Thymidine Kinase Activity Research Articles

The nanoparticulate Quillaja saponin KGI exerts anti-proliferative effects by down-regulation of cell cycle molecules in U937 and HL-60 human leukemia cells

Cancer cells are characterized by uncontrolled replication involving loss of control of cyclin dependent kinases (CDKs) and cyclins, and by abolished differentiation. In this study we introduce KGI, which is a nanoparticle with a Quillaja saponin as an active molecule. By the use of RNA array analysis and confirmation at the protein level, we show that KGI affects myeloid leukemia cells (in particular, the U937 monoblast cancer cell) by the following mechanisms: (A) ceasing cell replication via proteasome degradation, (B) down-regulation of key molecules at check points between G1/S and G2/M phases, (C) reduction of thymidine kinase activity, followed by (D) exit to differentiation and production of interleukin-8 (IL-8), eventually leading to apoptosis. Leukemia cell lines (U937 and HL-60 cells) were exposed to KGI for 8 h, after which the drug was removed. The cancer cells did not revert to replication over the following 10 days. Thus our findings suggest that the nanoparticle KGI inhibits proliferation and promotes differentiation in leukemic cells by interfering with the cell cycle process.

High Thymidine Kinase Activity and CD5 Positivity Predict Poor Prognosis Of DLBCL Treated With R-CHOP

IntroductionThymidine kinase (TK) activity has been investigated as a prognostic factor in hematological malignancies, and several studies have demonstrated that high TK activity correlates with the disease stage and provides prognostic information on overall survival (OS) and progression-free survival (PFS). We have reported that high thymidine kinase activity (TK) predicts poor prognosis for diffuse large B-cell lymphoma (DLBCL) patients treated with R-CHOP. In addition, CD5 positivity was reported as a poor predictor for DLBCL. The purpose of this retrospective study was to investigate the prognostic value of high TK activity and CD5 positivity compared with other laboratory findings in evaluating OS in patients undergoing R-CHOP for previously untreated DLBCL. MethodsWe retrospectively analyzed 176 patients newly diagnosed with DLBCL and treated with R-CHOP from September 2003 to October 2008 in our institute, and followed them until December 2012. The values of TK activity, CD5, non-germinal center type, C-reactive protein, lactate dehydrogenase, and beta2 microglobulin before R-CHOP were evaluated as prognostic factors for overall survival (OS). The cut-off of TK activity was defined as 14 IU/L because the median level of TK activity was 14.0 IU/L. CD5 positivity was defined if CD5 expression was detected by flow cytometry. Excluded were those positive for cyclin D1 or those with a history of chronic lymphocytic leukemia/small lymphocytic lymphoma. Germinal center type was defined by Hans classifier. The primary endpoint was OS. First, the OS and PFS were analyzed by the Kaplan-Meier method, and biological prognostic factors for OS were evaluated by Cox regression analysis. Second, we classified the patients into three risk groups due to significant poor predictors. All reported p-values were two-sided, and statistical significance was defined as p < 0.05. ResultsMedian age of the evaluable patients was 65.2 years old. The number of CD5+ patients was 19 (10.8%). The median levels of TK activity, CRP, LDH, and Hb were 14.0 IU/L (range, 3.0–1,100), 0.3 IU/L (range, 0.1–21.2), 254.5 IU/L (range, 111.0–44,432), and 13.1 g/dL (range, 7.7–17.0), respectively. Median follow-up was 60.0 months. Five-year OS rate of all patients was 51.1%. The OS was significantly worse in patients with high TK activity, CD5 positivity (CD5+), and high beta2 microglobulin level by univariate analysis. Five-year OS rates of the high-TK-activity arm and the low-TK-activity arm were 39.1% and 62.9%, respectively (p = 0.001). Five-year OS rates of the CD5+ arm and the CD5-negative arm were 21.1% and 53.8%, respectively (p = 0.002). The OS was significantly worse in patients with high TK activity and CD5+ by Cox regression analysis (hazard ratios 2.595 and 2.585; p = 0.044 and 0.026, respectively). We classified the patients into three groups based on the numbers with high TK activity and CD5+. There was a significant difference of OS in the three risk groups: five-year OS rates were 8.3% in the high-risk arm (two factors), 43.9% in the intermediate-risk arm (one factor), and 64.6% in the low-risk arm (zero factors) (p < 0.001). This prognostic model was suitable for PFS. Five-year PFS rates were 8.3% in the high-risk arm (two factors), 31.7% in the intermediate-risk arm (one factor), and 59.6% in the low-risk arm (zero factors) (p < 0.001). According to the chi square test and Fisher's exact test, there was no significant relationship between high TK and CD5+ (p = 0.153). ConclusionsHigh TK activity and CD5+ were strong predictors of short OS in patients with newly diagnosed DLBCL treated with R-CHOP. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

