From a biochemical perspective, a living cell is a collection of molecules jampacked into a confined space by a flexible barrier, called the plasma membrane. A diverse array of proteins embedded in the plasma membrane act as conduits between the cell interior and its external environment, conveying nutrients, metabolites, and information. The life of a cell—as well as that of any multicellular organism—depends on a cell's ability to communicate with its neighbors, both near and far. One way cells do this is with transmembrane receptors outfitted with both extracellular and intracellular domains that mediate information flow between the cell's external and internal environment. One class of transmembrane receptors, called integrin receptors, specializes in interacting with and binding to other cells and the extracellular matrix, a complex of molecules surrounding cells that provides structural support. By integrating various components of the extracellular matrix, integrins , play an important role in such diverse processes as cell differentiation, programmed cell death, wound healing, and metastasis.Integrins can be regulated by signals within the cell to bind to their ligands with either low or high affinity. While a multitude of integrin ligands have been identified and the general mechanics of both the extracellular and intracellular domains of these receptors are known, exactly how a signal crosses the receptor's transmembrane segment to regulate affinity has remained obscure. Now, Bing-Hao Luo, Timothy Springer, and Junichi Takagi have taken a mutational approach to shed light on the inner workings of the transmembrane segment and to explain how it transmits information.Much of what we know about the function of integrins has come from studying the crystal structures and models obtained from structural analysis. These analyses have generated information not only about the structure and composition of the extracellular and intracellular domains of integrins, but also about the conformational changes that accompany signaling events. Integrins contain a large extracellular domain, a transmembrane segment, and a relatively short intracellular “tail.” Integrins are heterodimers—molecules that contain two subunits composed of different amino acids—made up of an α chain and a β chain. Tight association of the two subunits is associated with an inactive, or low-affinity, state of the extracellular ligand-binding domain. Separation of the intracellular subunits is associated with a dramatic conformational change and activation of the extracellular domain, changing a bent structure with a downward-pointing ligand-binding site into an extended one with an outwardly stretched ligand-binding site. This mechanism differs from most transmembrane signaling molecules, which usually achieve activation through association with their target molecules.To investigate how the transmembrane segment mediates these changes, Luo, Springer, and Takagi systematically replaced amino acids in both the α and β transmembrane domains of the heterodimer with cysteines, creating the potential for binding interactions through a chemical reaction, disulfide bond formation, between the two subunits. By analyzing 120 possible cysteine pairs, the researchers not only confirmed the structure of the transmembrane region as helical but also mapped the proximal amino acid residues between the helices. To understand how the helical transmembrane domains transmit signals, the team introduced activating mutations in the amino acids of the α subunit cytoplasmic tail. Using this approach, they observed the loss of the contact between the subunits, indicating a separation of the transmembrane helices. Furthermore, when disulfide bond formation occurred, linking the transmembrane segments together, activation was suppressed. While previous models had proposed various modes of subunit movements, including hinge- and piston-like models, these results strongly support the notion that lateral separation of the subunits is the driving force behind the signal. As many diseases arise from defects in integrin adhesion, understanding the conformation and mechanism of integrin activation could suggest promising avenues for drug development aimed at correcting such defects.


In a series of 11 microsatellite stable (MSS) and 9 microsatellite unstable (MSI) colon cancer cell lines and primary colon carcinomas (25 MSS and 28 MSI) with known ploidy stem line and APC, KRAS, and TP53 mutation status, we analyzed the promoter methylation of the following genes: hMLH1, MGMT, p16INK4a (CDKN2A α-transcript), p14ARF (CDKN2A β-transcript), APC, and E-cadherin (CDH1). We compared the DNA methylation profiles of the cell lines with those of the primary tumors. Finally, we examined if the epigenetic changes were associated with known genetic markers and/or clinicopathological variables.Tumor cell lines are commonly used as experimental tools in cancer research, but their relevance for the hMLH1, MGMT, p16INK4a, p14ARF, APC, and E-cadherin, respectively, whereas 21%, 40%, 32%, 38%, 32%, and 40% of the primary tumors were methylated for the same genes. hMLH1 and p14ARF were significantly more often methylated in MSI than in MSS primary tumors, whereas the remaining four genes showed similar methylation frequencies in the two groups. Methylation of p14ARF, which indirectly inactivates TP53, was seen more frequently in tumors with normal TP53 than in mutated samples, but the difference was not statistically significant. Methylation of p14ARF and p16INK4a was often present in the same primary tumors, but association to diploidy, MSI, right-sided location and female gender was only significant for p14ARF. E-cadherin was methylated in 14/34 tumors with altered APC further stimulating WNT signaling.The cell lines and primary tumors generally showed similar overall distribution and frequencies of gene methylation. Among the cell lines, 15%, 50%, 75%, 65%, 20% and 15% showed promoter methylation for in vitro models, comparable with the in vivo situation, as the cell lines display many of the same molecular alterations as do the primary carcinomas. The combined pattern of epigenetic and genetic aberrations in the primary carcinomas reveals associations between them as well as to clinicopathological variables, and may aid in the future molecular assisted classification of clinically distinct stages.The present study shows that colon cancer cell lines are in general relevant  RB, p15INK4b, p16INK4a), the TP53 pathway (p14ARF), the WNT signaling pathway (APC, E-cadherin), DNA repair (MGMT, hMLH1, BRCA1), apoptosis (DAPK), and the metastasizing process (E-cadherin, TIMP3) (reviewed in [During the last decade, epigenetic changes have been reported in many cancers and they are now recognized to be at least as common as genetic changes . Aberraniewed in ,8).Development of colorectal cancer through various morphological stages has been linked to several genetic and epigenetic changes. The majority of carcinomas have several chromosomal aberrations, a phenotype often referred to as chromosomal instability. Approximately 15% of the tumors are near diploid but exhibit microsatellite instability (MSI), seen as genome-wide short nucleotide insertions and deletions . This phin vitro studies. Although many of the known genetic aberrations in colon cancer cell lines have been comprehensively described [Human cancer cell lines are important tools in cancer research. Their commercial availability and unrestrained growth make them well suited for escribed , severalThe frequencies of both methylation and gene mutation differ among various studies of cell lines and primary tumors. The genome characteristics, profiles of gene mutations, and methylation status are rarely reported in the same samples, let alone in large series. In the present report we address these potentially connected pathogenetic mechanisms by presenting methylation profiles of a set of genes in a series of MSI and microsatellite stable (MSS) colon cancer cell lines and primary colorectal carcinomas. The methylation profiles are compared with various known genetic and clinicopathological features of the same series.hMLH1, MGMT, p16INK4a, p14ARF, APC, and E-cadherin, respectively, whereas 0/11, 5/11, 8/11, 5/11, 2/11, and 1/11 of the MSS cell lines were hypermethylated for the same genes (Table The colon cancer cell line methylation-specific PCR (MSP) results are summarized in Table es Table , 2a. In Methylation status was assessable in more than 99% of the total number of analyses .hMLH1 promoter to 61% among the MSI tumors for the p14ARF gene  are shown in Table P = 0.231, Mann-Whitney test).Several of the primary tumor samples displayed widespread CpG island methylation Figure . Eighteep14ARF (P < 0.001) and hMLH1 (P = 0.015). Sixteen of 49 primary tumors harbored TP53 mutations, and all of the tumors with TP53 mutations also harbored unmethylated hMLH1 (P = 0.009). p14ARF hypermethylation was less common in tumors with mutated TP53 than in tumors with wild type TP53, although this was not statistically significant (P = 0.127). Four tumors displayed a G:C to A:T TP53 mutation and three of them simultaneously harbored a methylated MGMT gene. Four of 11 tumors with G:C to A:T KRAS (KRAS2) mutations were methylated at the MGMT promoter. Overall, the presence of KRAS mutations was not associated with the methylation status of the genes analyzed. Among the 20 tumors with p14ARF methylation, 10 were also methylated at the adjacent p16INK4a gene (P = 0.067). Finally, the APC promoter was methylated in 17/53 (32%) tumors, and 8/17 (47%) tumors displayed both APC mutation and methylation.The methylation status of the primary tumors was compared with genetic characteristics of the same tumors Table . In geneP = 0.080). We found no statistically significant associations between tumors with widespread methylation and presence of TP53, KRAS, or APC mutations.Among the tumors with widespread methylation (3 or more methylated genes), 13/18 (72%) tumors demonstrated MSI, whereas 5/24 (21%) were MSS (hMLH1 (P = 0.043) and p14ARF (P = 0.050). Tumors from patients younger than the mean age (68 years) had a lower methylation frequency for p16INK4a than did tumors from older patients, although this was not statistically significant (P = 0.074). There was a strong association between methylation and right-sided tumor location as 10/11 (91%) tumors methylated in hMLH1 and 12/19 (63%) of the tumors methylated in p14ARF were located in the right side of the colon (P < 0.001 and P = 0.005, respectively). There was no statistically significant association between methylation and histological grade. Most of the tumors with APC methylation (13/17, 76%) belonged to the Dukes' B group, but the differences were not statistically significant (P = 0.068).The clinicopathological features and methylation status of the primary tumors are summarized in Table P = 0.035). We saw no statistically significant associations between presence of widespread methylation and the remaining clinicopathological variables included in the present study.Tumors with widespread methylation (≥ 3 loci) are associated with right-sided localization; 10/17 (59%), versus 5/17 (29%) left-sided  have also shown similar frequencies of TP53, KRAS and APC mutations [in vitro system is a suitable experimental tool that closely reflect the in vivo situation.It has been debated for some time whether cell lines are more frequently methylated than primary tumors . Regardiutations  and ploiutations , which fhMLH1 primers we designed amplify a region of the promoter, in which methylation invariably correlates with the lack of hMLH1 expression [Previously reported variations in promoter hypermethylation frequencies of different tumor suppressor genes in colorectal cancer can be explained by various ratios of MSI versus MSS samples in the series analyzed, different