Bone morphogenetic proteins and Dickkopf-1 in ankylosing spondylitis.

OBJECTIVES: To determine bone morphogenetic proteins (BMPs) and Dickkopf homologue-1 (Dkk-1) levels in ankylosing spondylitis (AS). METHOD: Serum BMPs and Dkk-1 were measured in 72 AS patients and 30 healthy controls. For AS patients, we recorded the demographic data, disease activity, functional index, and global assessment with questionnaires, and image changes with roentgenography. We also measured human leucocyte antigen-B27 and systemic inflammatory reactants. RESULTS: BMPs were higher but Dkk-1 was significantly lower in AS patients than in controls. Dkk-1 was higher in AS patients who received non-steroidal anti-inflammatory drugs (NSAIDs) regularly in the past year (p = 0.001). Serum BMP-7 level and the BMP-7/Dkk-1 ratio correlated significantly with sacroiliitis severity, Bath Ankylosing Spondylitis Radiology Index (BASRI)-total, modified Stoke Ankylosing Spondylitis Spinal Score, and disease duration. There were also significant positive correlations among serum levels of BMP-2, -4, and -6, BASRI-total, and disease duration (p < 0.05). However, BMP-2/Dkk-1 was only significantly correlated with disease duration. The calculated area under the standard receiver operating characteristics curve suggested that BMP-2/Dkk-1 and serum BMP-2 are good indicators to predict disease activity, functional index, and patient global assessment in AS patients. CONCLUSION: BMPs and BMPs/Dkk-1 were significantly correlated with disease activity, and radiological and functional indices in AS patients. Dkk-1 was lower in AS patients than in controls. Among AS patients, Dkk-1 was higher in those taking NSAIDs regularly. BMP or Dkk-1 may be taken as a biomarker for disease severity or a treatment outcome predictor in AS, but this needs further study.

Presence of the Merkel cell polyomavirus in Merkel cell carcinoma combined with squamous cell carcinoma in a patient with chronic arsenism.

We present a case of Merkel cell carcinoma (MCC) coincident with squamous cell carcinoma (SCC) on the breast of a woman with chronic arsenism. This case demonstrates the distinct association of chronic arsenism with two different primary cutaneous carcinomas. Merkel cell polyomavirus (MCPyV) was identified in the lesional skin of the MCC but not in that of the SCC, suggesting there are different interactions of MCPyV in the pathogenesis of SCC and MCC related to arsenic. Physicians need to be vigilant in the occurrence of both SCC and MCC in patients with chronic arsenism. To our knowledge, this is the first study to show the presence of MCPyV in the MCC but not the SCC portion of an arsenic-induced tumour.

Wavelength-band-tuning photodiodes by using various metallic nanoparticles.

Wavelength-band tuning was easily achieved in this work by depositing various metallic nanoparticles (NPs) on silicon p-n junction photodiodes (PDs). The normalization spectrum of the PDs deposited with gold (Au) NPs reveals a high-wavelength pass characteristic; the PDs with silver (Ag) NPs coating behave as a low-wavelength pass, and the PDs with Au/Ag bimetallic NPs appear as a band-wavelength pass PD with a full width at half maximum of 450 âˆ¼ 630 nm. The issue of wavelength-band tuning is due to the different plasmonic resonance wavelengths associated with various metallic NPs. The extinction plot shows the Au NPs have a longer resonant wavelength of about 545 nm, leading to the incident light with a wavelength near or longer than 545 nm scattered by the Au NPs, hence a high-wavelength pass PD. The PDs with Ag NPs, due to the Ag NPs, exhibit a short resonant wavelength of 430 nm, and the short-wavelength incident light is absorbed near the silicon (Si) surface, where the Ag NPs is atop it. The shorter-wavelength incident light is enhanced by the plasmonic resonance of Ag NPs, making a low-wavelength PD. The Au/Ag NPs presents a resonant wavelength of 500 nm between the Au and Ag NPs. For the incident light with a wavelength close to 500 nm, a constructive interference causes a substantial increase in the local electromagnetic field, hence leading to a band-wavelength pass PD.