PET-MR bei Patienten mit Glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common and most aggressive primary tumor of the brain. In recent years newer therapeutic approaches have been developed. To allow for an optimized treatment planning it is important to precisely delineate necrotic tissue, edema and vital tumor tissue and to identify the most aggressive parts of the GBM. The magnetic resonance (MR) portion of an MR-positron emission tomography (PET) examination in patients with GBM should consist of both structural and functional sequences including diffusion-weighted and perfusion sequences. The use of (18)F-fluorodeoxyglucose ((18)F-FDG) is limited in patients with gliomas as glucose metabolism is already physiologically high in parts of the brain but (18)F-FDG is nevertheless a commonly used radiopharmaceutical for neuro-oncological questions. (18)F-fluorothymidine reflects the cellular activity of thymidine kinase 1 and correlates with the expression of KI-67 as an index of mitotic activity. The nitroimidazole derivatives (18)F-fluoromisonidazole and (18)F-fluoroazomycin arabinoside ((18)F-FAZA) allow the detection of hypoxic areas within the tumor. In recent years amino acid tracers, such as (18)F-fluoroethyltyrosine are increasingly being used in the diagnosis of gliomas. The simultaneous PET-MR image acquisition allows new approaches, e.g. motion correction by the simultaneous acquisition of MR data with a high temporal resolution and an improved quantification of the PET signal by integrating the results of functional MR sequences. Moreover, the simultaneous acquisition of these two time-consuming methods leads to reduced imaging times for this, often severely ill patient group.

Mitochondrial DNA damage induces apoptosis in senescent cells.

Senescence is a cellular response to damage and stress. The senescence response prevents cancer by suppressing the proliferation of cells with a compromised genome and contributes to optimal wound healing in normal tissues. Persistent senescent cells are also thought to drive aging and age-associated pathologies through their secretion of inflammatory factors that modify the tissue microenvironment and alter the function of nearby normal or transformed cells. Understanding how senescent cells alter the microenvironment would be aided by the ability to induce or eliminate senescent cells at will in vivo. Here, we combine the use of the synthetic nucleoside analog ganciclovir (GCV) with herpes simplex virus thymidine kinase (HSVtk) activity to create or eliminate senescent human cells. We show that low concentrations of GCV induce senescence through the accumulation of nuclear DNA damage while higher concentrations of GCV, similar to those used in vivo, kill non-dividing senescent cells via mitochondrial DNA (mtDNA) damage and caspase-dependent apoptosis. Using this system, we effectively eliminated xenografted normal human senescent fibroblasts or induced senescence in human breast cancer cells in vivo. Thus, cellular senescence and mtDNA damage are outcomes of synthetic nucleoside analog treatment, indicating that the GCV–HSVtk combination can be used effectively to promote the targeted formation or eradication of senescent cells.

Serum thymidine kinase activity compared with CA 15-3 in locally advanced and metastatic breast cancer within a randomized trial