methods for analyzing methylation, the inter-individual variation in scoring of methylated samples, incomplete bisulphite modification, tumor heterogeneity, and the fact that different parts of the gene promoter region in question have been analyzed. In the present study, we used primer sets known to only detect methylation in tumor cells, never in normal tissues from the same patients ,31,38-42pression ,43,44. Mpression ,43,44.hMLH1 and p14ARF genes, whereas the four additional genes analyzed revealed similar methylation frequencies in the MSS and MSI groups. Promoter methylation of the hMLH1 gene was, not surprisingly, found only in tumors and cell lines with MSI, not in the MSS samples. The MSS tumors and cell lines per definition contain functional hMLH1 protein, and transcriptional silencing of hMLH1 by hypermethylation is known to be the main cause of MSI in sporadic CRC [p14ARF methylation may have a specific role in MSI tumors, since it seems to be most often inactivated in tumors with wild type TP53 (see below). However, the relatively high methylation frequencies of the remaining analyzed genes, and also their overall similar frequency in MSI and MSS samples, imply that they are important in colorectal carcinogenesis independently of tumor site and MSI status.As expected, the MSI primary tumors showed more methylation overall than did the MSS group. However, this was only significant for the adic CRC ,28,45. Ap16INK4a, have been reported to be methylated consistently in most tumor types analyzed [p16INK4a methylation frequencies vary from 18% [p16INK4a and p14ARF are more commonly methylated in tumors with MSI than in MSS [p14ARF is higher than that for p16INK4a in MSI colorectal carcinomas.Inactivation of tumor suppressor genes by promoter hypermethylation has been recognized to be at least as common as gene disruption by mutation in tumorigenesis . Indeed,analyzed . In colofrom 18%  to 50 % from 18%  with mosfrom 18% ,46,49-51n in MSS ,11,51-536-guanine by an irreversible transfer to an internal cysteine residue [6-guanine has a tendency to base pair with thymine during replication, thereby introducing a G:C to A:T transition mutation in the DNA [MGMT gene has previously been reported to be associated with G:C to A:T mutations in the tumor suppressor gene TP53 [KRAS [TP53 but seemingly not for KRAS, although no certain conclusions can be drawn from the limited number of samples with G:C to A:T mutations.The DNA repair protein MGMT is able to remove promutagenic alkyl groups from O residue . Left un the DNA . Inactivene TP53  and the 53 [KRAS ,58. Our ARF protein interacts in vivo with the MDM2 protein, neutralizing MDM2's inhibition of TP53 [p14ARF in tumors with mutated TP53 than in tumors with wild type TP53 has been reported previously [TP53 mutation in colorectal carcinomas [p14ARF gene in MSI tumors with few TP53 mutations is in agreement with a recent study [The p14 of TP53 . Less hyeviously . Additiorcinomas -62. The APC gene is frequent in colorectal and other gastrointestinal carcinomas, usually by truncating mutations [APC methylation in the upper range of what has been seen in previous studies [APC are common in colorectal cancer [APC mutations in our study were also methylated. We have not looked at allele-specific mutation, but methylation and mutation in the same tumor might reflect one mutated allele and methylation of the other, in accordance with Knudson's two hit hypothesis. This has previously been demonstrated for APC in colorectal cancer samples by Esteller et. al [MYC and CCND1 (cyclin D1) further stimulating cell proliferation [APC caused by methylation (n = 17) and/or gene mutation (n = 26). The E-cadherin gene was also methylated in 14/34 tumors with altered APC, presumably further stimulating WNT signaling [APC methylation seemed to be more common in Dukes B stage tumors.Inactivation of the utations ,64. An a studies ,65,66. Sl cancer ,68 and, l cancer ,22,69, aferation . Among tignaling . IntereshMLH1 in sporadic carcinomas is associated with proximal tumor location in the large bowel [hMLH1 promoter were taken from the right side of the colon. An association between sporadic proximal colon carcinomas and methylation has also been reported for p16INK4a and p14ARF [p14ARF. However, p16INK4a demonstrated the same tendency. Both hMLH1 and p14ARF are strongly associated with MSI and MSI is in turn strongly associated with proximal tumor location [The present study confirms that methylation of ge bowel ,21,45,70location ,72, hencp14ARF does not exclude p16INK4a methylation, is in accordance with previous studies [p16INK4a or p14ARF methylation with female gender and increased age has been described in some studies [p14ARF and hMLH1, but not of p16INK4a. We also found a weak association between p16INK4a methylation and increasing age. This potential age-specific methylation was not confirmed for any of the other genes studied. The gender-associated methylation of hMLH1 has previously been described [When it comes to gene methylation and its association with other clinicopathological features, contradictory results have been reported. Our observation that methylation of  studies ,24. Corr studies  but not  studies ,21,24. Wescribed ,74 and met. al [Like Toyota et. al , we founin vivo situation. The methylation profile of specific genes, in particular hMLH1 and p14ARF, has strong associations with genetic and clinicopathological features and might be related to biologically distinct subsets of colorectal tumors.The data presented here demonstrate that multiple genes are methylated in colorectal carcinomas. This underlines the important role epigenetic inactivation of tumor suppressor genes plays during the process of tumor development. Epigenetic changes in colon cancer cell lines are overall comparable with those of primary carcinomas of the large bowel, which make the cell lines relevant models for the TP53, KRAS and APC [et. al [Fifty-three primary colorectal carcinomas from 52 patients, including 25 MSS tumors and 28 MSI tumors, were submitted to methylation analyses. One of the tumors was from a patient with hereditary non-polyposis colorectal cancer (HNPCC), whereas the rest of the cases were sporadic . The tum and APC ,64,77. T and APC ,78-80, 3 and APC , and tot [et. al . The pri [et. al . The DNAhMLH1, MGMT, p16INK4a, p14ARF, APC and E-cadherin by MSP, a method that distinguishes unmethylated from methylated alleles of a given gene [Promoter methylation was studied in ven gene . After bMGMT [p16INK4a [p14ARF [APC [E-cadherin fragments [hMLH1 fragments were designed in accordance with hMLH1 promoter methylation and gene expression studies [One or two μg DNA from each sample was modified as described . PreviouMGMT ,82, p16Ip16INK4a ,82, p14A [p14ARF , APC [39ARF [APC  and E-caragments  , 200 μM dNTP (Amersham Pharmacia Biotech Products Inc., Piscataway, NJ), and 0.625 U HotStarTaq DNA Polymerase (QIAGEN). PCR products were loaded onto 7.5% polyacrylamide gels, stained with ethidium bromide, and visualized by UV illumination. An independent second "methylated reaction" of the MSP was performed for all the samples included in the present study. In cases with diverging results from the two rounds of MSP, we did a third independent MSP round.All the PCRs were carried out in a total volume of 25 μl containing 1 × PCR Buffer (15mM MgClin vitro with SssI methyltransferase (New England Biolabs Inc., Beverly, MA) was used as a positive control for MSP of methylated alleles, whereas DNA from normal lymphocytes was used as a control for unmethylated alleles. Water was used as a negative PCR control in both reactions.Human placental DNA  treated 2 test. Two of the statistically significant cross-tables analyzed by the Pearson χ2 had cells with expected count less than 5, with a minimum count of 2.96 (Table P values are derived from two tailed statistical tests using the SPSS 11.5 software.All 2 × 2 contingency tables were analyzed using Fisher's exact test. Three × 2 tables were analyzed by the Pearson χ96 Table . The ManAPC mutation status in the cohort. GIM and TOR have collected the series of human primary tumors and provided all clinical and pathological information. RH provided all cell lines and information about them. ME contributed with scientific discussions of the results and participated in the writing of the manuscript. RAL conceived the study, was responsible for its design and coordination, and contributed in the evaluation of the results and in preparation of the manuscript. All authors have read and approved of the final manuscript.GEL cultured and isolated DNA from all cell lines and carried out the MSP analyses of these and of the patient samples. GEL interpreted the results, performed the statistics and drafted the manuscript. LT participated in the study design, scored the MSP results independently of author 1, and contributed to manuscript preparation. TL was responsible for the update of the Additional file 1 lists the MSP primers used in the present study.Click here for file


Totally implantable central venous access devices (intraports) are commonly used in cancer patients to administer chemotherapy or parenteral nutrition. Rupture of intraport is a rare complication.During 3 years period, a total of 245 intraports were placed in cancer patients for chemotherapy. Four of these cases (two colon cancer and one each of pancreas and breast cancer) had rupture of the intraport catheter, these forms the basis of present report.insitu for intraports was 164∀35 days. Median follow-up time was 290 days and total port time in situ was 40180 days. The incidence of port rupture was 1 per 10,000 port days.Mean time Three of the 4 cases were managed by successful removal of catheters. In two of these the catheter was removed under fluoroscopic control using femoral route, while in the third patient the catheter  was removed surgically. One of the catheters could not be removed and migrated to right ventricle on manipulations.Port catheter rupture is a rare but dreaded complication associated with subcutaneous port catheter device placement for chemotherapy. In case of such an event the patient should be managed by an experienced vascular surgeon and interventional radiologist, as in most cases the ruptured catheter can be retrieved by non operative interventional measures. Totally implantable central venous access devices (intraports) are commonly used in patients with cancer to administer chemotherapy, blood and blood products, antibiotics, parenteral nutrition and to obtain blood samples for laboratory analysis. The catheter is usually placed in the subclavian vein under local anesthesia. There are many complications associated with this technique like hemothorax, pneumothorax, pocket infection, infection of the tunnel or the port, bleeding, hematoma and thrombosis of the catheter or the vein. A very rare complication of the intraport catheter is rupture inside the subclavian vein .The literature regarding common complications is abound, however there is very little information on port ruptures. Some attempts has been made to correlate these with type of port used, site of placement, type of chemotherapy, duration of catheter use etc ,3. Howev® with titanium portal and detachable silicon rubber catheter  were used in all cases. All patients requiring removal or replacement of the device, prior to completion of the intended treatment were identified from the operative room and hospital records.Between June 1995 and May 1998, 245 patients underwent intraports insertions at the "Agii Anargiri" Anticancer Hospital of Kifissia, Athens, Greece, and were followed up for possible device-related morbidity. Baseline demographic information and the indication for the placement were obtained from the retrospective review of patients' medical and operative reports. Follow-up continued till the device was removed