxifen therapy. Single-nucleotide polymorphisms (SNPs) in The percentage increase in BMD was greater in postmenopausal women and in those patients who had been treated with chemotherapy. No significant associations between tested SNPs and either baseline BMD or change in BMD with 1 year of tamoxifen therapy were detected.The evaluated SNPs in ESR and CYP2D6 do not seem to influence BMD in tamoxifen-treated subjects. As mortality from breast cancer has been decreasing , has been the standard adjuvant hormonal treatment for early-stage hormone receptor-positive breast cancer for more than two decades . It has CYP2D6 is a highly polymorphic gene. Indeed, more than 80 different alleles have been identified, many of which confer a decreased or absent CYP2D6 activity. As has been previously shown in the cohort of patients described in this report, there is an association between decreased CYP2D6 activity and decreased serum concentrations of endoxifen, one of the main active metabolites of tamoxifen  P450 enzymes, primarily by CYP2D6 . Single-nucleotide polymorphisms (SNPs) have been identified in both ESR1, ESR2, and CYP2D6 on BMD, we initiated a prospective study to evaluate the changes in BMD in both pre- and postmenopausal women who were recommended tamoxifen either as adjuvant therapy for newly diagnosed breast cancer or for chemoprevention. Baseline BMD and change in BMD with 1 year of therapy were tested for associations with polymorphisms in ESR1, ESR2, and CYP2D6.To further analyse the associations between genetic variants in www.ClinicalTrials.gov (NCT0022893).Eligible patients were recruited into a prospective, observational, open-labelled, multicentre registry study conducted by the Consortium on Breast Cancer Pharmacogenomics (COBRA) from 2000 to 2006. Participating COBRA institutions included the Lombardi Comprehensive Cancer Center at Georgetown University Medical Center, Washington, DC; the Indiana University Cancer Center, Indianapolis, Indiana; and the Breast Oncology Program at the University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan. The analysis presented in this report is a secondary aim of the main study, and was specifically designed to test relationships between polymorphisms in the genes that encode ER and CYP2D6 and change in BMD after 1 year of tamoxifen therapy. Results from this cohort regarding other genotypes and phenotypes have been previously reported . Patients were excluded if they were pregnant or lactating. No restrictions were placed on the use of bisphosphonates for trial enrolment, although subjects taking bisphosphonates were excluded from the BMD analyses, as described below. Use of supplemental calcium and vitamin D was encouraged. The protocol was approved by the institutional review boards of all three participating study sites. All enrolled patients provided written informed consent.Pre- and postmenopausal women 18 years or older, who had oestrogen and/or progesterone receptor-positive breast cancer, were starting adjuvant treatment with tamoxifen, had a history of ductal carcinoma in situ were enrolled in the trial after completion of primary surgical resection of breast cancer, and after any indicated adjuvant chemotherapy and/or radiation therapy. Before starting tamoxifen, a complete medical history, comprehensive list of medications, physical examination, and baseline laboratory studies were obtained from each patient. Menopausal status was determined on the basis of self-reported menstrual history. Women who were 60 years or older, who were amenorrhoeic for 12 months before enrolment, or had undergone previous bilateral oophorectomy were considered postmenopausal. Women with regular menses before adjuvant chemotherapy or tamoxifen were considered premenopausal. All other patients were considered perimenopausal. Patients were followed up in the outpatient clinic 1, 4, 8, and 12 months after the initiation of treatment with tamoxifen at 20 mg per day. During each follow-up visit, changes in medical history and medication use were reviewed.Participants with invasive breast cancer or ductal carcinoma g. All samples (plasma and whole blood) were then transferred to cryogenic vials  and stored at −80 °C pending analysis.Blood samples (∼10 ml) were collected at baseline in heparinised Vacutainer tubes  for plasma isolation and genomic DNA extraction for genotyping analysis. Plasma was separated within 1 h of blood collection by centrifugation at 2060 http://www.pharmgkb.org with the accession ID PS207749.Standard dual-energy X-ray absorptiometry (DXA) scans were performed to measure BMD immediately before initiation of tamoxifen and at the completion of 1 year of therapy. The BMD values  of the left femoral