The primary objective was to estimate serum thymidine kinase 1 (TK1) activity, reflecting total body cell proliferation rate including cancer cell proliferation, in women with loco regional inoperable or metastatic breast cancer participating in a prospective and randomized study. Secondary objectives were to analyze TK1 in relation to progression-free survival (PFS), overall survival (OS), therapy response and other tumour characteristics, including CA 15-3, widely used as a standard serum marker for disease progression. TK1 and CA 15-3 were analysed in 198 serum samples collected prospectively from women included in the randomized TEX trial between December 2002 and June 2007. TK1 activity was determined by the ELISA based DiviTum™ assay, and CA 15-3 analyses was generated with the electrochemiluminescence immunoassay Cobas Elecsys CA 15-3 II. High pre-treatment TK1 activity predicted shorter PFS (10 vs. 15months p=0.02) and OS (21 vs. 38months, p<0.0001), respectively. After adjustment for age, metastatic site and study treatment TK1 showed a trend as predictor of PFS (p=0.059) and was an independent prognostic factor for OS, (HR 1.81, 95% confidence interval (CI) 1.26-2.61, p=0.001). There was a trend of shortened OS for women with high CA 15-3 (p=0.054) in univariate analysis, but not after adjustment for the above mentioned covariates. Both TK1 (p=0.0011) and CA 15-3 (p=0.0004) predicted response to treatment. There were statistically different distributions of TK1 and CA 15-3 in relation to the site of metastases. TK1 activity measured by DiviTum™ predicted therapy response, PFS and OS in loco regional inoperable or disseminated breast cancer. These results suggest that this factor is a useful serum marker. In the present material, a prognostic value of CA 15-3 could not be proven.

Thymidine kinase and mtDNA depletion in human cardiomyopathy: epigenetic and translational evidence for energy starvation

This study addresses how depletion of human cardiac left ventricle (LV) mitochondrial DNA (mtDNA) and epigenetic nuclear DNA methylation promote cardiac dysfunction in human dilated cardiomyopathy (DCM) through regulation of pyrimidine nucleotide kinases. Samples of DCM LV and right ventricle (n = 18) were obtained fresh at heart transplant surgery. Parallel samples from nonfailing (NF) controls (n = 12) were from donor hearts found unsuitable for clinical use. We analyzed abundance of mtDNA and nuclear DNA (nDNA) using qPCR. LV mtDNA was depleted in DCM (50%, P < 0.05 each) compared with NF. No detectable change in RV mtDNA abundance occurred. DNA methylation and gene expression were determined using microarray analysis (GEO accession number: GSE43435). Fifty-seven gene promoters exhibited DNA hypermethylation or hypomethylation in DCM LVs. Among those, cytosolic thymidine kinase 1 (TK1) was hypermethylated. Expression arrays revealed decreased abundance of the TK1 mRNA transcript with no change in transcripts for other relevant thymidine metabolism enzymes. Quantitative immunoblots confirmed decreased TK1 polypeptide steady state abundance. TK1 activity remained unchanged in DCM samples while mitochondrial thymidine kinase (TK2) activity was significantly reduced. Compensatory TK activity was found in cardiac myocytes in the DCM LV. Diminished TK2 activity is mechanistically important to reduced mtDNA abundance and identified in DCM LV samples here. Epigenetic and genetic changes result in changes in mtDNA and in nucleotide substrates for mtDNA replication and underpin energy starvation in DCM.

Synthesis and antiviral activities of methylenecyclopropane analogs with 6-alkoxy and 6-alkylthio substitutions that exhibit broad-spectrum antiviral activity against human herpesviruses.

Methylenecyclopropane nucleosides have been reported to be active against many of the human herpesviruses. The most active compound of this class is cyclopropavir (CPV), which exhibits good antiviral activity against human cytomegalovirus (HCMV), Epstein-Barr virus, both variants of human herpesvirus 6, and human herpesvirus 8. CPV has two hydroxymethyl groups on the methylenecyclopropane ring, but analogs with a single hydroxymethyl group, such as the prototypical (S)-synguanol, are also active and exhibit a broader spectrum of antiviral activity that also includes hepatitis B virus and human immunodeficiency virus. Here, a large set of monohydroxymethyl compounds with ether and thioether substituents at the 6 position of the purine was synthesized and evaluated for antiviral activity against a range of human herpesviruses. Some of these analogs had a broader spectrum of antiviral activity than CPV, in that they also inhibited the replication of herpes simplex viruses 1 and 2 and varicella-zoster virus. Interestingly, the antiviral activity of these compounds appeared to be dependent on the activity of the HCMV UL97 kinase but was relatively unaffected by the absence of thymidine kinase activity in HSV. These data taken together indicate that the mechanism of action of these analogs is distinct from that of CPV. They also suggest that they might be useful as broad-spectrum antiherpesvirus agents and may be effective in the treatment of resistant virus infections.