or the patient died. Follow-up time ranged from 1 month to over 3 years. Port-A-CathAll intraports were inserted by percutaneous access technique to the subclavian vein. The tip of the catheter was positioned in the superior vena cava or the right atrium. The ports were then placed within a subcutaneous pocket created on the anterior chest wall. The position was documented by immediate intraoperative chest roentgenogram. Four of these patients had port rupture of which three were complete and one partial, one of these ruptures was accidental.Routine removal of the subcutanous intraport was carried out in the operating room under local anesthesia with xylocaine 1% (10 cc). An incision was placed on the skin in the area over the drum of the device. Then the drum was prepared by cutting off the tissues that surround the port. The port and the catheter were caught with Kocher forceps. The tissues around the catheter were dissected and the catheter was slowly pulled out. The catheter was cut into three pieces and was sent for culture. Patients were discharged after two hours of observation.in situ was 40180 days, while mean (SD) port time in situ was 164 (35) days.Over a three year period between 1995 and 1998 a total of 245 port devices were fitted in cancer patients . Mean age of the patients was 58 ± 6.3 years. Total port time After a median follow-up of 290 days (range 30–690 days) four ports ruptured, thus bringing the number of fractures per 1000 port days to 0.01% or one rupture every 10,000 port days. Two patients had cancer of the colon and one each had cancer of breast and pancreas. Both patients with colonic cancer received 5 flurouracil and leukovorin, patient with breast cancer received epirubicin and paclitaxal and patient with pancreatic cancer received gemcitabine, paclitaxal and cisplatinum. No correlation was observed with type of chemotherapy or site of disease. Three of the port ruptures were full and one partial one of the ruptures was accidental (case 1). The detail of the cases has been provided in next section. Three of the patients survived for two years after the catheter removal, while the fourth patient with accidental port rupture and whose catheter was left behind in the right ventricle died of progressive disease two month later.enoxaparin sodium, 1 mg/kg/12 h for 5 days and 40 mg/day for further 14 days in order to prevent thromboembolic event. The patient died two months later due to progressive disease without obvious complications related to the retained catheter.A 65-years-old female with colonic cancer, received intraport for chemotherapy administration. After 6 months due to the poor response to chemotherapy it was decided to remove the catheter. During the manipulations for removal, the catheter, accidentally ruptured at the point of its entrance in to subclavian vein. The peripheral part of the catheter remained in the vein, while only the central part could be removed. Another attempt to uncover the subclavian vein till superior vena cava failed. Patient underwent thoracotomy for removal of remaining catheter two days later. Superior vena cava was opened and catheter removal was attempted, however, during this process the catheter slipped into right atrium and further attempts to retrieve it were abandoned. Patient was started on anticoagulant treatment using et al [A 68-year-old female with colonic cancer received intraport for administration of chemotherapy. After one and a half year of treatment it was decided to remove the catheter as the catheter has thrombosed due to non heparinization. At the time of its removal the catheter ruptured at the point of its entry to subclavian vein. The peripheral part of the catheter remained in the vein. An unsuccessful attempt was made to expose subclavian vein till superior vena cava. Later this catheter migrated to right ventricle. The catheter was removed using the technique of Yedlicka et al , throughA 74-years-old female suffering from breast cancer underwent intraport insertion for administration of chemotherapy. After fourteen months of treatment it was decided to remove the catheter as the catheter had thrombosed. On attempted removal the catheter was found to be ruptured at its entry to subclavian vein (Figure Next day, the broken part of the catheter was removed successfully under fluoroscopic control using the technique described above for case 2 (Figure n Figure . Next da2 Figure .In a 56-year-old female patient with pancreatic carcinoma underwent an intraport placement for chemotherapy. Eight months later, the patient complained of pain in the back during the administration of chemotherapy. A fluoroscopic examination showed partially broken catheter in the vein while the other part was lying in the subcutaneous tissue. The catheter was removed from the subclavian vein carefully to avoid complete rupture of the catheter. Similar to case 3 above, the biomedical examination showed a significant reduction in the elasticity of the catheter material.Since Aubaniac first described the percutaneous central venous catheterization in 1952, insertion of central venous access devices for fluid administration has increased rapidly. The total complication rate associated with central venous catheter devices ranges from 0.4 to 29% ,4,6.et al, in 1997 [Spontaneous rupture of the port catheters appears to be a very rare and dreaded event. Biffi  in 1997 .The biomechanical analysis, of ruptured catheters in our series showed a significant decrease in the elasticity of the material. No correlation between changes of mechanical properties of the material and specific chemotherapy administrated through the port has been established so far ,8. Even In case 1 the port catheter rupture was due to wrong manipulations during catheter removal and accidental cutting of peripheral part without first holding it in clamp. The further catheter movement was induced by negative intra-thoracic pressure during respiration . Other pet al [Ballarini et al , suggestet al ,10-12. IThere is no controversy that the foreign bodies inserted in the systematic circulation must be removed. This is best achieved under fluoroscopy with specific catheters and snare loops. Removal of intravascular material by means of minimally invasive techniques presents excellent results, while at the same time it minimizes morbidity and mortality ,14. If sIntravascular rupture of subcutaneous port catheters is a rare complication. Etiology still remains elusive, however wrong placement has been advocated as the most important cause. Other causes include catheter material faults and alterations of the material's mechanical properties, probably due to the administered substances; however, there is no data to support the effect of administered substances. Ruptured catheters are best removed by minimally invasive radiological techniques.The authors declare that they have no competing interests.DF participated in the operations, and drafted the manuscript.CT participated in the operations, patients follow-up search of literature and preparation of draft.GF conducted the follow-up and helped to draft the manuscript.AN and SR participated in the operations, design of the study and preparation of manuscript for publication.All authors read and approved the final manuscript.


As part of a long-term initiative to improve cancer surveillance in New York State, small area maps of relative risk, expressed as standardized incidence ratios (SIRs), were produced for the most common cancers. This includes prostate cancer, the focus of this paper, since it is the most common non-dermatologic malignancy diagnosed among men and the second leading cause of cancer deaths for men in the United States.ZIP codes were chosen as mapping units for several reasons, including the need to balance between protecting personal privacy and public demand for fine geographic resolution. Since the population size varies greatly among such small mapping units, hierarchical Bayes spatial modelling was applied in this paper to produce a map of smoothed SIRs. It is further demonstrated how other characteristics of the large sample from the stationary posterior distribution of SIRs can be mapped to investigate various aspects of the statewide spatial pattern of prostate cancer incidence.Thematic mapping of the median and 95 percentile range of SIRs provided, respectively, a map of spatially smoothed values and the uncertainty associated with these smoothed values. Maps were also produced to identify ZIP codes expressing a 95% probability, in the Bayesian paradigm, of being less than or greater than the null value of 1.The model behaved as expected since areas that were statistically elevated coincided with areas identified by the spatial scan statistic, plus the relative uncertainty increased as a ZIP code's population decreased, with an exaggerated effect for low population ZIP codes on the edge of the state border.The overall smoothed pattern, along with identified high and low areas, may reflect difference across the state with respect to socio-demographics and risk factors; however, this is confounded by potential differences in screening and diagnostic follow-up. Nevertheless, the Bayes modelling approach is shown to provide not only smoothed results, but also considerable other information from a large empirical distribution of outcomes associated with each mapping unit. Geographic surveillance of chronic disease is central to understanding spatial or spatial-temporal patterns that may help to identify discrepancies in disease burden among different regions or communities. As part of ongoing efforts in New York State to understand spatial patterns of cancer and to help implement cancer prevention and control programs, small area maps of cancer relative risk, expressed as standardized incidence ratios (SIRs), have been produced and shared with the public  for the Prostate cancer, the focus of this paper, was included because it is the most common non-dermatologic malignancy diagnosed among men and the second leading cause of cancer deaths for men in the United States (US) . AlthougResults for prostate cancer  are reproduced in Figure It is well recognized that the stability of population-based statistics like the SIRs in Figure y, as a random variable that has arisen from a probability distribution with expectation θ. This expectation is modeled, via an appropriate link g(·), as a linear function g(θ) = α + β x'+ ε, for a common value α, explanatory covariates β x'and a random effect ε that captures unexplained variation. If the random effect is associated with exchangeable spatial heterogeneity, estimates are smoothed towards a global mean, whereas if the random effect is associated with local spatial autocorrelation, estimates are smoothed towards a local neighborhood mean, which is typically more meaningful in geographic epidemiology. There are different approaches to modelling local spatial dependence, and section 6.3 of Cressie [The parametric approach of generalized linear modelling  treats t Cressie  presents Cressie .Estimation of model parameters can proceed by maximum likelihood ; howeverFully Bayesian modelling assigns hyperprior distributions to these hyperparameters, so that every parameter of the hierarchical model is allowed to vary over a prior distribution and no single point estimate is used to represent an unknown parameter value. Furthermore, the fully Bayesian approach allows the convolution model that incorporates both a heterogeneous and spatially structured random effect , thus alSince there are no closed form analytical solutions for parameter estimates of a fully Bayesian model, nor likelihood profiles to maximize, Markov Chain Monte Carlo (MCMC) methods are used to generate large samples from the posterior distributions of all stochastic nodes of the hierarchical model, given the likelihood describing the original data distribution and