neck (hip) and total lumbar spine were measured at the University of Michigan using a Lunar DPX-L bone densitometer  and at Indiana University and Georgetown University using Hologic 4500 bone densitometers . Baseline and 12-month DXA assessments were performed on the same bone densitometer for all but 17% of subjects at a single study site . The BMD data set can be found on ESR1) SNPs (XbaI=rs 9340799 and PvuII=rs 2234693) and two ER-β (ESR2) SNPs (ESR2_01=rs 1256049 and ESR2_02=rs 4986938) was performed in duplicate by Taqman assays as previously described by the NCI CGAP project (snp500cancer.nci.nih.gov). Amplification and analysis were performed using the iCycler real-time thermocycler . Genotyping for CYP2D6 variants was performed as previously described (http://www.pharmgkb.org) with accession IDs ESR1 (PS204992 and PS204997), ESR2 , and CYP2D6 .Genotyping for two ER-α  . Differences in each BMD outcome among genotypes, including age- and centre-adjusted analysis, were also examined using a general linear model. For post hoc comparisons, we compared the adjusted means between all pairs of three genotypes while controlling for overall alpha using the Tukey–Kramer method. For all analyses, a P-value of <0.05 was considered to be statistically significant.The primary end point for this study was to test the association between menopausal status, previous chemotherapy, genotype, and mean percentage change in BMD over 12 months. Because of similarities in bone biology in peri- and postmenopausal women, data from peri- and postmenopausal patients were combined and compared with data from premenopausal patients in this analysis . The ESR2 haplotypes were also constructed but were few because of the low frequency of ESR2_01 variants. Haplotype associations with baseline BMD and percentage change in BMD were tested with a generalised estimating equation approach through its online implementation (http://www.mayo.edu/statgene) , significantly outlying values of percentage BMD change (n=2), use of raloxifene at the time of study enrolment (n=1), and failure to initiate protocol-directed treatment (n=1) . All 276 remaining patients were included in the baseline BMD analyses. Of these 276 patients, 11 did not have any baseline DXA measurements, 3 did not have baseline lumbar spine DXA measurements, and 9 did not have baseline hip DXA measurements. Therefore, 95% of analysed subjects had baseline lumbar spine DXA measurements, and 93% had baseline hip DXA measurements. Baseline characteristics of the analysed cohort are described in P<0.0001) and all were postmenopausal.Of the 297 patients who enrolled in the clinical trial, 21 were excluded from BMD analyses because of concomitant bisphosphonate use during the 1-year study duration (n=17), premature discontinuation of study participation for disease progression (n=1), toxicity (n=18), a switch to aromatase inhibitor (n=2), relocation (n=2), non-compliance or withdrawal of consent (n=8), and unknown (n=10). Data on hip BMD were missing on seven patients at one or both time points either because of machine malfunction or previous bilateral hip replacement. Thus, a total of 211 patients  had both baseline and 12-month DXA assessment at the hip, and 218 patients  had both DXA assessments at the lumbar spine. These subjects are included in the analyses evaluating percentage change in BMD with 1 year of tamoxifen therapy. There were no differences between the baseline characteristics of patients with and without 12-month DXA assessments.In total, 58 patients did not have matched baseline and 12-month DXA assessments for the following reasons: missing baseline and/or 12-month measurements at either the hip or the spine (α (ESR1 PvuII and XbaI) and ER-β (ESR2_01 and _02) were determined for 260 patients . Similar results were noted for the baseline T score at the lumbar spine . No statistically significant differences in either the BMD or T score were noted between subjects treated and not treated with chemotherapy.Mean baseline BMD for all patients eligible for analysis divided by their menopausal and chemotherapy status is listed in The percentage change in BMD was calculated for each patient with matched baseline and 12-month BMD determinations. The percentage change in hip and lumbar spine BMD for all patients in aggregate, as well as for patients divided by chemotherapy and/or menopausal status, is presented in P=0.002), but only a trend was noted at the hip (P=0.065).Similar to previously reported results, postmenopausal women had an increase in BMD at the hip (+0.8%) and stability of BMD at the lumbar spine (−0.1%) with 1 year of tamoxifen therapy . The difference in the percentage change in BMD between chemotherapy-treated