Development of &lt;sup&gt;18&lt;/sup&gt;F-Labeled PET Probes for Imaging Cell Proliferation

In recent years, two different methods have been developed to image cell proliferation with the functional imaging technique, Positron emission Tomography (PET), proliferation rate and proliferative status. Proliferation rate is a measure of the tumor doubling time and uses radiolabeled analogs of the DNA precursor thymidine. This approach measures the activity of the enzyme thymidine kinase 1 (TK1) and provides a pulse label of the S phase fraction of a tumor. Proliferative status provides a measure of the ratio of proliferating (P) and quiescent (Q) cells in a tumor. This imaging approach for measuring proliferative status involves measuring the sigma-2 (σ(2)) receptor status of a tumor, the only protein which has been validated for making this measurement in vivo with PET. This article provides an overview of the biological information obtained from these different imaging strategies, and the development of radiotracers for imaging proliferation rate and proliferative status.

Thymidine Kinase-Mediated Shut Down of Bone Morphogenetic Protein-4 Expression Allows Regulated Bone Production

Bone morphogenetic Proteins (BMPs) are growth factors also involved in ossification and chondrogenesis that have generated interest for their efficiency in inducing bone neo-synthesis. BMPs expression in engineered cells has been successful in stimulating osteoblastic differentiation and ectopic and orthotopic bone formation in vivo. We have previously shown that an adenoviral vector expressing bone morphogenetic protein type-4 (BMP-4) is able to efficiently drive bone formation in a rabbit model of discontinuous bone lesions. However, unregulated secretion of BMPs has also been implicated in bone overproduction and exostosis. We have constructed a replication-defective first generation adenoviral (FG-Ad) vector containing a cassette for the expression of BMP-4 associated with the Herpes Simplex virus thymidine kinase (TK) gene (FG-B4TK) in order to shut down BMP-4 expression and, therefore, regulate bone production. TK expression does not interfere with BMP-4 ability to induce ectopic bone formation in athymic nude mice. Administration of ganciclovir blocks ectopic bone production in quadriceps muscle transduced with the FG-B4TK with no effect on the contralateral muscle transduced with a vector expressing only BMP-4. Histological findings confirmed the pro-apoptotic activity of TK and the reduction of mineralized areas in the quadriceps transduced with FG-B4TK in mice treated with ganciclovir. We have generated a system to block BMP-4 secretion by inducing apoptosis in transduced cells therefore blocking unwanted bone formation. This system is an additional tool to generate regulated amount of bone in discontinuous bone lesions and can be easily coupled with biomaterials capable of recruiting cells and generating a local bioreactor.

63P - Serum Thymidine Kinase Activity Compared with CA 15-3 in Locally Advanced and Metastatic Breast Cancer as Part of a Prospective and Randomized Study

63P - Serum Thymidine Kinase Activity Compared with CA 15-3 in Locally Advanced and Metastatic Breast Cancer as Part of a Prospective and Randomized Study

Abstract 2658: 18F-FLT PET imaging for noninvasive, early assessment of tumor response to MDM2-p53 disruption using a novel MDM2 inhibitor.