all appropriate prior and hyperprior distributions of the likelihood parameters . EstimatIn what follows, the fully Bayesian approach is applied to simulating large samples from the posterior distribution of prostate cancer relative risk in each of 1412 ZIP codes in New York State. Various aspects of these distributions are then mapped to reveal information on the geographic patterns of prostate cancer.th – 2.5th percentile) of the 1000 values sampled from the posterior distribution of SIRs for each ZIP code.The model defined by equations 1–5 was applied to simulate a sample of 1000 independent observations from the stationary posterior distribution of standardized incidence ratios for each ZIP code. Summary statistics and graphical analysis of these empirical distributions indicated that they arose from generally symmetric posterior distributions. Since the sample mean, median and mode were very similar for each ZIP code, the median was chosen to represent central tendency and is mapped in Figure The posterior distributions can also be used to identify ZIP codes where a specified mass of the distribution of relative risk is greater or less than the null value. For example, Figure The Poisson model applied in this paper is a particular application of hierarchical Bayes spatial generalized linear models for the exponential family of likelihoods . Particuwij = 1 if a mapping unit j shares a common border with unit i, and wij = 0 otherwise. This weighting scheme actually has its roots in image analysis, for which this type of modelling was developed [Most applications to date use the first order binary weighting scheme where eveloped , and it ninj/dij for neighboring mapping units separated by geographic distance dij and of population sizes ni and nj. These authors applied such a weight to prostate cancer mortality mapping; however, this gravity-type weighting may be better suited for infectious disease, not chronic disease.A proper approach may be to define weights as a decay function of geographic distance between population-weighted centroids of the mapping units. This function may be obtained by fitting a model correlogram to residuals that are obtained from a model that does not include a random effect to account for spatial autocorrelation. Cressie and Chan  used theIf a decay function is fit from the data, the varying stability of disease rates among the mapping units presents a challenge. Since the Bayesian model is designed to adjust for varying stability, perhaps a hierarchical model like the one applied in this paper can be extended so that the weights in Equation (4) are defined as a decay function whose unknown parameters can be assigned "hyperprior" distributions.Maps like in Figure Viewing Figures We also see that many edge ZIP codes in less populated areas tend to have greater uncertainty relative to their non-edge neighbors because there are fewer neighbors to borrow strength from. For the heterogeneous Poisson model applied in this paper, Lawson et al  suggest The smoothed pattern in Figure Although the patterns seen in Figures Some areas appear elevated that are in popular vacation spots, such as the eastern forks of Long Island and the "north country" of New York State including the Thousand Islands area along the Saint Lawrence River , the number of cases within each unit, Yi, conditional on location i, is defined as a Poisson random variable with expectation Eiθi, where Ei equals the age- and race-adjusted expected number of prostate cancer cases, and θi equals the area-specific relative risk. Given an observed response yi, note that the maximum likelihood estimate of relative risk is  = yi / Ei, the standardized incidence ratio (SIR).Letting the geographic domain (New York State) be subdivided into θi is assigned a log-normal prior distribution, log(θi) ~ N(μi, ), where the expectation and variance are defined by a linear function of a common value (intercept), α, and two independent random effects, a heterogeneous component, ui, that does not depend on geographic location (exchangeable) and an autocorrelated component, vi, that reflects local spatial structure by incorporating the influence of neighboring geographic units. Prior distributions are then assigned to these linear terms and consequent hyperprior distributions are assigned to the variance terms, thus creating a 4-level hierarchical model as follows.The relative risk parameter Yi ~ Poisson(Eiθi)     (1)Level 1, define the likelihood: θi) = α + ui + vi     (2)Level 2, link to a linear function: log(α ~ N(0,0.0001), noting that 0.0001 is the precision, thus defining a vague prior,Level 3, assign prior distributions: δi with respect to unit i and wij is a weight defining the relationship between geographic unit i and its neighbor j. The weight is defined simply as wij = 1 if ZIP codes i and j are adjacent (share a common border) and wij = 0 otherwise.for a neighborhood of geographic units Level 4, assign hyperprior distributions to precision terms:τu = 1 /  ~ Gamma(a,b) and τv = 1 /  ~ Gamma(c,d)     (5)for shape parameters a and c, and inverse scale parameters b and d.vi, is defined according to the conditional auto-regressive model (CAR) [vi is conditional on geographic location, whereby its expectation equals a local neighborhood average. The Bayesian model puts increasing emphasis on this term as the underlying population at location i decreases.This is the convolution model originally proposed by Besag, York and Mollie , where tel (CAR) . Note thθi = exp(α + ui + vi).Although covariates can be incorporated into the log-linear expression at the second level of the model, our interest is with estimating and mapping the relative risk, α in Equation (2) , the mosh et al . For theFinal smoothed results from each model are compared in Figure α, and heterogeneous random effect, ui, zero (0) was used to initiate one chain, plus/minus four standard deviations of the statewide log(SIR) were used to initiate the other respective chains. Zero is the statewide average log(SIR) that provides a point estimate for α, plus it is the expected value of ui. For the random effect associated with local spatial clustering, vi, the initial values were based on the average plus/minus four standard deviations of the log of first order neighborhood average SIRs. For the precision terms τu and τv, the inverse of the sample variances of the log(SIR) and the log of the first order neighborhood average SIRs were used respectively for one chain, plus lower and higher values were chosen for the other two chains in order to be well dispersed from the middle value, but not wildly so with respect to what is reasonably expected based on the observed spatial variability.WINBUGS 1.4  was usedConvergence of relative risk for the three independent chains was confirmed by graphing their traces and observing random mixing of all chains, which revealed white noise variation around a common value, with no trend. This was supported by observing Brooks-Gelman-Rubin diagnostics that clearly satisfied convergence criteria . After adeviance node in WINBUGS. The mean deviance was then calculated as D(y, μ) = 2 [l(y, y) - l(y, μ)], where l(y, y) is the mean maximum achievable log likelihood, obtained for a saturated model where a parameter is assigned to each datum, and l(y, μ) is the mean log likelihood obtained for the model in question. This takes the conventional assessment of deviance for generalized linear models [Variations of the model defined above were compared by evaluating the mean deviance of 1000 iterations chosen from the three independent Markov Chains after burn-in. This was done by obtaining the mean of -2(log likelihood) for each iteration, as provided by the r models  and applr models .Incorporating a random effect associated with local spatial structure (CAR term) provides much stronger prior information than the exchangeable random effect alone Table , which a


The insect exoskeleton or cuticle is a bi-partite composite of proteins and chitin that provides protective, skeletal and structural functions. Little information is available about the molecular structure of this important complex that exhibits a helicoidal architecture. Scores of sequences of cuticular proteins have been obtained from direct protein sequencing, from cDNAs, and from genomic analyses.Most of these cuticular protein sequences contain motifs found only in arthropod proteins.Drosophila melanogaster and the Anopheles gambiae genomes, that have been predicted to be cuticular proteins, based on a Pfam motif (PF00379) responsible for chitin binding in Arthropod cuticle. The total number of the database entries is 445: 370 derive from insects, 60 from Crustacea and 15 from Chelicerata. The database can be accessed from our web server at .cuticleDB is a relational database containing all structural proteins of Arthropod cuticle identified to date. Many come from direct sequencing of proteins isolated from cuticle and from sequences from cDNAs that share common features with these authentic cuticular proteins. It also includes proteins from the CuticleDB was primarily designed to contain correct and full annotation of cuticular protein data. The database will be of help to future genome annotators. Users will be able to test hypotheses for the existence of known and also of yet unknown motifs in cuticular proteins. An analysis of motifs may contribute to understanding how proteins contribute to the physical properties of cuticle as well as to the precise nature of their interaction with chitin. One particular family of cuticular proteins constitutes one of the largest multigene families known in insects . UnrelatThe database nomenclature is based either on the names given by those who deposited the sequences or on codes assigned by genome projects. Thus, we have retained the existing names/codes for the convenience of the users.cuticle, exoskeletal, carapace) to the Protein databases of Entrez and Uniprot (release 1.8) [Anopheles gambiae and Drosophila melanogaster, from Ensembl [The data collection has been basically done in two ways. First, by submitting appropriate keywords ase 1.8)  we colle Ensembl  and EBI, Ensembl . This mo Ensembl  and most  we coll Ensembl . A short Ensembl , and, as Ensembl , was wid Ensembl ,3and refIn order to ensure that our data collection is complete, we scanned all protein sequences of Uniprot (release 1.9) for PF00379. Again manual filtration was required. In addition to PF00379, other motifs have been described in cuticular proteins, some are found along with PF00379 while others define other families of cuticular proteins ,3. All rThe data for our database was obtained by parsing the fields Definition, Accession, GI from Version, Organism and Origin from the Entrez entries. From the Uniprot entries we used Primary accession number, Protein name, Origin of the protein, Cross-references and Sequence information. This retrieval was done with Perl scripts. Additional information, concerning temporal and local expression of the proteins or corresponding mRNAs, was drawn from literature.The data have been organised based on a relational model and is stored in a PostgreSQL database system. The user has supervisory access through our Apache web-server. The database is managed by an interferential software, written in Java, which tends to settle any web-server's query. Also, it implements a homemade computational tool that performs motif search as described below.The main page of cuticleDB includes the following interfaces: Introduction, Data Retrieval, User manual and Contact. On clicking the Data Retrieval icon, users are presented with the search interface of the database. The query can be done in two ways: either by searching in fields or by gathering a set of proteins Figure .The separate fields in which the user may search are Protein name, Taxonomy, references in other databases (the user may