and -untreated subjects remained when patients were subdivided by menopausal status (vs −1.5% P<0.001) and hip (+1.8 vs −0.1% P=0.025), and for premenopausal women at the hip (−1.1 vs −3.6% P=0.030) but not at the lumbar spine.Patients who had received chemotherapy before initiation of tamoxifen therapy had an increase in both lumbar spine (+0.5%) and hip (+1.3%) BMD, whereas those who did not receive chemotherapy had a decrease in BMD at both sites , differences that were statistically significant (lumbar spine: l status . DiffereESR1 (PvuII and XbaI) and two ESR2 (ESR2_01 and ESR2_02) polymorphisms and baseline BMD. No association was noted between baseline BMD in the overall patient cohort or in any patient subgroup for the four polymorphisms studied .Because we previously showed that serum concentrations of endoxifen, a key active metabolite of tamoxifen, are lower in patients with In this prospective clinical trial of pre- and postmenopausal women initiating therapy with tamoxifen, the subjects underwent BMD assessment at baseline and at 1 year to determine the effects of tamoxifen therapy on change in BMD. Approximately half of the patients in this trial had previously received chemotherapy, which was fairly evenly divided between the menopausal groups. Our data regarding the general effects of tamoxifen as a SERM are consistent with previously reported results in which premenopausal women have a decrease in BMD with tamoxifen therapy that is greater at the lumbar spine than at the hip  compared with that reported by −2). Finally, a third potential source of bias is the replacement of a DXA machine at one institution during the conduct of this study. However, the effect of this change on the outcome of the study is likely to be minimal, as all subjects at that institution had their baseline DXA scans performed on the same machine, and only 17% had their baseline and 12-month DXA assessments performed on different machines. In addition, this may more closely reflect standard clinical practice, in which patients are unlikely to undergo serial bone density assessment on the same machine. Although bone turnover markers could be assessed to confirm the changes noted on DXA scanning, we unfortunately do not have sufficient remaining serum or urine samples for this evaluation.In postmenopausal women who did not receive chemotherapy, we did not find as great an increase in BMD with tamoxifen therapy as has previously been reported (ESR1 and ESR2) on baseline BMD and change in BMD with tamoxifen therapy. However, no associations were detected between any of the four polymorphisms evaluated and either baseline BMD or change in BMD with tamoxifen therapy, or within ESR1 or ESR2 haplotypes. This finding is consistent with an earlier report of tamoxifen therapy in postmenopausal Japanese women, in whom no association was noted between change in BMD and either of the two ESR1 genotypes . Thus, it is possible that small single gene effects are present, but were not detected. In addition, because of the complexity of gene–gene interactions, it is possible that no single genotype–phenotype association is sufficiently strong to be evident, but rather a combination of genotypes may be required for a meaningful effect. Given the difficulties with multiple gene comparisons, a cohort of this size would be insufficient to arrive at meaningful conclusions if the effect sizes are small. Although it is possible that a comprehensive haplotype-tagging approach may engender useful associations, it is clear that no large effect due to these germline variants, which have previously been shown to influence BMD, is evident in this study. It is possible that a pathway approach involving an examination of genetic variants in genes that code for other elements within the oestrogen signalling pathways may reveal valuable mechanistic and predictive data, and that a genome-wide variant analysis might also be useful, but such examinations are beyond the scope of this study.In summary, our data are consistent with previously reported data on the change in BMD with tamoxifen therapy in pre- and postmenopausal women. No inherited gene variants were found to be statistically significantly associated with baseline or change in BMD during tamoxifen therapy. The finding that previous chemotherapy may influence BMD response to tamoxifen is provocative and worthy of further study.