Abstract Introduction: Potent, selective, and orally available small molecule inhibitors of the p53 regulator MDM2 represent a promising class of therapeutics. When applied to p53WT tumors, MDM2 inhibitors disrupt the MDM2-p53 interaction, thereby activating the p53 pathway and inducing cell cycle arrest and apoptosis. The aim of this study was to evaluate two clinically available PET tracers for their ability to monitor early response to treatment with a novel MDM2 inhibitor. Methods: 2’-Deoxy-2’-18F-fluoro-D-glucose (FDG) is used to measure glucose transport and hexokinase activity, and its use in cancer detection and response is based on the preferential glycolytic metabolism of tumor cells. 3′-Deoxy-3′-18F-fluoro-L-thymidine (FLT), an analog of thymidine, can measure nucleoside transport and thymidine kinase activity, which is upregulated in the S-phase of mitosis and can serve as a surrogate for cellular proliferation. Two p53WT in vivo tumor models were evaluated: (1) MDM2-amplified SJSA-1 human osteosarcoma xenografts which exhibit tumor regression upon MDM2 inhibitor treatment, and (2) MDM2-WT HCT116 human colon carcinoma xenografts which exhibit tumor stasis upon treatment. FDG and FLT PET scans (separate cohorts) were acquired (1) 24 hr before treatment with MDM2 inhibitor or vehicle (i.e., baseline), (2) after 6 hr and 72 hr of treatment, and (3) 72 hr after cessation of treatment (i.e., wash-out). Results: MDM2 inhibitor treatment resulted in comparable decreases in FDG and FLT tumor standardized uptake values (SUV) in SJSA-1 tumor xenografts at 6 hr post-treatment (-23% and -9% relative to baseline, respectively; P&amp;lt;0.01) that persisted throughout the 72 hr treatment (-52% and -34%, respectively; P&amp;lt;0.001) and 72 h wash-out period (-54%; P&amp;lt;0.0005 and -28%, respectively). In HCT116 xenografts, MDM2 inhibitor treatment significantly reduced FLT tumor SUV at 6 hr post-treatment (-32% relative to baseline, P&amp;lt;0.005) and persisted throughout the 72 hr treatment (-70%; P&amp;lt;0.001), and rebounded sharply by the end of the wash-out period in HCT116 xenografts (-32%; P&amp;lt;0.005). FDG tumor SUV was not significantly reduced at any timepoint in HCT116 xenografts. Conclusions: FDG and FLT PET imaging were roughly equivalent in ability to monitor efficacy of MDM2 inhibitor treatment in a SJSA-1 tumor regression model; however only FLT PET imaging revealed a treatment effect in the HCT116 tumor stasis model. FLT PET imaging was successfully used as a noninvasive tool to provide quantitative measurement of tumor response to treatment with a novel MDM2 inhibitor. These results suggest that FLT PET imaging could have utility as an early clinical pharmacodynamic biomarker. Citation Format: Tim Kazules, Becky Bryant, Matt Silva, Jude Canon, Charles Glaus. 18F-FLT PET imaging for noninvasive, early assessment of tumor response to MDM2-p53 disruption using a novel MDM2 inhibitor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2658. doi:10.1158/1538-7445.AM2013-2658

Prognostic value of high thymidine kinase activity in patients with previously untreated diffuse large B-cell lymphoma treated by rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone

The purpose of this study was to investigate prognostic factors for overall survival (OS) among patients with previously untreated diffuse large B-cell lymphoma (DLBCL) treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP). We evaluated four biological parameters, including thymidine kinase (TK) activity. This study included 183 patients. The median level of TK was 14.0 IU/L, which we chose as the cut-off. After a median follow-up time of 50.0 months, the OS rate at 4 years in the high and low TK arm were 46.7% and 66.7%, respectively (p = 0.001). By multivariate analysis, OS was significantly inferior in the high TK arm (hazard ratio 2.705; p = 0.045). The complete response (CR) rate in the high TK arm was significantly worse than in the low TK arm. OS was significantly better in patients who had achieved CR than in those with partial response or less. In conclusion, high TK activity was a strong predictor for short OS and poor response among patients with previously untreated DLBCL treated with R-CHOP.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Monitoring metabolic responses to chemotherapy in single cells and tumors using nanostructure-initiator mass spectrometry (NIMS) imaging

BackgroundTissue imaging of treatment-induced metabolic changes is useful for optimizing cancer therapies, but commonly used methods require trade-offs between assay sensitivity and spatial resolution. Nanostructure-Initiator Mass Spectrometry imaging (NIMS) permits quantitative co-localization of drugs and treatment response biomarkers in cells and tissues with relatively high resolution. The present feasibility studies use NIMS to monitor phosphorylation of 3′-deoxy-3′-fluorothymidine (FLT) to FLT-MP in lymphoma cells and solid tumors as an indicator of drug exposure and pharmacodynamic responses.MethodsNIMS analytical sensitivity and spatial resolution were examined in cultured Burkitt’s lymphoma cells treated briefly with Rapamycin or FLT. Sample aliquots were dispersed on NIMS surfaces for single cell imaging and metabolic profiling, or extracted in parallel for LC-MS/MS analysis. Docetaxel-induced changes in FLT metabolism were also monitored in tissues and tissue extracts from mice bearing drug-sensitive tumor xenografts. To correct for variations in FLT disposition, the ratio of FLT-MP to FLT was used as a measure of TK1 thymidine kinase activity in NIMS images. TK1 and tumor-specific luciferase were measured in adjacent tissue sections using immuno-fluorescence microscopy.ResultsNIMS and LC-MS/MS yielded consistent results. FLT, FLT-MP, and Rapamycin were readily detected at the single cell level using NIMS. Rapid changes in endogenous metabolism were detected in drug-treated cells, and rapid accumulation of FLT-MP was seen in most, but not all imaged cells. FLT-MP accumulation in xenograft tumors was shown to be sensitive to Docetaxel treatment, and TK1 immunoreactivity co-localized with tumor-specific antigens in xenograft tumors, supporting a role for xenograft-derived TK1 activity in tumor FLT metabolism.ConclusionsNIMS is suitable for monitoring drug exposure and metabolite biotransformation with essentially single cell resolution, and provides new spatial and functional dimensions to studies of cancer metabolism without the need for radiotracers or tissue extraction. These findings should prove useful for in vitro and pre-clinical studies of cancer metabolism, and aid the optimization of metabolism-based cancer therapies and diagnostics.