submit Entrez GenInfo Identifier, Entrez Accession Number, Uniprot AC, Flybase ID, Ensembl code, Interpro AC or Pfam AC as a query) and the protein sequence. The protein sequence can be searched against any pattern according to the user's imagination and, therefore, hypotheses for novel motifs can be tested. This is performed by a separate, homemade tool that has been integrated in cuticleDB and which gives the user the opportunity to detect new motifs in cuticular proteins. The integration of this tool is of importance especially in a database such as this, given the significance of motifs not only in cuticular proteins, but in structural proteins in general.Users can gather all protein entries from a single species (35 species are included in cuticleDB) or all protein entries whose protein sequence contains one of a series of motifs. However, this series of motifs has been pre-selected by the constructors of the database and cannot be modified by the user. The selection criterion was the frequency of appearance of these motifs in the literature. The most commonly found motifs were searched against all protein sequences of the database and have properly been assigned to each entry.A typical cuticleDB entry contains the following fields: Protein Name, References to other databases (Entrez Protein Database, Uniprot, Interpro, Pfam, Flybase, Ensembl), Taxonomy, Expression Details, Protein Sequence and its Length, Database-Source of the sequence and the method by which the sequence was obtained Figure . The fieD. melanogaster, A. gambiae). The only verified cuticular proteins are those where the complete protein sequence or a unique N-terminal region was determined from a protein extracted from a cleaned cuticle or where a specific antibody reacted with proteins in cuticle or extracted from it. Finding mRNA in the epidermis is presumptive evidence that a protein is cuticular. The majority of cuticular proteins in this database were designated as cuticular proteins based on their sequence similarity to authentic cuticular proteins. Such proteins where sequence is the sole criterion for assignment are marked as "putative" in the database. Furthermore, at present, the annotation of the proteins of A. gambiae is preliminary. Many proteins are missing signal peptides, other clearly have been incorrectly assembled. Such sequences are marked as preliminary as well as putative. This database will be continuously updated at regular intervals to accommodate annotation.Taxonomic data are taken from Entrez. The total number of entries in cuticleDB is 445. These proteins are distributed in the three large taxa: Insecta (370 entries), Crustacea (60 entries) and Chelicerata (15 entries). The database includes entries from 6 orders of the class Insecta: Diptera (258 entries), Lepidoptera (39 entries), Orthoptera (37 entries), Hemiptera (6 entries), Coleoptera (22 entries), Dictyoptera (8 entries). The large number of proteins in Diptera is due to the inclusion of cuticular proteins from the two genomes . Both oSubsequently, we used these Profile Hidden Markov Models as a prediction tool for classifying the cuticular proteins into two groups RR1, RR2. The prediction was in agreement with the literature as far as the known RR1 and RR2 proteins are concerned. The total number of RR1 and RR2 proteins in cuticleDB are 132 and 148, respectively. The start and end positions of the two motif-types are shown in the corresponding entry of each protein. A smaller class, RR-3, with 75 conserved residues was also identified by Andersen [We have also studied the appearance of another motif: AAP(A/V). This small, hydrophobic tetrapeptide has been found to occur mainly in proteins of hard cuticles ,3, whereThe most severe problem of genome projects to date is that of correct annotation. So, accurate and specialized databases as cuticleDB with its description of highly conserved motifs will be of help to genome annotators. Therefore, cuticleDB can be used as a basis for annotating new cuticular proteins by similarity in future Arthropod genome projects.cuticleDB can also be utilized in molecular research as well, due to its focus on motif appearance. Cuticular proteins, as is the case with all structural proteins are marked by the presence of characteristic motifs. Some motifs are repeated within a protein sequence, whereas others appear only once. cuticleDB has been designed in such a way that the user can have a complete view of motif occurrence in the sequence of each protein entry. First, each entry shows the exact position of the most common cuticle motifs in the protein sequence. Secondly, the user is given the opportunity to search the sequence for novel motifs and therefore, test hypotheses for the existence of new patterns. Subsequently, hypotheses for possible interactions between cuticle macromolecules (either proteins with chitin or proteins with proteins) can be tested. Moreover, our results of the RR1 and RR2 predictions can be used as a guide for identifying a certain protein as coming from either soft or from hard regions of the cuticle. Most importantly, the information about the RR1 and RR2 distinction can be used for studies of cuticle's mechanical properties. As RR1 and RR2 proteins appear in soft and hard cuticles respectively, which means that the former interact with chitin more loosely than the latter, one can gain an insight in cuticle's molecular construction combining our data on the sequences of RR1 and RR2 proteins with some experimental work. Moreover, one could use the Expression Details, namely where and when each protein is expressed, when studying the differential construction of the cuticle among different developmental stages or among different regions of a single cuticle.The goal of cuticleDB constructors was the collection of all cuticular protein sequences that have appeared to date and their detailed and correct annotation. The better the organisation of the data, the easier the work will be for researchers dealing with cuticle and structural proteins in general. cuticleDB will help them to answer questions like : 'What kind of proteins appear in hard cuticles?' 'Why do RR2 proteins interact with chitin more tightly than RR1 proteins?' 'Which motifs contribute to protein-protein interaction in the cuticle?' 'From which stage can a certain protein be extracted?' Furthermore, it is hoped that, detection of common properties of these proteins, as well as recognition of important differences that are responsible for cuticle's complexity and important functions will be facilitated by its existence.Last but not least, it is hoped that this database will be of help to genome annotators in the near future as more arthropod genomes become available.. An e.mail biodb@biol.uoa.gr may also be used for comments, corrections and further data (sequence) submission.cuticleDB was created and is maintained in the Department of Cell Biology and Biophysics, Faculty of Biology of the National and Kapodistrian University of Athens. It is freely available at the URL: RR1: The extended Rebers and Riddiford Consensus, type IRR2: The extended Rebers and Riddiford Consensus, type IIHMM: Hidden Markov ModelCKM performed the data collection, and test procedures, and also participated in the design and the implementation of the databaseICS carried out the design of the algorithms and the database,. implemented all the algorithms, and also created the web interfaceVAI supervised the data collection and the testsJHW compiled the first draft of known cuticular proteins, provided a critique of the data base during its constructionSJH coordinated and supervised the whole project, suggesting the general directions and innovative features of the databaseAll authors have read and accepted the final manuscript.


Coronary artery bypass grafting surgery is an effective treatment modality for patients with severe coronary artery disease. The conduits used during the surgery include both the arterial and venous conduits. Long- term graft patency rate for the internal mammary arterial graft is superior, but the same is not true for the saphenous vein grafts. At 10 years, more than 50% of the vein grafts would have occluded and many of them are diseased. Why do the saphenous vein grafts fail the test of time? Many causes have been proposed for saphenous graft failure. Some are non-modifiable and the rest are modifiable. Non-modifiable causes include different histological structure of the vein compared to artery, size disparity between coronary artery and saphenous vein. However, researches are more interested in the modifiable causes, such as graft flow dynamics and wall shear stress distribution at the anastomotic sites. Formation of intimal hyperplasia at the anastomotic junction has been implicated as the root cause of long- term graft failure.Many researchers have analyzed the complex flow patterns in the distal sapheno-coronary anastomotic region, using various simulated model in an attempt to explain the site of preferential intimal hyperplasia based on the flow disturbances and differential wall stress distribution. In this paper, the geometrical bypass models (aorto-left coronary bypass graft model and aorto-right coronary bypass graft model) are based on real-life situations. In our models, the dimensions of the aorta, saphenous vein and the coronary artery simulate the actual dimensions at surgery. Both the proximal and distal anastomoses are considered at the same time, and we also take into the consideration the cross-sectional shape change of the venous conduit from circular to elliptical. Contrary to previous works, we have carried out computational fluid dynamics (CFD) study in the entire aorta-graft-perfused artery domain. The results reported here focus on (i) the complex flow patterns both at the proximal and distal anastomotic sites, and (ii) the wall shear stress distribution, which is an important factor that contributes to graft patency.The three-dimensional coronary bypass models of the aorto-right coronary bypass and the aorto-left coronary bypass systems are constructed using computational fluid-dynamics software (Fluent 6.0.1). To have a better understanding of the flow dynamics at specific time instants of the cardiac cycle, quasi-steady flow simulations are performed, using a finite-volume approach. The data input to the models are the physiological measurements of flow-rates at (i) the aortic entrance, (ii) the ascending aorta, (iii) the left coronary artery, and (iv) the right coronary artery.The flow field and the wall shear stress are calculated throughout the cycle, but reported in this paper at two different instants of the cardiac cycle, one at the onset of ejection and the other during mid-diastole for both the right and left aorto-coronary bypass graft models. Plots of velocity-vector and the wall shear stress distributions are displayed in the aorto-graft-coronary arterial flow-field domain. We have shown (i) how the blocked coronary artery is being perfused in systole and diastole, (ii) the flow patterns at the two anastomotic junctions, proximal and distal anastomotic sites, and (iii) the shear stress distributions and their associations with arterial disease.The computed results have revealed that (i) maximum perfusion of the occluded artery occurs during mid-diastole, and (ii) the maximum wall shear-stress variation is observed around the distal anastomotic region. These results can enable the clinicians to have a better understanding of vein graft disease, and hopefully we can offer a solution to alleviate or delay the occurrence of vein graft disease. The complex anatomy of coronary vessels has made the investigation of coronary flow and hemodynamics one of the most difficult and challenging studies until today. Leonardo Da Vinci , developed insightful sketches (some 550 years ago) of the anatomy of the heart and its blood vessels, as shown in