The Association of Immune Markers with Cognitive Performance in South African HIV-Positive Patients.

Dysregulated expression of neuro-immune markers has previously been linked to HIV-associated neurocognitive impairment. We undertook an exploratory approach in a HIV clade-C cohort, investigating the association between eight immune markers and neurocognitive performance in 99 HIV+ and 51 HIV- participants. Markers were selected on preliminary and putative evidence of their link to key neuro-immune functions. Cognitive performance was established using a battery of tests sensitive to HIV-associated neurocognitive impairment, with domain-based scores utilized in analysis. The markers Thymidine phosphorylase (TYMP) and Neutrophil gelatinase-associated lipocalin (NGAL) were significantly higher while Matrix Metalloproteinase (MMP)9 was significantly lower in HIV+ participants. Our results further showed that in the HIV+ group, worse psychomotor processing speed was associated with higher TYMP and NGAL levels and worse motor function was associated with higher NGAL levels. Future studies should explore the underlying mechanisms of these markers in HIV-associated neurocognitive impairment. Graphical Abstract The association of peripheral immune markers with neurocognitive performance in South African HIV-positive patients.

Development and validation of a quantitative method for thymidine phosphorylase activity in peripheral blood mononuclear cells.

The enzyme thymidine phosphorylase (TP) is important for activation of capecitabine and 5-fluorouracil. Assessment of TP phenotype might be suitable for identification of patients at risk of fluoropyrimidine-induced toxicity. In this paper, we describe the development and validation an assay for TP activity in peripheral blood mononuclear cells (PBMCs). The assay was based on ex vivo conversion of the TP substrate thymidine to thymine. The amount of thymine formed was determined by high-performance liquid chromatography - ultraviolet detection (HPLC-UV) with 5-bromouracil as internal standard. Lymphocytes and monocytes were purified from isolated PBMCs to examine cell-specific TP activity. TP activity in PBMCs demonstrated Michaelis-Menten kinetics. The lower limit of quantification was 2.3 µg PBMC protein and assay linearity was demonstrated up to 22.7 µg PBMC protein. Within-day and between-day precisions were ≤9.2% and ≤6.0%, respectively. Adequate stability TP activity was demonstrated after long-term storage of PBMC dry pellets and lysates at -80 °C. In monocytes, TP activity was approximately 3 times higher than in lymphocytes. Clinical applicability was demonstrated in samples that were collected from five cancer patients. A simple, precise and sensitive HPLC-UV assay for quantification of TP activity in PBMCs was developed that can be applied for clinical research.

Poor Prognosis Associated with High Levels of Thymidine Phosphorylase and Thrombocytosis in Patients with Renal Cell Carcinoma.

PURPOSE: The study aimed to investigate the expression of thymidine phosphorylase (TP) in renal cell carcinoma (RCC) and its correlation with clinicopathological features and thrombocytosis, and also to determine their prognostic significance. PATIENTS AND METHODS: TP expression in 127 RCC specimens was evaluated with immunohistochemistry assays. Platelet (PLT) counts of patients before surgery were recorded. Correlations among TP expression, PLT and the clinicopathological features of the patients and their prognostic values were studied statistically. RESULTS: Sixty-eight patients of high TP expression (54%) and 59 of low TP expression (46%) were detected. There were 25 patients with thrombocytosis (20%). High TP expression was significantly associated with tumor stage (p = 0.004), histological grade (p = 0.001) and thrombocytosis (p = 0.012). The 5-year overall survival rate was 88.1% in patients with low TP expression, whereas it was 61.8% in patients with high TP expression (p < 0.001). The 5-year survival rates for patients with and without thrombocytosis were 16.0% and 88.2%, respectively (p < 0.001). The multivariate analysis showed that high TP expression and thrombocytosis would play a role as independent prognostic factors in RCC patients. CONCLUSION: High TP expression and thrombocytosis can be regarded as independent prognostic factors of poor survival in patients with RCC.

Can thymidine phosphorylase be a predictive marker for gemcitabine and doxifluridine combination chemotherapy in cholangiocarcinoma?: case series.

Unresectable cholangiocarcinoma is poorly responded to chemotherapy, especially for the case refractory to gemcitabine and cisplatin. Here, we tested whether high expression of thymidine phosphorylase (TP) can be a predictive biomarker for the indicator for gemcitabine and doxifluridine combination chemotherapy in the cholangiocarcinoma refractory to gemcitabine and cisplatin. Immunohistochemical staining for TP was performed with a biopsy specimen. We accepted the result as positive when more than 10% of cancer cells were stained with moderate intensity. Here, we report 2 cases of TP-positive cholangiocarcinoma well controlled with gemcitabine and doxifluridine combination chemotherapy, which had been refractory to the first line treatment with gemcitabine and cisplatin combination chemotherapy.