Enterohaemorrhagic Escherichia coli O157:H7 Shiga-like toxin 1 is required for full pathogenicity and activation of the p38 mitogen-activated protein kinase pathway in Caenorhabditis elegans.

Enterohaemorrhagic Escherichia coli (EHEC) causes life-threatening infections in humans as a consequence of the production of Shiga-like toxins. Lack of a good animal model system currently hinders in vivo study of EHEC virulence by systematic genetic methods. Here we applied the genetically tractable animal, Caenorhabditis elegans, as a surrogate host to study the virulence of EHEC as well as the host immunity to this human pathogen. Our results show that E. coli O157:H7, a serotype of EHEC, infects and kills C. elegans. Bacterial colonization and induction of the characteristic attaching and effacing (A/E) lesions in the intact intestinal epithelium of C. elegans by E. coli O157:H7 were concomitantly demonstrated in vivo. Genetic analysis indicated that the Shiga-like toxin 1 (Stx1) of E. coli O157:H7 is a virulence factor in C. elegans and is required for full toxicity. Moreover, the C. elegans p38 mitogen-activated protein kinase (MAPK) pathway, an evolutionarily conserved innate immune and stress response signalling pathway, is activated in the regulation of host susceptibility to EHEC infection in a Stx1-dependent manner. Our results validate the EHEC-C. elegans interaction as suitable for future comprehensive genetic screens for both novel bacterial and host factors involved in the pathogenesis of EHEC infection.

Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection.

Although maternal human immunodeficiency virus type 1 (HIV-1) transmission occurs during gestation, intrapartum and postpartum (by breast-feeding), 50-70% of all infected children seem to acquire HIV-1 shortly before or during delivery. Epidemiological evidence indicates that mucosal exposure is an important aspect of intrapartum HIV transmission. A simian immunodeficiency virus (SIV) macaque model has been developed that mimics the mucosal exposure that can occur during intrapartum HIV-1 transmission. To develop immunoprophylaxis against intrapartum HIV-1 transmission, we used SHIV-vpu+ (refs. 5,6), a chimeric simian-human virus that encodes the env gene of HIV-IIIB. Several combinations of human monoclonal antibodies against HIV-1 have been identified that neutralize SHIV-vpu+ completely in vitro through synergistic interaction. Here, we treated four pregnant macaques with a triple combination of the human IgG1 monoclonal antibodies F105, 2G12 and 2F5. All four macaques were protected against intravenous SHIV-vpu+ challenge after delivery. The infants received monoclonal antibodies after birth and were challenged orally with SHIV-vpu+ shortly thereafter. We found no evidence of infection in any infant during 6 months of follow-up. This demonstrates that IgG1 monoclonal antibodies protect against mucosal lentivirus challenge in neonates. We conclude that epitopes recognized by the three monoclonal antibodies are important determinants for achieving substantial protection, thus providing a rational basis for AIDS vaccine development.

Synergistic action of oncolytic herpes simplex virus and radiotherapy in pancreatic cancer cell lines.

BACKGROUND: Despite much research in chemotherapy and radiotherapy, pancreatic adenocarcinoma remains a fatal disease, highly resistant to all treatment modalities. Recent developments in the field of herpes simplex virus (HSV) engineering have allowed the generation of a number of promising virus vectors for treatment of many cancers, including pancreatic tumours. This study examined the use of one such virus, NV1023, in combination with radiation therapy in pancreatic cancer cell lines. METHODS: HSV therapy in combination with radiotherapy was investigated in pancreatic cancer cell lines Hs766T, Panc-1 and MIA PaCa-2. Multiple therapy effect analysis was performed by computerized simulation. Mechanisms underlying synergy, such as virus replication and apoptosis, were investigated. RESULTS: The combination of NV1023 and radiation yielded a synergistic oncolytic effect in all tested pancreatic cancer cell lines, with the greatest effect achieved in MIA PaCa-2. This effect was not mediated by an increase in rapid viral replication, but by a substantial increase in apoptosis. CONCLUSION: The synergistic oncolytic actions of HSV and radiotherapy observed in pancreatic cancer cell lines encourage further testing of this multimodality treatment.