Figure The major cause of death in both the developed and developing countries is cardiovascular disease. The study of coronary arterial circulation is important, because it is crucial in maintaining heart perfusion and function. The process of arteriosclerosis involves the formation of atherosclerotic plaques in the coronary tree. With time, this results in stenosis of the blood vessel and in turn decreases coronary flow. When it cannot match the oxygen demand of the myocardium, it results in ischemia and thereafter in an infarct. In order to overcome this problem of diminished perfusion of the affected myocardium, CABG is one of the treatment options that are being used to improve coronary perfusion. In this procedure, new routes around narrowed and blocked arteries are constructed with both arterial and venous conduits, allowing sufficient blood flow to be delivered to the ischemic heart muscles.In spite of CABG being an effective surgical technique to revascularize the myocardium, 20–50% of bypass grafts fail due to the formation of intimal hyperplasia -3. PreviFlow regimes in end-to-side anastomosis, provide pertinent information concerning wall shear stress gradient which affects intimal hyperplasia thickening. The three- dimensional experimental studies of flow in an end-to-side anastomosis by Ojha et al  have revAlthough wall shear stress distribution is a major factor in the onset of coronary diseases it cannot be measured directly and is hence calculated from velocity profiles. Even though these velocity profiles can be obtained in vivo using magnetic resonance (MR) or color-flow Doppler ultrasound (CDU), there are some limitations to measurements due to the small dimensions of the arteries. Thus the alternative to measure the wall shear stress is to use CFD to simulate flow in CABG geometry and thereafter compute the wall shear stress.Unfortunately, most of the modeling work in graft-flow is limited to only a part of the total bypass conduit geometry, namely the anastomosis site. As regards to the anastomosis geometry, Song MH et al  developeEven three-dimensional CFD simulations have been performed on end-to-side anstomosis of a stenosed coronary bypass, by Bertolotti and Deplano . Herein,An important factor is the effect of proximal artery flow condition on the hemodynamics at the distal end-to-side anastomosis. This effect has been analysed by Kute and Vorp , using CThe combined use of imaging techniques and CFD may be necessary for meaningful clinical studies. With the availability of imaging techniques like coronary angiography, magnetic resonance angiogram (MRA), computed tomography (CT), several researchers have analysed the flow in realistic bypass graft models. There have also been some studies combining experimental measurements and CFD analyses -17.Doppler analysis is generally preferred since it is safe, accurate and rapid when compared to angiography. Even though angiography certainly gives a definite answer about graft patency, there is more risk involved and it is time consuming and requires the assistance of a cardiologist. Doppler methods have been used by Bertolotti et al  and Lin It is clear from literature review that an important step towards realistic flow simulations concerns the generation of appropriate coronary vasculature geometry (involving the entire flow domain from the aorta to the perfused artery) with physiological boundary conditions. Unfortunately, a lot of work in these areas has been carried out on subsections of this bypass flow domain ,15,16,18The bypass models simulating the flow field of the anastomosis in the right and left aorto-saphenous bypass grafts are illustrated in Figures In Figures μ) of 0.00408 Pa and a density (ρ) of 1050 kg/m3. The blood vessel walls are assumed to be rigid and impermeable. For a quasi-steady, three-dimensional and laminar flow, the Navier-Stokes equations (for mass and momentum conservation) governing fluid motion is written as follows:The blood is assumed to be incompressible, with a Newtonian behaviour having dynamic viscosity , the flow field is automatically updated during each time-interval, by adopting the time-varying input data of the aorta  at the inlet to the aorta, from the left ventricle (LV). The velocity magnitude is computed from the physiologically representative stroke volume over the ejection period, based on the flow wave form  shown inDuring systole, the inputs to the model consist of (i) monitored time-varying flow-rate waveform at the inlet of the aorta,  the calculated uniform velocity at the ascending aorta, and (ii) the flow conditions at the left and right coronaries obtained from the monitored time-varying input flow-rate waveforms Figures  &3d.The fluid dynamics simulations are performed by using a control-volume-based technique, implemented in the computational fluid dynamics (CFD) code Fluent . The comThe geometry of the aorto-coronary bypass graft models is constructed in Gambit, using the dimensions provided by our cardiac-surgeon joint-author (TYS). The elements employed to mesh the computational domain consisted primarily of regular structured hexahedral elements as well as wedge elements wherever necessary. In order to carry out the mesh sensitivity analysis, numerical simulations were carried out by varying the number of mesh elements in the computational domain. Initially, the domain was discretized into 120974 hex/wedge elements. The accuracy of the simulation results was then improved by employing a finer mesh that contained 419765 elements. This number was further increased to 623138, but resulted in no significant improvement in the results. Thus to maintain a balance between the computational cost and the numerical accuracy, we concluded (based on the mesh independence test) that the appropriate number of elements for our study is 419765.In the solution algorithm used by Fluent, the governing equations are solved sequentially. Because the governing equations are non-linear (and coupled), several iterations of the solution loop need to be performed before a converging solution is obtained. Using this approach, the resultant algebraic equations for the dependent variables in each control volume are solved sequentially by a point implicit (Gauss Seidel) linear equation solver, in conjunction with an algebraic multi-grid (AMG) method. The governing equations are solved iteratively until convergence of all flow variables is achieved. The solutions of all the flow variables are deemed to have converged once their residuals computed from two successive iterations are below the set desired convergence criteria of 10-3. The study was also carried out by setting the convergence criteria as 10-4; this did not alter the results obtained earlier.For these numerical simulations we have incorporated the unsteady flow character by dividing the cardiac cycle into a number of small time interval, and analyzing for steady flow within these time intervals. To observe the velocity distribution features of the entire flow field, the computed velocity vectors are illustrated in the plane of symmetry at two different instants of the cardiac cycle.At the onset of ejection, at t = 0.13 sec, Figure The computed wall shear stress  is depicted in Figure At the mid-diastolic instant (at t = 0.7 sec), when the aortic valve is closed, Figure In the anastomosis domain, a strong region of recirculation is observed near the occluded end of the artery, which forces the flow to move into the perfused right coronary  artery as indicated in Figure The flow pattern variations give us insight into the wall-shear distribution. The high velocity gradients in the anastomosis give rise to large spatial variations in the resulting wall shear stress. The largest value of wall shear stress is seen in Figure The distribution of velocity-vectors in the flow field at the onset of ejection, t = 0.13 sec is shown in Figure During early ejection, as the left coronary vessel is embedded in the myocardium, the myocardium is subject to intramyocardial pressure which causes some reverse flow. The effect of LV contraction on the graft flow is incorporated by adopting the measured flow profile in the left coronary vessel at a point prior to its anastomosis with graft. In this way, we simulate the reversal of flow in the graft at the onset of ejection.A parabolic velocity profile of the back-flow in the graft is seen in Figure The velocity vectors plots at mid-diastole are displayed in Figure In the anastomosis domain, a strong recirculation region is seen in the heel region of the anastomosis at the distal end of the bypassed vessel, Figure In the anastomosis heel region  there is negligible shear stress. However, elevated shear stress (of magnitude 59.43 Pa) is seen in the anastomotic toe region  both the proximal and distal anastomotic regions are included, while other works ,15,16,18Our results for the aorto-right coronary bypass model and the aorto-left coronary bypass model at two different instants of the cardiac cycle, clearly reveal the following features: (i) at the onset of ejection, in the aorto-right coronary bypass model, very little flow enters the graft with a velocity magnitude around 0.0495 m/s; the maximum flow velocity inside the graft is around 0.2 m/s; (ii) at the mid-diastolic instant, the flow profile in the graft is skewed towards the outer wall, with the peak velocity increasing as it travels downstream; close to the graft exit, the maximum flow velocity attained is around 1 m/s; (iii) at the onset of ejection, in the aorto-left coronary bypass model, there is a backflow from the left coronary artery into the graft; the peak velocity of flow at the entrance to the graft is 0.0788 m/s, and very little perfusion is given to the host artery; (iv) at the mid-diastolic instant, the graft perfusion is maximum, with peak velocity magnitude of 1.1 m/s.Our study confirms that blood flow through the coronary artery bypass graft primarily occurs only during the diastolic phase of the cardiac cycle. This is in agreement with the physiological observation of coronary blood flow. There is however some difference between the flow patterns in the right and left coronary graft at the onset of ejection, with some backflow from the left coronary artery into the bypass graft which is not obtained in the case of the right coronary arterial bypass. The phenomenon  can be explained by the predominant intra-cardiac course of the left coronary artery system. This aspect is also an original feature of our work.Lastly, our study has also shown (i) a low wall shear-stress region near the heel region of the anastomosis domain, and (ii) a high wall shear-stress in the toe region of the anastomosis domain, making it prone to intimal hyperplasia. This may have some clinical significance. We should take this into consideration in designing a coronary anastomotic device, so as to minimize biomechanical injuries to the coronary arterial wall. In doing so, we can alleviate or retard the development of intimal hyperplasia, which is the Achilles heel of the effectiveness of coronary artery surgery with saphenous vein.The computed results have revealed that (i) maximum perfusion of the occluded artery occurs during mid-diastole, and (ii) the maximum wall shear stress variation was observed around the toe of the anastomotic region. According to our cardiac surgeon joint author (TYS), this preliminary result can enable the clinicians to have a better understanding of vein graft disease, and hopefully we can offer a solution to alleviate or delay the occurrence of vein graft disease.MS carried out the computational fluid dynamic studies and drafted the manuscript. DNG and LPC guided the study, helped in interpretation of results and critically reviewed the manuscript. YST provided the surgical aspect of the study, and the dimensions for the model. All authors have read and approved the final manuscript.