High grade gliomas (HGG) including glioblastomas (GBM) are the most common and devastating primary brain tumours. Despite important progresses in GBM treatment that currently includes surgery combined to radio- and chemotherapy, GBM patients' prognosis remains very poor. Immunotherapy is one of the new promising therapeutic approaches that can specifically target tumour cells. Such an approach could also maintain long term antitumour responses without inducing neurologic defects. Since the past 25 years, adoptive and active immunotherapies using lymphokine-activated killer cells, cytotoxic T cells, tumour-infiltrating lymphocytes, autologous tumour cells, and dendritic cells have been tested in phase I/II clinical trials with HGG patients. This paper inventories these cellular immunotherapeutic strategies and discusses their efficacy, limits, and future perspectives for optimizing the treatment to achieve clinical benefits for GBM patients. Glioblastoma is the most common primary brain tumour in humans  and has  Until recently, it was generally assumed that immune reactions do not occur in the brain because of the blood-brain barrier (BBB) and the specific features of the brain such as the absence of conventional lymphatic vessels or the low level of circulating T cells. It is now known however that the central nervous system maintains a two-way communication network with the immune system. Infectious or experimental autoimmune encephalomyelitis animal models allow better understanding of how the immune system operates in the brain.α4/β7 integrins and therefore exhibit tropism for the brain ,–72,54–72β, TNFα, PGE2, or IFNγ [6 and 1010. The number of injections and the frequency of the injections were also highly variable. In most studies, DC were prepared using a standard method, that is, from peripheral monocytes cultivated in the presence of GM-CSF and IL4. In six studies, they were matured using different cocktails with IL1 or IFNγ , 71, 72. or IFNγ . Vaccine or IFNγ . In one, or IFNγ . The numin vitro tests or DTH) was observed in more than half of patients. For the twenty-four patients who relapsed after vaccination and had an analysable tumour, lymphocyte infiltrations, particularly CD8 cells, were found in 15 patients.  Tolerance appeared to be acceptable with only one grade IV neurotoxicity (stupor) event being reported for all of these studies . This paγ synthesis (before versus after vaccination using in vitro PBMC stimulation) compared with non-responders. In addition, this study showed that responders to vaccination exhibited a better response to chemotherapy delivered in a second phase [β and with better survival. The opposite was observed for three other patients without T-cell infiltration . One study showed that patients vaccinated with mature DC had better survival than those vaccinated with immature DC and that conjoint administration of DC in the peripheral blood and intracranially gave a superior response than peripheral injection alone [ The radiological response was described in 10 studies with 6 CR (4 GBM), 11 PR (5 GBM), 7 MR (6 GBM), and 27 SD (14 GBM) among the 130 patients. Thirteen studies have reported a beneficial effect in terms of survival compared with historical cohorts or nonrandomized control groups , 67–72. nd phase . In theind phase  had fouron alone . Summarizing, active immunotherapy appears to have a beneficial effect in some patients, particularly those with a small tumour, without major toxicity. Both clinical trials using ATC and DC demonstrate induced immune responses  and some clinical responses. Once again, due to the large variability of protocols tested in these trials, that is, the source of ATC, the type of DC used, the cell number injected, the number of injection, the type of adjuvants, it is almost impossible to recommend a particular approach. One can just point out that for DC, no dose-related toxicity or efficacy has been demonstrated . In addi There were 76 objective responses (18 CR and 58 PR) in the 396 HGG patients described in the trials reported in this paper. Forty-nine and 27 responses were reported for adoptive and active immunotherapy, given objective responses rates of 23% and 15% respectively. Only patients with residual disease at the time of the vaccination can be evaluated for CR or PR; patients who undergo a gross total resection can only achieve stable disease as their best radiographic outcome. For other solid tumours, similar criteria based on radiological tumour measurements (RECIST-based criteria) are used. Using these criteria, a 3.3% overall objective response rate was reported for 1306 patients with different metastatic cancer vaccinated with peptides, pox viruses, tumour cells, or dendritic cells . Higher  The interaction between injected or induced immune cells and tumour cells can lead to an equilibrium rather than a destruction. Objective criteria based on changes in the tumour masses may therefore not be well adapted to assess this type of response. That is why survival is often