Positive culture for Klebsiella pneumoniae in relevant sputum samples as a predictor of rapidly fatal outcome in septic patients at medical intensive care units.

Sixty-five patients with main diagnosis of sepsis, who were directly admitted to the emergency department (ED) and had fatal outcomes after transfer to the medical intensive care units (MICU), were included. Patients who died within 48 hours of MICU transfer were defined as having rapidly fatal outcomes (RFO). The following clinical variables, including diagnosis of infection source; results of blood, sputum, and urine cultures; management for sepsis in the ED and MICU and survival time, were analyzed. There were 30 (46%) patients with RFO. The median survival time in the RFO group was 22.6 hours in MICU. Klebsiella pneumoniae was the most common pathogen isolated from blood (7/65, 10.7%) and relevant sputum samples (7/45, 15.5% ). Multivariate analysis revealed that age, gender and positive sputum culture for K. pneumoniae (hazard ratio, 11.898, p < 0.001) were independently associated with RFO in septic patients. The median survival times for patients with positive and negative K. pneumoniae sputum culture were 17 hours and 66.8 hours (p < 0.001, by the log rank test), respectively. This study found that positive sputum culture of K. pneumoniae was an important independent predictive factor of RFO in septic patients admitted to the MICU.

Ribavirin antagonizes the effect of azidothymidine on HIV replication.

Azidothymidine and ribavirin both inhibit replication of human immunodeficiency virus in vitro and show promise of clinical utility in patients infected with this virus. In this study, the possible interactions of these drugs were examined in vitro, and a reproducible antagonism between azidothymidine and ribavirin was found to occur under a variety of experimental conditions. The mechanism responsible for this antagonism appeared to be inhibition of azidothymidine phosphorylation by ribavirin. Because similar effects may occur in vivo, clinical trials of these two drugs in combination must be performed only under carefully controlled conditions.

Reappraisal of HLA antibody analysis and crossmatching in kidney transplantation.

Enzyme-linked immunosorbent assay (ELISA) and flow cytometric techniques have been introduced to overcome the limited sensitivity and specificity of the CDC assay. This retrospective study used lambda antigen tray-mixed screening and Luminex HLA class I and II specificity assays to re-examine: (1) the accuracy with which detection of HLA antibody and specificity by ELISA predicts pretransplantation National Institutes of Health (NIH)/Centers for Disease Control and Prevention (CDC) crossmatch; and (2) a comparison of Luminex and ELISA methods to detect HLA antibodies. Sera from 481 patients awaiting kidney transplantation were tested using the ELISA method lambda antigen tray-mixed and using NIH-CDC to determine how well HLA antibodies detected using ELISA predicted crossmatches using CDC. Pretransplantation sera from 48 patients with follow-up data were retested using both ELISA lambda antigen tray-mixed and Luminex to compare the efficacy of the 2 methods.

Eighteen-year follow-up of a retrospective study of HLA antibody on kidney graft survival.

An increasing number of studies have demonstrated adverse graft survival in patients who have anti-HLA antibodies, whether preformed or developed posttransplantation. This retrospective study used Lambda antigen tray-mixed (LAT-M) screening and Luminex HLA class I and II specificity assay to re-examine the impact of pretransplantation HLA antibody on long-term graft survival. In this study, pretransplantation sera from 288 renal patients were tested using the enzyme-linked immunosorbent assay (ELISA) method, LAT-M. Among the 234 of the patients who did not have pretransplantation antibodies, 85% enjoyed 5-year functional graft survival, 76% 10-year functional graft survival, and 56% 15-year functional graft survival. The corresponding functional graft survival for the 54 patients who tested HLA antibody-positive was 65%, 53%, and 28%, respectively (P = .0021).