Utility of Serum Thymidine Kinase Activity Measurements for Cases of Bovine Leukosis with Difficult Clinical Diagnoses

This study evaluated the clinical usefulness of serum thymidine kinase (TK) activity for diagnosing bovine leukosis cases for which clinical diagnosis was difficult ('BL with difficult diagnosis'). Median TK activity values in 24 'BL with difficult diagnosis' and 36 cattle for which BL was clinically confirmed by cytology findings of enlarged superficial lymph nodes ('clinically confirmed BL') were 36.8 and 39.4 U/l, respectively (no significant difference). The percentage with positive TK activity (> 5.4 U/l) was also similar in both groups (83.3% for 'BL with difficult diagnosis' and 97.2% for 'clinically confirmed BL'). TK activity was significantly higher in cows with 'BL with difficult diagnosis' compared to those with other tumors (N = 13) and those with inflammatory diseases (N = 14). Maximum TK activity in cows with other tumors and inflammatory diseases was not high (< 10 U/l). Median TK activities in cows with other tumors and those with inflammatory diseases were 1.8 and 1.4 IU/l, respectively. Positive TK activity was found in a significantly higher percentage of cows with 'BL with difficult diagnosis' (83.3%) relative to the percentages of cows with other tumors (15.3%) and inflammatory diseases (21.4%). Thus, TK activity is an appropriate marker for detecting BL onset in cows with 'BL with difficult diagnosis' as well as 'clinically confirmed BL' group. While the specificity of TK activity required for BL diagnosis is not clear, simultaneous evaluation of serum lactate dehydrogenase activity may assist in the differential diagnoses of other tumors and inflammatory diseases from BL.

Role of Thymidinekinase and Thymidine Phosphorylase as Enzymatic Test for Efficacy of Oncological Patients Treatment

The treatment of breast cancer has been undergoing an incredible sequence of changes. The role of individual treatment of women with breast cancer is not clearly defined. Since last century the drug 5-fluorouracil with another anticancer agents and prodrugs from group of fluoropyrimidines are widely used. Thymidine phosphorylase is the enzyme which can also catalyze the interconversion of uracil as well as several fluoropyrimidines. Thymidine Phosphorylase (TP) and Thymidine Kinase (TK) also play an important role in thymidine homeostasis and thus in DNA synthesis. Thymidine kinase is used as cells proliferation marker. Thymidine phosphorylase is similar to the platelet-derived endothelial cells growth factor. High expression of the enzyme is related to malignant angiogenesis and invasion. That is why we investigated the activity of TK and TP both in healthy women and patients with breast cancer (BC) of the I–IV stages and of the same age. Analysis of biochemical and morphological data of these patients revealed that the patients are initially divided according to the activity of blood plasma TP also had different morphological structure of the tumor. In the 1-st group the initial activity of TP was sharply reduced and in the 2nd group TP activity ranged normally. During the treatment of women from the 1st group the activity of TP was not changed, but the activity of TK was increased 3 times. In patients from the 2nd group the activity of TP was authentically increased, but TK activity was reduced. The follow-up analysis showed that four-year lethality in the 1-st group constituted 25%, and there was no lethality in the the 2nd group. Therefore the studied enzymes activity may be used as the informative test for monitoring of patients with breast cancer and optimization of drug treatment.