regarded as a better endpoint after immunotherapy. Several studies reported in this paper have shown better survival for patients treated with immunotherapy compared to patients from historical cohorts or nonrandomized groups. All are phase I/II trials, with a limited number of patients, which must lead to a cautious interpretation. Lower tumour grade, extent of resection, younger age, good performance status, mutation of IDH1, as well as an intact neurological function are recognised as favourable prognostic factors for HGG . These f The immune therapy was in these trials well tolerated, with few grade III/IV adverse events. Cerebral oedemas were often detected in active immunotherapy using effector cells directly infused in the tumour site. However this side effect is difficult to separate from oedema usually observed after surgery. Alternatively, IL2 and GM-CSF used as adjuvant, might lead to vascular perturbations leading in edema formation. Therefore, an MRI before this type of treatment is necessary to distinguish the increased cerebral edema due to surgery from immunotherapy. The lack of other major side effects could be explained by the selectivity of the treatment aiming at destroying specifically tumour cells. Alternative explanations could be the relative inefficacy of the treatment or the frequently use of steroids in glioma that could prevent some major side effects. Up to now, major toxicity has not been reported, except for one patient with bulky residual tumour, for whom an overwhelming peritumoural inflammatory reaction was observed, after injection of DCs . Phase I Treatment for newlydiagnosed GBM begins with surgery when feasible, followed by focal radiotherapy (RT). For patients up to 70 years, the current standard of care comprises also a temozolomide (TMZ) chemotherapy during and after RT. At recurrence, repeat surgery, as well as new lines of chemotherapy like nitrosourea or bevacizumab (+/− irinotecan) may prolong survival in some patients. Despite this multimodal treatment, mean overall survival remains below 16 months . Most stexvivo expanded TIL plus IL2. The addition of the lymphodepletion preparative regimen consisting in a chemotherapy (cyclophosphamide and fludarabine) and whole body irradiation resulted in objective response rates of 52% and 72% in two trials with both 25 patients, with increased survival compared to patients who received T cells without lymphodepletion. One of the key improvements is the persistence of infused cells, which is highly associated with objective response. Lymphodepletion could act by eliminating competition for homeostatic cytokines, in particular IL15, for which high levels can be detected only after treatment. Other factors, like elimination of T reg cells could also play major roles [in vitro tests, such as phenotyping of effector cells or killing assays against tumour cells, should help to predict the treatment efficiency on a particular patient. However, the phenotype of the effector cells used in the different clinical trials with adoptive therapies varies between patients and has no clear impact on the clinical outcome that is, the presence of T-LAK (CD3+) and LAK cells (CD3− CD56+) in LAK trials or the ratio of CD4/8 cells in CTL trials. Moreover, most of the studies using LAK and CTL cells could not correlate the high cytotoxicity activity of the effector cells observed in vitro against autologous tumour cells and the in vivo efficiency of the treatment. This highlights the need to integrate the recent advances in the field in order to improve the quality of the injected cells. As the killing activity of LAK is not specific, it seems better to use CTL. CTL can be expanded from TIL repeatedly stimulated in vitro. This technique works very well for melanoma, a tumour wellknown to induce significant numbers of CTL during the natural course of the disease. Though Quattrocchi et al., in the only trial using this approach, expanded up to 3 × 109 lymphocytes from tumours [in vivo is a major point for the success of adoptive immunotherapy, it is interesting to note that persistence of effector cells at the site of the tumour was often transient as observed on biopsies or tumour samples analysed after relapse in the reported studies. In the addition to improve the injected cells, working on this point could therefore lead to great improvements. Instead of proposing heavy treatments inducing lymphodepletion, the use of alternative cytokines to IL2, like IL15 could be tested as well as the combination of an active vaccination. In an animal model, the concomitant adoptive transfer of T cells and a vaccination improves substantially the anti-tumour efficacy of the treatment [ Up to now, the best results