Enhanced nitric oxide and cyclic GMP formation plays a role in the anti-platelet activity of simvastatin.

BACKGROUND AND PURPOSE: It has been found that 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) exert various vascular protective effects, beyond their cholesterol-lowering property, including inhibition of platelet-dependent thrombus formation. The objective of the present study was to determine whether the nitric oxide (NO)/cyclic GMP-mediated processes in platelets contribute to the anti-aggregatory activity of simvastatin. EXPERIMENTAL APPROACH: After rabbit platelets were incubated with simvastatin for 5 min, aggregation was induced and the platelet aggregation, nitric oxide synthase activity, guanylyl cyclase activity, NO and cyclic GMP formation were measured appropriately. KEY RESULTS: Treatment with simvastatin concentration-dependently inhibited platelet aggregation induced by collagen or arachidonic acid with an IC(50) range of 52-158 microM. We also demonstrated that simvastatin (20-80 microM) concentration-dependently further enhanced collagen-induced NO and cyclic GMP formation through increasing NOS activity (from 2.64+/-0.12 to 3.52+/-0.21-5.10+/-0.14 micromol min(-1) mg protein(-1)) and guanylyl cyclase activity (from 142.9+/-7.2 to 163.5+/-17.5-283.8+/-19.5 pmol min(-1) mg protein(-1)) in the platelets. On the contrary, inhibition of platelet aggregation by simvastatin was markedly attenuated (by about 50%) by addition of a nitric oxide synthase inhibitor, a NO scavenger or a NO-sensitive guanylyl cyclase inhibitor. The anti-aggregatory effects of simvastatin were significantly increased by addition of a selective inhibitor of cyclic GMP phosphodiesterase. CONCLUSIONS AND IMPLICATIONS: Our findings indicate that enhancement of a NO/cyclic GMP-mediated process plays an important role in the anti-aggregatory activity of simvastatin.

Does immunosuppressive pharmacotherapy affect isoagglutinin titers?

OBJECTIVE: Preoperative reduction of isoagglutinins leads to successful ABO-incompatible (ABOi) renal transplantation. The strategy includes pretransplantation plasmapheresis, more potent immunosuppressive drugs, splenectomy, and anti-CD20 antibody. It has been reported that low isoagglutinin antibody titers posttransplant were observed among ABOi renal transplants with favorable outcome. The isoagglutinin titers may increase slightly when plasmapheresis is discontinued; however, it never returns to the pretreatment level under immunosuppressive therapy. This raises the question of what occurs to the isoagglutinin titer in ABO-compatible renal transplants under maintenance immunosuppressive pharmacotherapy. METHODS: We analyzed 10 renal transplant recipients, including seven living and three cadaveric donors. Patients were treated with basiliximab (20 mg) intravenously on day 0 and day 4. Maintenance immunosuppressive therapy involved a calcineurin inhibitor, mycophenolate mofetil, and steroid. Anti-human globulin isoagglutinin titers were routinely examined 1 day before and day 0 and 1, 2, 3, 4, 8, 12, and 24 weeks posttransplant. No ALG or intravenous immunoglobulin or plasmapheresis treatment was provided in the follow-up period. RESULTS: Our preliminary data showed nearly no influence on isoagglutinin titer levels in 6-month follow-up under maintenance immunosuppressive therapy. In addition, no significant difference in isoagglutinin titer was observed between tacrolimus and cyclosporine groups. CONCLUSION: Maintenance immunosuppressive pharmacotherapy did not affect isoagglutinin titer levels in ABO-compatible kidney transplants. Further study is needed to investigate the mechanisms of persistent low-level isoagglutinin titers among successful ABOi renal transplantation patients.