In contrast, glioma patients treated with HSV-TK/ GCV did not show significant treatment benefit, most likely due to insufficient transgene delivery to tumor cells. Therefore, this study aimed at developing a strategy for real-time noninvasive HSV-TK gene was fused to the firefly luciferase (Luc) gene and the fusion construct HSV-TK-Luc was expressed in U87MG human malignant glioma cells. Nude mice with subcutaneous gliomas stably expressing HSV-TK-Luc were subjected to GCV treatment and tumor response to therapy was monitored in vivo by serial bioluminescence imaging. Bioluminescent signals over time were compared with tumor volumes determined by caliper.The in vivo.Transient and stable expression of the HSV-TK-Luc fusion protein in U87MG glioma cells demonstrated close correlation of both enzyme activities. Serial optical imaging of tumor bearing mice detected in all cases GCV induced death of tumor cells expressing the fusion protein and proved that bioluminescence can be reliably used for repetitive and noninvasive quantification of HSV-TK/ GCV mediated cell kill in vivo evaluation of gene therapy strategies for treatment of malignant disease.This approach may represent a valuable tool for the  Presently, the standard method for assessing delivery of therapeutic transgenes to tumors relies on ex vivo analysis of explanted tumor tissue [Treatment with the suicide gene/ prodrug activating system herpes simplex virus type I thymidine kinase/ ganciclovir (HSV-TK/ GCV) is highly efficient in animal models of malignant glioma -3. In cor tissue ,9,10. Tir tissue -13.Luc) from the North American firefly Photinus pyralis as a reporter has several advantages compared to other imaging methods: (1) the technique is very sensitive (possibly 10-15 – 10-17 mole of luciferase/L are detectable in vivo, [Bioluminescence imaging (BLI) using luciferase (in vivo, ) and detin vivo, ,15, (2) in vivo, , backgroin vivo monitoring of the activity of a therapeutic transgene by fusing the bioluminescent reporter gene Luc to the bioactivating "suicide" gene HSV-TK. We investigated whether this fusion construct could be used to monitor HSV-TK mediated cytotoxicity in malignant glioma by serial optical imaging in vivo. Noninvasive real time evaluation of localization, activity and persistence of a therapeutic gene in living animals may represent an important step towards optimization of gene therapy protocols.This study aimed at generating a sensitive tool for noninvasive HSV-TK cDNA from the retroviral vector G1Tk1SvNa ([Luc) gene from the pGL3 vector (Promega) were ligated into pCDNA 3.1(-) (Invitrogen). For the fusion construct, EGFP in the pEGFPLuc vector (BD Biosciences) was exchanged for HSV-TK cDNA, which had been amplified from G1Tk1SvNa by PCR. The resulting HSV-TK-Luc fusion gene contained a humanized form of the firefly Luc gene to ensure high expression in mammalian cells [HSV-TK cDNA was inserted in frame upstream of the Luc cDNA, and both genes were separated by a linker sequence of 33 nucleotides. All transgenes were expressed under the control of the CMV promoter. The correct sequence of the fusion construct HSV-TK-Luc was confirmed by DNA sequencing.The Tk1SvNa (, kind gian cells . The ful5 cells/ well 16 to 24 h prior to transfection. Cells were transfected under serum-free conditions with the indicated amounts of DNA and Lipofectamine (Invitrogen) according to the manufacturer's protocol. For selection of stable clones transfected cells were replated at low density 48 h after transfection and incubated with 1 mg/ml  geneticin  for 4 weeks. Colonies were picked and analyzed for transgene expression.The human glioblastoma cell lines U87MG, T98G, LN18, U343, LN-Z308 and human embryonic kidney 293 cells were cultured under standard conditions. Cells were seeded in 6-well plates at a density of 3 – 5 × 103 cells/ well in a 96-well plate. GCV was added at final concentrations of 0 – 10 μg/ml and cells were incubated at 37°C/ 5% CO2 for 4 days. MTT (Sigma, Deisenhofen, Germany) was added at a final concentration of 0.5 mg/ml for 2 h. Absorbance was measured in a microplate reader (Victor2, Perkin Elmer Life Sciences, Turku, Finland) at 590 nm (reference 660 nm). Experiments were performed in quadruplicates and repeated at least twice. Results are reported along with the standard deviation (SD).Transiently or stably transfected U87MG cells were seeded at 4 × 10Transiently transfected U87MG cells were lysed in CCLR lysis buffer (Promega) 2 days after transfection. Stably transfected cells were lysed in the same buffer when they had reached ~90% confluence. Protein content of all cell lysates was determined by the Bradford Protein assay (Bio-Rad, Munich, Germany). Equal amounts of protein were analyzed luminometrically for luciferase activity with a microplate reader (Victor2) using the Luciferase Assay System reagent (Promega). All experiments were repeated at least twice and mean values are reported along with the SD.For bioluminescence imaging of intact cells HSV-TK-Luc expressing U87 glioma cells were transferred to a black microtiter plate in order to minimize light scattering, and MTT assay was performed in quadruplicates as described above. On day 4 after addition of GCV, D-Luciferin was added to a final concentration of 500 μM to the culture medium. Cells were placed in a dark box and light emission was imaged using a cooled CCD camera (Visiluxx Imager, Visitron). Light emitted from a region of interest (ROI) drawn over each well was quantified and mean values from quadruplicate measurements were compared with MTT results.Immunohistochemistry on paraffin sections using a rabbit polyclonal anti-Luc antibody (CR2029RAP, Europa Bioproducts) was performed essentially as described by Lee et al . HE stai6 human U87MG glioma cells stably expressing the HSV-TK-Luc fusion protein. When xenografts had reached a size of ~5 mm in diameter, in general on days 7 to 9 post tumor implantation GCV therapy was initiated. Mice were injected twice daily i.p. with 30 mg/ kg GCV for 14 days. Control mice with xenografts (n = 3) received saline injections. Tumor size was measured every 2 to 4 days by caliper. Tumor volume was calculated according to the formula 0.52 × width2 × length.All animal protocols were approved by the Animal Care and Use Committee at Martin-Luther-University Halle-Wittenberg. Six week old male NMRI nu/nu mice (Charles River) were injected s.c. at four sites, each with 2 × 10For BLI animals were anesthetized with ketamine/xylazine and injected i.p. with 150 mg/ kg D-Luciferin. Approximately 8 minutes after D-Luciferin injection mice were placed in a dark box and a grayscale image was acquired at low light (exposure time 2 seconds). Bioluminescence was measured in the dark by a CCD camera cooled to -120°C (VisiLuxx Imager), using an acquisition time of 15 min and binning 6. Bioluminescent signals were displayed in pseudocolors and superimposed on the grayscale image using Metamorph software (Visitron). Mice receiving GCV were imaged at least on days 7, 15, 22, 29, and 56 post tumor implantation (corresponding to start and day 8 of GCV therapy, as well as days 1, 8, and 35 after end of GCV therapy), while untreated control animals were subjected to BLI on days 7, 22, 29 and 35. In each animal a region of interest (ROI) was drawn over a single tumor or over all tumors as indicated in the text. Integrated as well as maximum light units (= counts) within this area were calculated after background subtraction. Final values are reported as the mean of the integrated or maximum counts obtained from all mice within one group. The CCD camera in use has a quantum efficiency approaching 90% at wavelengths between 550 and 770 nm, indicating that one photon is converted to ~0.9 electrons. One photoelectron corresponds to 4.52 counts. For serial quantification of light emission the conditions for image acquisition (e.g. exposure time, time between D-Luciferin application and image acquisition, stage position) were kept constant.Statistical analysis was performed using the ANOVA and Student's t test . A p value of <0.05 was considered significant.HSV-TK cDNA was fused in frame with the Luc cDNA in 2 ways: one fusion protein contained HSV-TK N-terminally, in the other construct Luc preceded the HSV-TK moiety. Both constructs were expressed under control of the CMV promoter. Several human glioma cell lines as well as 293 cells were transiently transfected with these constructs. In general, Luc activity was found to be up to 50-fold higher in cells expressing the HSV-TK-Luc construct compared to cells expressing the Luc-HSV-TK construct (data not shown). Therefore, all further studies were performed with the HSV-TK-Luc fusion construct.To achieve a strictly equimolar coexpression of a therapeutic and a reporter gene, the HSV-TK, Luc, or HSV-TK-Luc transgenes, respectively. As all 3 vectors were of equal size, equal amounts of DNA corresponded to equimolar amounts of plasmid. Cytotoxic activity as measured by MTT assay was compared to luminometrically determined light production and found to be tightly correlated in HSV-TK-Luc transfected cells . Photon emission above background levels was not detectable in cells that had been transfected with HSV-TK only, while no cytotoxic activity was conferred to cells expressing only Luc.In order to characterize this fusion construct more thoroughly, both transient and stable transfection experiments were performed using the human U87MG glioma cell line Figures  and 2. FHSV-TK or Luc alone. The overall cytotoxic activity of the fusion construct proved to be 60% of that measured in cells transfected with HSV-TK alone. Representative curves for 2 μg and 0.1 μg of transfected DNA are shown in Figure HSV-TK-Luc was 22% of that seen in cells transfected with Luc only  of y Figure . A tight2 = 0.79; p < 0.001, data not shown).Having demonstrated in transient transfection experiments that Luc could be employed as a reporter for monitoring the therapeutic effect of HSV-TK, U87MG cell clones stably expressing the HSV-TK-Luc fusion protein were generated by selection of transfected cells with geneticin. Comparison of 18 of these clones for Luc and cytotoxic activity revealed a good correlation between both enzymatic activities . Photon emission from as few as 500 intact cells expressing the fusion construct was detectable by the CCD camera while the lower detection limit for the Luc expressing U87MG cell clone was 125 cells.Enzymatic activity in the U87MG clone with both the highest Luc and HSV-TK activity was compared with U87MG clones expressing unfused HSV-TK or Luc. Cells stably expressing HSV-TK did not luminesce upon addition of D-Luciferin while Luc expressing cell clones were resistant to GCV mediated cell killing (data not shown). The dual function fusion protein compared favorably to the respective clones with the highest HSV-TK or Luc activity. Light production in the HSV-TK-Luc expressing cell clone was ~41% of that seen in Luc expressing U87MG cells while cytotoxic activity of the HSV-TK-Luc labeled U87MG clone was ~84% of that seen with the most active HSV-TK expressing U87MG clone (data not shown). Photon emission determined luminometrically was found to be linearly correlated with cell number over a range of at least 5 orders of magnitude . These data demonstrate that both enzyme activities were also preserved in U87MG cells stably expressing the HSV-TK-Luc fusion construct.We further examined whether the cytotoxic activity of the HSV-TK moiety could be visualized by monitoring light emission from 3, 2 × 104 and 2 × 105 cells were injected s.c. on the back of the animals. Although not palpable, 2 × 104 cells expressing the fusion construct were detected by the CCD camera immediately after injection (= day 0), either when injected alone or mixed with 1.8 × 105 (90%) non-luminescent parental U87MG cells prior to injection, while 2 × 103 cells injected s.c. were not seen (data not shown). This high level of detectability by BLI proves the