Extracorporeal continuous portal diversion plus temporal plasmapheresis for “small-for-size” syndrome

To investigate the effect of plasmapheresis via the portal vein for "small-for-size" syndrome (SFSS) aided by extracorporeal continuous portal diversion (ECPD). Extensive or total hepatectomy in the pig is usually adopted as a postoperative liver failure (PLF) or SFSS model. In this study, animals which underwent 85%-90% hepatectomy were randomized into either the Systemic group (n = 7) or the Portal group (n = 7). In the Systemic group, all pigs received temporal plasmapheresis (PP) via the extracorporeal catheter circuit (systemic to systemic circulation) from 24 to 30 h post-hepatectomy (PH); in the Portal group, all pigs received ECPD to divert partial portal vein flow (PVF) to the systemic circulation after hepatectomy, then converted to temporal PP from 24 to 30 h PH, and subsequently converted to ECPD again until 48 h PH. In the Portal group, the PVF was preserved at 3.0-3.3 times that of the baseline value, similar to that following 70% hepatectomy, which was regarded as the optimal PVF to the hypertrophic liver remnant. At 48 h PH, all pigs were re-opened and the portal vein pressure (PVP), PVF, and HAF (hepatic artery flow) were measured, and then diversion of the portal venous flow was terminated. After 1 h the PVP, PVF, and HAF were re-measured. The portal hemodynamic changes, liver injury, liver regeneration and bacterial/lipopolysaccharide (LPS) translocation were evaluated in the two groups. The PVP in the Portal group was significantly lower than that in the Systemic group during the time period from 2 to 49 h PH (P < 0.05). Serum alanine aminotransferase (ALT), total bilirubin (TB) and ammonia were significantly reduced in the Portal group compared with the Systemic group from 24 to 48 h PH (P < 0.05). The Portal group may have attenuated sinusoidal endothelial injury and decreased the level of HA compared with the Systemic group. In the Systemic group, there was significant sinusoidal dilation, hydropic changes in hepatocytes and hemorrhage into the hepatic parenchyma, and the sinusoidal endothelial lining was partially destroyed and detached into the sinusoidal space. CD₃₁ immunostaining revealed significant destruction of the endothelial lining. In the Portal group, there was no intraparenchymal hemorrhage and the sinusoidal endothelial cells and hepatocytes were well preserved. CD₃₁ immunostaining was mild which indicated less destruction of the endothelial lining. HA was significantly decreased in the Portal group compared with the Systemic group from 2 to 48 h PH. The rate of liver remnant regeneration was elevated, while apoptosis was attenuated in the Portal group compared with the Systemic group. Thymidine kinase activity was much higher in the Portal group than in the Systemic group at 48 h PH. The PCNA index was significantly increased and the apoptotic index was significantly decreased in the Portal group compared with the Systemic group. Bacterial translocation and endotoxin, as well as the inflammatory response, were significantly attenuated in the Portal group compared with the Systemic group. LPS, tumor necrosis factor-α and interleukin-6 levels were all significantly decreased in the Portal group compared with the Systemic group from 24 to 48 h PH, while bacterial DNA level was significantly decreased from 2 to 48 h PH. PP plus ECPD via the portal vein can attenuate toxic load and hyperperfusion injury, and should be undertaken instead of PP via the systemic circulation in SFSS or PLF.

Inhibition of Mycoplasma pneumoniae growth by FDA-approved anticancer and antiviral nucleoside and nucleobase analogs.