Influence of preoperative allograft function (effective renal plasma flow) on the short-term outcome following living donor kidney transplantation.

OBJECTIVES: Predonation kidney function is supposed to be an important factor affecting graft outcome. Controversial evidence suggests that higher predonation glomerular filtration rate (GFR) positively correlated with posttransplant graft outcome. The purpose of this study was to examine the relationship between living donor graft kidney function as measured by effective renal plasma flow (ERPF) and short-term graft function. METHODS: We performed a retrospective analysis of 45 patients who underwent living donor renal transplantation at our institution from 2001 to 2007. The comprehensive nuclear medicine evaluation of donors' ERPF was performed before laparoscopic nephrectomy. The preoperative absolute ERPF-recipient body surface area (F/BSA) ratio and absolute ERPF-recipient body weight (F/Wt) ratio were determined for each donor-recipient pair. Posttransplant graft function was estimated by the four-variable Modification of Diet in Renal Disease (Chinese MDRD) equation. RESULTS: Estimated GFR correlated with F/BSA ratio at 3 months and 6 months (Pearson r = .495, P = .001 and r = .441, P = .012). Estimated GFR correlated with F/Wt ratio at 3 months and 6 months (r = .567, P < .001 and r = .453, P = .009). The correlations between the estimated GFR at 3 months and other variables were investigated. However, in the final multivariate model, F/BSA ratio and F/Wt ratio were the independent predictors of graft function. CONCLUSION: Preoperative ERPF can be used to calculate F/BSA and F/Wt ratios before living donor kidney transplantation. Our study provided evidence that F/BSA and F/Wt ratios may be considered predictive indices for short-term outcomes. An extreme discrepancy should be avoided between preoperative allograft function (absolute ERPF) and recipient body surface area or body weight.

Five-year experience of adoption and evolution of laparoscopic living donor nephrectomy: results from a center without large volume of patients.

OBJECTIVES: Despite the advantages of laparoscopic living donor nephrectomy (LDN), this technique is known to have a steep learning curve that makes worldwide adoption challenging, especially in institutions without a large patients volume. Herein, we have reviewed our 5-year experience of adoption and evolution of this surgical technique, examining the donor and recipient outcomes. METHODS: Between September 2002 and June 2007, 40 LDNs were performed consecutively. Our surgical technique was mainly derived from the University of California San Francisco method. We retrospectively reviewed the donor demographics, operative characteristics, perioperative complication of donors/recipients, and outcomes of donors and recipients. RESULTS: Among the 40 cases, 36 (90.0%) were left-sided LDNs. Mean operative time was 335.1 +/- 66.9 minutes, blood loss was 303.9 +/- 333.2 mL, and warm ischemia time was 243.2 +/- 127.0 seconds. Multiple renal arteries required bench arterial reconstruction in 7 (17.5%) donor kidneys. Three renovascular injuries occurred intraoperatively, and 2 (5.0%) required open conversion. The overall postoperative complication rate was 20.0%. Postoperative donor serum creatinine was 1.5 times higher than preoperative serum creatinine. All but one recipient was discharged with adequate renal function. Graft function continues in 36 of the 38 harvested kidneys (94.7%) during the follow-up period. One (2.5%) recipient developed ureteral necrosis, and no recipients developed vascular thrombosis. CONCLUSIONS: LDNs can be performed with careful adoption and evolution in institutions without a large patient volume. The intraoperative complication rate of LDN can be reduced with experience.

High-throughput gender identification of Accipitridae eagles with real-time PCR using TaqMan probes.