for patients with metastatic melanoma have been obtained with adoptive therapy involving lymphodepletion followed by the intravenous injection of or roles . In case tumours  it could tumours . As persreatment . It is l Most studies suggest that cancer vaccine could have the most benefit in state of low tumour burden. This is also observed in a series of 56 recurrent GBM patients vaccinated with DC. Total resection before vaccination was the only predictor of a better survival . In contin vitro growth is not sufficient to allow vaccination. The delay between the beginning of the culture and the time when vaccine preparation is available can be very long. Median expansion times range between 4.5 and 30 weeks [in vitro  [ Vaccination strategy can consist of inoculation of inactivated autologous tumour cells. The first limitation step of the approach is the culture of the tumour cells. It is not always possible to establish primary cell lines from malignant gliomas. In the study of Parney, on 116 malignant gliomas, culture was successful in 61% of cases . In some30 weeks , 51, 53,n, 2002) , which cn, 2002) –92. Howen, 2002) . In the n, 2002) . Effectsn, 2002) . A possin, 2002) . This hihttp://www.mmri.mater.org.au/). Many recent reviews have focused on the use of DC as cancer vaccine [α2, YKL-40, and GP100 [ Most of vaccination protocols for HGG patients have used DC, the most potent APC. This reflects the popularity of this vaccine approach: up to now, more than 3000 patients suffering from varied cancers, enrolled in more than 200 trials have received DC  are superior to immature DCs (imDC) [α and IL1β and patients receiving only DC matured with TNFα, IL1β, and PGE2 [α, TNFα, IL1β and IFNγ) allows the generation of fully mature DC, with high migratory responsiveness to lymphoid organ chemokines and an ability to produce significant levels of IL12p70 after CD40 ligation (which mimics the interaction between DC and CD40L-activated cells in lymph nodes) [α-type-1 polarized DCs, pulsed with peptide antigens can migrate into draining lymph nodes after SC injection and induce antigen-specific CTL, allowing a prolong survival. The secretion of CXCL10 by these DC seems to play a major role in the induction of specific CTL and their homing to the brain [α-DC loaded with glioma-associated antigen epitopes, in combination with the administration of poly-ICLC, is currently tested in recurrent malignant gliomas [ About the best way to generate DC s (imDC) . They cas (imDC) . Van Gooand PGE2 . The math nodes) . In a mohe brain . As the  gliomas , the tra The relevance of intravenous DC injection has never been tested for HGG patients. It is worth noting that the only autologous cellular immunotherapy approved by the US FDA is based on the intravenous administration of PBMC cultured with recombinant PAP-GM-CSF for the treatment of metastatic prostate cancer . In mostβ, PGE2, and IL10 [Whatever the immunotherapy approach, one fundamental element to take into consideration is the corrupted tumour microenvironment which favors tumour development. Different mechanisms developed by glioma cells can suppress anti-tumour immune responses. Several defects at the systemic level have been reported in glioma patients such as decreased T-cell responsiveness , 112, inand IL10 –123. β1-3 [β2, IDO, and PDL-1 on GBM immunosuppressive properties on T-cell functions in a tumour-specific T-cell model [ Beside these systemic effects, glioma cells develop several molecular strategies to inhibit directly the immune cell effectors at the tumour site. Several intracellular, membrane and soluble molecules have been described as taking part in this phenomenon such as CD70 , 125, Faβ1-3 . We havell model . Differell model . GIM arell model . Furtherll model . As descll model , 137, alll model . One recll model . These cβ, IDO, and PD1 are also being tested clinically. Preclinical studies in glioma models have shown the interest to combine vaccination with inhibition of TGFβ [ A better efficacy could certainly associate optimized standard immunotherapy protocols with treatments designed to hamper this immunomodulation. The early results using immunomodulator molecules have been encouraging (as an example CTLA4 blockade in melanoma patients ) and one of TGFβ  or deple of TGFβ . In conclusion, immunotherapy can be considered as a potential new interesting weapon for treatment of patients with HGG and one should expect many improvements of this type of approach over the next years. However, there is a need for well-designed trials with higher numbers and more homogenous patients, taking into account the current recognized prognostic or predictive factors like and leading to phase III randomized studies.