BackgroundMycoplasma pneumoniae (Mpn) is a human pathogen that causes acute and chronic respiratory diseases and has been linked to many extrapulmonary diseases. Due to the lack of cell wall, Mpn is resistant to antibiotics targeting cell wall synthesis such as penicillin. During the last 10 years macrolide-resistant Mpn strains have been frequently reported in Asian countries and have been spreading to Europe and the United States. Therefore, new antibiotics are needed. In this study, 30 FDA-approved anticancer or antiviral drugs were screened for inhibitory effects on Mpn growth and selected analogs were further characterized by inhibition of target enzymes and metabolism of radiolabeled substrates.ResultsSixteen drugs showed varying inhibitory effects and seven showed strong inhibition of Mpn growth. The anticancer drug 6-thioguanine had a MIC (minimum inhibitory concentration required to cause 90% of growth inhibition) value of 0.20 μg ml-1, whereas trifluorothymidine, gemcitabine and dipyridamole had MIC values of approximately 2 μg ml-1. In wild type Mpn culture the presence of 6-thioguanine and dipyridamole strongly inhibited the uptake and metabolism of hypoxanthine and guanine while gemcitabine inhibited the uptake and metabolism of all nucleobases and thymidine. Trifluorothymidine and 5-fluorodeoxyuridine, however, stimulated the uptake and incorporation of radiolabeled thymidine and this stimulation was due to induction of thymidine kinase activity. Furthermore, Mpn hypoxanthine guanine phosphoribosyl transferase (HPRT) was cloned, expressed, and characterized. The 6-thioguanine, but not other purine analogs, strongly inhibited HPRT, which may in part explain the observed growth inhibition. Trifluorothymidine and 5-fluorodeoxyuridine were shown to be good substrates and inhibitors for thymidine kinase from human and Mycoplasma sources.ConclusionWe have shown that several anticancer and antiviral nucleoside and nucleobase analogs are potent inhibitors of Mpn growth and that the mechanism of inhibition are most likely due to inhibition of enzymes in the nucleotide biosynthesis pathway and nucleoside transporter. Our results suggest that enzymes in Mycoplasma nucleotide biosynthesis are potential targets for future design of antibiotics against Mycoplasma infection.

Screening of herpes simplex virus type 1 isolates for acyclovir resistance using DiviTum® assay

Rapid alternative methods are required to evaluate easily acyclovir (ACV) sensitivity of clinical herpes simplex virus (HSV) isolates. The objective of this study was to screen 54 ACV-sensitive and 41 ACV-resistant clinical HSV-1 isolates, well characterized by phenotypic and genotypic methods, for the phosphorylation activity of the viral thymidine kinase (TK) using a commercially available and modified non-radioactive DiviTum® test on the basis of an indirect enzyme linked immunosorbent assay. The ACV-sensitive HSV-1 isolates had high TK activity values between 31.5±6.4 DiviTum® Units per liter (DU/L) and 487.4±60.1 DU/L. The mean activity of all ACV-sensitive isolates was calculated as 212.3±15.7 DU/L. By contrast, the mean activity of all ACV-resistant HSV-1 isolates was significantly lower at 5.5±1.3 DU/L. Out of the 41 ACV-resistant HSV-1 isolates, 38 had no or very low phosphorylation activities of the viral TK between 0 DU/L and 9.3±3.2 DU/L. The remaining three ACV-resistant viral isolates had TK activities between 44.6±5.1 DU/L and 80.9±13.3D U/L. In conclusion, the modified DiviTum® test can be used to screen HSV-1 isolates for their sensitivity to ACV. Acyclovir-sensitive HSV-1 isolates show TK activities >30 DU/L and ACV-resistant isolates have activity values <10 DU/L. However, single ACV-resistant HSV-1 isolates can have TK activity values >30 DU/L. These strains are most likely ACV-resistant TK-altered mutants, but no evidence was provided for an alteration of the TK.

Toll-like Receptor 11 (TLR11) Prevents Salmonella Penetration into the Murine Peyer Patches

Toll-like receptors (TLRs) are key molecular sensors used by the mammalian innate immune system to detect microorganisms. Although TLR functions in colonic immune homeostasis and tolerance to commensal bacteria have been intensively researched, the precise roles of different TLRs in response to pathogen infection in the gut remain elusive. Peyer patches are the major entrance of Salmonella infection and antigen transportation in intestine. Here, we report that, in contrast to TLR5 as a "carrier of Salmonella," TLR11 works as a "blocker of Salmonella" to prevent highly invasive Salmonella from penetrating into the murine Peyer patches and spreading systemically. TLR11 plays an important role in mediating TNF-α induction and systemic inflammation in response to Salmonella infection. Remarkably, in mice lacking TLR11, apparent hemorrhages at Peyer patches are induced by highly invasive Salmonella, a phenotype resembling human Salmonella infection. Therefore, our results indicate a potentially important role for TLR11 in preventing murine intestinal infection and modulating antigen transportation in the gut and imply an important role for various TLRs in cooperation with tight control of pathogens penetrating into Peyer patches. The TLR11 knock-out mouse can serve as a good animal model to study Salmonella infection.