The objective was to develop high-throughput gender identification of eagles. Based on BLAST and alignment analyses, the CHD-Z and CHD-W sequences of nine species of eagles were highly homologous with Spilornis cheela hoya (S. c. hoya); therefore, TaqMan probes were designed to target their CHD-ZW-common and CHD-W-specific regions. In S. c. hoya, genders were identified using TaqMan-based, real-time PCR (amplified by P2/P8 primers); this method was validated with anatomically confirmed controls (one of each gender). Both genders had high intensities of the HEX-labeled (CHD-ZW-common) probe, whereas only females had high intensity of the FAM-labeled (CHD-W-specific) probe. The sequence of the CHD-W-specific probe designed for S. c. hoya was completely homologous with the CHD-W-specific region in Circaetus gallicus, Gyps indicus, and Gyps bengalensis, and was only one nucleotide different from those of Accipiter nisus, Spizaetus nipalensis, Aquila chrysaetos, Circus spilonotus, and Milvus migrans. For the CHD-ZW-common probe, all species listed were completely conserved. Using real-time PCR software, we established auto-calling of the genders of 15 individuals of S. c. hoya. In conclusion, this method provided accurate, high-throughput gender identification for S. c. hoya, and has considerable potential for identifying the gender of several related species of eagles.

Melanopsin in cells of origin of the retinohypothalamic tract.

All known eukaryotic organisms exhibit physiological and behavioral rhythms termed circadian rhythms that cycle with a near-24-hour period; in mammals, light is the most potent stimulus for entraining endogenous rhythms to the daily light cycle. Photic information is transmitted via the retinohypothalamic tract (RHT) to the suprachiasmatic nucleus (SCN) in the hypothalamus, where circadian rhythms are generated, but the retinal photopigment that mediates circadian entrainment has remained elusive. Here we show that most retinal ganglion cells (RGCs) that project to the SCN express the photopigment melanopsin.

Synergistic apoptosis of MCF-7 breast cancer cells by 2-methoxyestradiol and bis(ethyl)norspermine.

2-Methoxyestradiol (2ME) is an estradiol metabolite with anti-tumor and anti-angiogenic properties. We studied the effect of 2ME on apoptosis of MCF-7 breast cancer cells and explored a combination therapy using 2ME and a polyamine analogue, bis(ethyl)norspermine (BE-3-3-3). Determination of viable cells on day 4 of treatment with 2ME/BE-3-3-3 combinations showed synergistic effects by Chou-Talalay analysis. APO-BRDU analysis showed that there was only 1.5+/-0.5% apoptosis at 200 nM 2ME and 3.7+/-1.7% in the presence of 2.5 microM BE-3-3-3. Combination of 200 nM 2ME and 2.5 microM BE-3-3-3 resulted in 52.2+/-2.6% apoptosis. Up to 90% of the cells underwent apoptosis in the presence of 1000 nM 2ME and 2.5 microM BE-3-3-3. Combination treatments resulted in total disruption of microtubules and depletion of putrescine, spermidine and spermine. In addition, phosphorylation of Akt and nuclear localization of cyclin D1 were altered by 2ME/BE-3-3-3 combination. Our results suggest an important strategy to induce apoptosis of breast cancer cells, with potential applications in therapy.

The sleep switch: hypothalamic control of sleep and wakefulness.

More than 70 years ago, von Economo predicted a wake-promoting area in the posterior hypothalamus and a sleep-promoting region in the preoptic area. Recent studies have dramatically confirmed these predictions. The ventrolateral preoptic nucleus contains GABAergic and galaninergic neurons that are active during sleep and are necessary for normal sleep. The posterior lateral hypothalamus contains orexin/hypocretin neurons that are crucial for maintaining normal wakefulness. A model is proposed in which wake- and sleep-promoting neurons inhibit each other, which results in stable wakefulness and sleep. Disruption of wake- or sleep-promoting pathways results in behavioral state instability.

Computerized quantitation of synergism and antagonism of taxol, topotecan, and cisplatin against human teratocarcinoma cell growth: a rational approach to clinical protocol design.