usefulness of the HSV-TK-Luc construct as a highly sensitive reporter in vivo.The above high expresser U87MG cell clone was used for xenograft experiments in nude mice. For sensitivity testing, 2 × 106 HSV-TK-Luc labeled U87MG cells at four different sites on the back and the flanks, respectively  measured on day 7 were 122961 ± 22155. Serial measurements (during and after GCV therapy) of tumor volumes and integrated light intensity units within a region of interest (ROI) including all tumors were plotted against each other , thus confirming our cell culture data . In this mouse a significant correlation between light emission and tumor volume could be demonstrated, when tumor volumes were plotted against the maximum light emission within a ROI  instead of integrated light units. In general, integrated light units within a ROI correlated closely with maximum light emission from this ROI: correlation coefficients (R2) for all tumors in treated mice varied between 0.97 and 0.99, all p values were <0.003.Regarding therapeutic efficacy in these mice on an individual basis, photon emission and tumor volumes showed a significant correlation, with RFive weeks after end of GCV therapy (day 56) light emission was no longer detectable in 5 of the 7 GCV-treated mice, while in 4 of them small residuums at the tumor site were still visible. Two mice still showed very weak light emission from one of their flank tumors which also disappeared in subsequent imaging studies. All GCV treated mice survived and tumor recurrence was not observed until closure of the study at day 90 post tumor implantation.2 = 0.98; p = 0.010; Figure The 3 untreated control mice were imaged on days 7, 22, 29, and 35 post tumor implantation and had to be sacrificed on weeks 5 to 6 post cell injection due to massive tumor growth. Although 2 tumors with relatively strong light emission on day 7 post tumor implantation regressed within 4 weeks in one of the control mice, overall tumor growth in all control animals plotted against light emission from these tumors still showed a tight correlation , R2 was 0.90 (p = 0.050) for this large tumor. When serially determined maximum light emission was used for quantification in this particular tumor, R2 dropped to 0.37 (p = 0.4).The control mouse shown in Figure When control mice were sacrificed, tumors were explanted, and immediately reimaged. Bioluminescence imaging confirmed reduced light emission from hemorrhagic and necrotic areas in large tumors Figure . ImmunohHSV-TK/ GCV in cell culture and in vivo. The HSV-TK-Luc fusion protein was successfully used in a brain tumor animal model for serial and sensitive real time quantification of the cytotoxic effect of HSV-TK by BLI.This study demonstrates that firefly luciferase is a valuable tool for monitoring noninvasively the efficacy of the prodrug activating system Fusion of two enzymes is the only way to guarantee stoichiometric, and thus correlated expression of both fusion partners. We chose this approach because coexpression of two separate transgenes from either one or separate promoters has been reported to result in severely impaired gene expression, e.g. due to inefficient internal ribosome entry site (IRES)-mediated translation  or to prThe HSV-TK protein contains several nuclear targeting signals and is usually located predominantly in the nucleus . The enzRenilla Luc [cytotoxic activity of HSV-TK was not examined in the study. Renilla Luc activity in the above construct was found to be ~6 – 8-fold higher than seen with its unfused counterpart. As this enzyme is structurally unrelated to firefly Luc and has a lower molecular weight (36 kDa vs. 62 kDa), the study cannot be directly compared to our data. Notably, the authors mention that their attempt to fuse HSV-TK to firefly Luc resulted in a "poorly active" fusion protein [A decrease in HSV-TK activity of up to 80% compared to unfused HSV-TK has also been observed by Ray et al. when fusing the enzyme to illa Luc . N2A neu protein .Renilla Luc [in vivo with bioluminescence, fluorescence, and PET. Cytotoxicity generated by enzymatic conversion of prodrug by HSV-TK was however not measured in either of the above studies.Recently, the generation of several triple fusion proteins for imaging with different modalities was reported by two groups ,25. Thescorrelated expression of the three reporters within the triple fusion protein (HSV-TK-EGFP-Luc), nor were the activity levels of the different fusion partners compared to those of their unfused counterparts. Despite these limitations, the study confirms that Luc remains functional if fused N-terminally to other proteins, and that enzymatic activity is sufficient for in vivo BLI.Ponomarev et al.  did not Renillla or firefly Luc at the NH2-terminus, followed by a fluorescent protein and a mutated HSV-TK enzyme. This orientation of the fusion partners as well as the use of mutated HSV-TK optimized for use with PET limits the direct comparison of the presented data to our results. Bioluminescent activity of the 4 fusion constructs containing firefly Luc was reduced to 22 – 63% of the activity of unfused Luc, which is similar to our findings when expressing HSV-TK-Luc in U87MG glioma cells. One of the 4 constructs (Luc-mRFP-mutant HSV-TK) fully retained HSV-TK PET reporter activity while in the others HSV-TK activity (as assessed by intracellular radiotracer accumulation) was reduced to 30 – 61% of the activity of the corresponding unfused enzyme. This is in line with our findings when expressing HSV-TK-Luc transiently in U87MG glioma cells.Ray et al.  comparedRenilla Luc: (1) light emission of Renilla Luc peaks at 480 nm and thus shows only limited tissue penetration, (2) coelenterazine, the Renilla Luc substrate is prone to autoluminescence, resulting in high background if injected i.p. [Although it seems attractive to perform BLI with different luciferase enzymes, the following facts argue in favor of firefly Luc instead of ted i.p. , (3) coeted i.p. , and (4)HSV-TK and Luc genes residing on different plasmids. The time point of peak activity of both reporters differed by ~19 hours, most likely due to differences in half lives of the two enzymes. This finding supports our approach of expressing both enzymes as one molecule as this should greatly diminish differences in protein stability.Iyer et al.  examinedAttenuation of both enzymatic activities is most likely a result of steric hindrance and might be substantially reduced by selecting another linker sequence. Longer intervening sequences as well as introduction of flexible polyglycine linkers may contribute to an increase in enzyme activity ,29.2 = 0.86). In contrast to our study, GCV treatment in vivo resulted in a decrease in light emission while the tumors continuously grew in size. Most likely, this reflects the relatively poor cytotoxic activity of the mutant HSV-TK used in these experiments, implying that engineered HSV-TK optimized for use as a PET reporter may not retain its full cytotoxic activity when substrates such as GCV are used. Data on the cytotoxic potential of the virus construct in cell culture were not presented.Recently, De et al.  introducWe show here for the first time that a HSV-TK-Luc fusion protein in conjunction with GCV treatment can confer a curative effect on glioma bearing animals. While HSV-TK-Luc expressing glioma cells in culture were not killed completely when using GCV concentrations of up to 10 μg/ml, xenografts consisting of these cells were fully eliminated in all GCV treated mice. It has been shown by several groups that HSV-TK expressing tumor cells can elicit an antitumor immune reaction even in immunocompromised animals such as nude mice, most likely mediated by natural killer (NK) cells, activated in vivo by GCV induced cell killing ,31. We s6 cells intracerebrally in nude mice (data not shown), confirming the high sensitivity of BLI. Indeed, it has already been demonstrated in a murine orthotopic pituitary tumor model that bioluminescent light can travel through skull [Our study used a subcutaneous glioma model for "proof of concept" to allow for simultaneous bioluminescence imaging and measurement of tumour size by caliper. HSV-TK-Luc expressing U87MG glioma cells were also detected by the CCD camera after inoculation of 2 × 10gh skull .The cooled CCD camera system we used allows for quantification of emitted light. Some authors suggested that the level of transgene expression could be more reliably quantified by maximum light emission than by integrated light units within a ROI . Althougintegrated light signals emitted from tumor ROIs seem to be the measure of choice for serial imaging of transgene expression in growing tumors. The fact that tumor volume and photon emission are less tightly correlated in large tumors as compared to smaller ones implies that photon emission reflects mainly the presence of viable tumor cells within a tumor and is a more precise measure for cytotoxic efficacy than tumor size, as has also been suggested by others [If maximum and integrated light units within a ROI over single tumors were compared, we consistently found that both parameters tightly correlated with tumor size in tumors up to ~1 cm in diameter. In larger tumors (maximum diameter examined = 2 cm) the increase in size was in general less closely correlated with both integrated and maximum light units, but light emission from a ROI drawn over the entire tumor was still far more accurately mirroring tumor growth than maximum light units emitted from this region. With the increase in tumor thickness, maximum light emission from the tumor core is reduced due to necrosis, light scattering, and reduced supply of oxygen and D-Luciferin to tumor cells. On the other hand, the increase in tumor length and width is better reflected by light signals integrated over the entire tumor area, while a concomitant change in maximum light signal does not necessarily have to occur. Therefore, y others ,35.-15 – 10-17 vs. 10-11 – 10-12 mole/L of reporter probe are detectable, [Herpes simplex virus type I thymidine kinase has also been used as a reporter gene for monitoring therapeutic success with PET ,36. Howeectable, ) in combin vivo by BLI, when the bioluminescent reporter luciferase is fused in frame to the therapeutic gene HSV-TK. We used a clonal human glioma cell line stably expressing the HSV-TK-Luc fusion construct, thus guaranteeing high level transgene expression. Despite the somewhat attenuated activity of both fusion partners, a high degree of cytotoxicity by HSV-TK mediated GCV bioactivation as well as strong bioluminescent signals upon administration of D-luciferin were consistently demonstrated. In order to mirror more closely in vivo gene therapy of malignant brain tumors, experiments are underway to insert the HSV-TK-Luc fusion gene (and an improved version of it) into appropriate viral vectors and subsequently use them for treatment of orthotopically established gliomas in mice. Serial assessment of transduction levels, transgene localization and time course of fusion gene expression in living animals by BLI can aid in developing more potent gene therapy vectors for treatment of malignant glioma.We showed that therapeutic efficacy of a suicide gene/ prodrug activating system can be accurately monitored CCD, charged coupled device; CMV, cytomegalovirus; cps, counts per second; EGFP, enhanced green fluorescent protein; GCV, ganciclovir; HSV-TK, herpes simplex virus type 1 thymidine kinase; Luc, luciferase; mRFP, monomeric red fluorescent protein, PET, positron emission tomography; ROI, region of interest; SD, standard deviation, SEM, standard error of the mean.None declared.AS constructed the plasmids, performed the animal experiments together with CT and set up the in vitro assays. CT and SJ participated in the cell culture experiments and enzymatic assays. SJ and AS carried out the immunohistochemical studies. NGR and AS designed the experiments and evaluated the data. All authors have read and approved the manuscript.