BACKGROUND: Cisplatin-based induction chemotherapy may achieve a complete response (i.e., no sign of tumor following treatment) in 70%-80% of patients with germ cell tumors. However, only a minority of patients in whom the firstline regimens fail are cured with the salvage regimens. PURPOSE: The aim of these studies was to identify new agents or new regimens for the treatment of germ cell tumors by carrying out quantitative assessment in vitro of two promising new antitumor agents (paclitaxel [Taxol] and topotecan) and three more established agents (cisplatin, vincristine, and etoposide). These agents were used singly or in two- and three-drug combinations and were selected because they represent five distinct categories of antineoplastic mechanisms. METHODS: The combination index-isobologram method, which is based on the median-effect principle developed by Chou and Talalay, was used for computerized data analysis. This method was selected because it takes into account both the potencies of each drug and combinations of these drugs and the shapes of their dose-effect curves. RESULTS: Synergism against the growth of teratocarcinoma cells resistant to cisplatin (833K/64CP10 cells) was greater than against the growth of parent 833K cells. The degrees of synergism were in the following order: cisplatin + topotecan > or = paclitaxel + cisplatin + topotecan > paclitaxel + topotecan > or = paclitaxel + etoposide > paclitaxel + cisplatin + etoposide > paclitaxel + cisplatin. All other combinations showed nearly additive effects or moderate antagonism. The degrees of antagonism were as follows: cisplatin + etoposide > or = paclitaxel + vincristine > paclitaxel + cisplatin + vincristine > cisplastin + vincristine. The combination of paclitaxel and cisplatin was synergistic against 833K/64CP10 cells and moderately antagonistic against 833K cells. Since the combination of paclitaxel, cisplatin, and topotecan and the two-component combinations of these drugs (cisplatin + topotecan and paclitaxel + topotecan) showed synergism stronger than that of other combinations, these three drugs were selected for illustrating detailed data analysis, using a computer software program developed in this institute. CONCLUSIONS AND IMPLICATIONS: Our findings suggest that, as a result of synergy, the doses of these agents needed to achieve an antitumor effect may be reduced by twofold to eightfold when these agents are given in combination. The present quantitative data analyses for synergism or antagonism provide a basis for a rational design of clinical protocols for combination chemotherapy in patients with advanced germ cell tumors.

Synergistic anticancer effects of ganciclovir/thymidine kinase and 5-fluorocytosine/cytosine deaminase gene therapies.

BACKGROUND: A bacterial enzyme, Escherichia coli cytosine deaminase, which converts the prodrug 5-fluorocytosine into the toxic drug 5-fluorouracil, and a viral enzyme, herpes simplex virus thymidine kinase, which converts ganciclovir from an inactive prodrug to a cytotoxic agent by phosphorylation, are being actively investigated for use in gene therapy for cancer. The purpose of this study was to determine whether combining these prodrug-activating gene therapies might result in enhanced anticancer effects. METHODS: Rat 9L gliosarcoma cells were transfected with plasmids containing the E. coli cytosine deaminase gene (9L/CD cells), with plasmids containing the herpes simplex virus thymidine kinase gene (9L/TK cells), or with both expression plasmids (9L/CD-TK cells). The drug sensitivities of the cell lines were evaluated; in addition, the sensitivities of 9L and 9L/CD-TK cells mixed in varied proportions were measured. The effects of prodrug treatment on 9L/CD-TK tumor growth (i.e., size and volume) in nude mice were monitored. The isobologram method of Loewe and the multiple drug-effect analysis method of Chou-Talalay were used to measure the interaction between the two prodrug-activating gene therapies. To elucidate the mechanism of interaction, the phosphorylation of ganciclovir in 9L/CD-TK cells after varying prodrug treatments was studied. RESULTS AND CONCLUSIONS: The presence of transfected cytosine deaminase and thymidine kinase genes in 9L gliosarcoma cells reduced cell survival, both in vitro and in vivo, following treatment with the relevant prodrugs; the effects of the two components appeared to be synergistic and related mechanistically to the enhancement of ganciclovir phosphorylation by thymidine kinase following 5-fluorouracil treatment.