Thymidine phosphorylase participates in platelet signaling and promotes thrombosis.

RATIONALE: Platelets contain abundant thymidine phosphorylase (TYMP), which is highly expressed in diseases with high risk of thrombosis, such as atherosclerosis and type II diabetes mellitus. OBJECTIVE: To test the hypothesis that TYMP participates in platelet signaling and promotes thrombosis. METHODS AND RESULTS: By using a ferric chloride (FeCl3)-induced carotid artery injury thrombosis model, we found time to blood flow cessation was significantly prolonged in Tymp(-/-) and Tymp(+/-) mice compared with wild-type mice. Bone marrow transplantation and platelet transfusion studies demonstrated that platelet TYMP was responsible for the antithrombotic phenomenon in the TYMP-deficient mice. Collagen-, collagen-related peptide-, adenosine diphosphate-, or thrombin-induced platelet aggregation were significantly attenuated in Tymp(+/-) and Tymp(-/-) platelets, and in wild type or human platelets pretreated with TYMP inhibitor KIN59. Tymp deficiency also significantly decreased agonist-induced P-selectin expression. TYMP contains an N-terminal SH3 domain-binding proline-rich motif and forms a complex with the tyrosine kinases Lyn, Fyn, and Yes in platelets. TYMP-associated Lyn was inactive in resting platelets, and TYMP trapped and diminished active Lyn after collagen stimulation. Tymp/Lyn double haploinsufficiency diminished the antithrombotic phenotype of Tymp(+/-) mice. TYMP deletion or inhibition of TYMP with KIN59 dramatically increased platelet-endothelial cell adhesion molecule 1 tyrosine phosphorylation and diminished collagen-related peptide- or collagen-induced AKT phosphorylation. In vivo administration of KIN59 significantly inhibited FeCl3-induced carotid artery thrombosis without affecting hemostasis. CONCLUSIONS: TYMP participates in multiple platelet signaling pathways and regulates platelet activation and thrombosis. Targeting TYMP might be a novel antiplatelet and antithrombosis therapy.

Diagnosis of mitochondrial neurogastrointestinal encephalopathy disease in gastrointestinal biopsies.

A 14-year-old boy with mitochondrial neurogastrointestinal encephalopathy (MNGIE) disease had a lifelong history of failure to thrive and gastrointestinal symptoms including vomiting, pain, and diarrhea, leading to progressive cachexia. At the age of 9 years, after an extensive workup, the diagnosis of Crohn disease was strongly suspected, and he underwent colonoscopy with multiple biopsies. At 11 years of age, vision change and poor balance lead to a diagnosis of leukodystrophy by magnetic resonance imaging. Investigations for metachromatic leukodystrophy, adrenal leukodystrophy, and globoid cell leukodystrophy were all negative. A diagnosis of MNGIE disease was suspected when he continued deteriorating with gastrointestinal symptoms, multiple neurologic deficits, and encephalopathy. Markedly diminished thymidine phosphorylase activity and increased thymidine plasma levels confirmed the diagnosis of MNGIE. At autopsy, megamitochondria were observed by light microscopy in submucosal and myenteric ganglion cells and in smooth muscle cells of muscularis mucosae and muscularis propria, along the entire gastrointestinal tract from the esophagus to the rectum. Megamitochondria in ganglion cells were also observed in a retrospective review of the endoscopic intestinal biopsies taken at age 9 and 13 years and in the appendectomy specimen obtained 1 month before his demise. This study corroborates the presence of megamitochondria in gastrointestinal ganglion cells in MNGIE disease, better illustrates their detailed morphology, and describes for the first time similar structures in the cytoplasm of gastrointestinal smooth muscle cells. Pathologists should be able to recognize these structures by light microscopy and be aware of their association with primary mitochondriopathies.

Activity of thymidilate "salvage pathway" enzymes in human gastric cancer and blood serum: correlation with treatment modalities.

UNLABELLED: A comparative study of enzyme activity features of thymidilate "salvage pathway" synthesis in blood serum and tissues of different age patients with gastric cancer (T(3-4)N(0-x)M(0)) was carried out. AIM: To evaluate the diagnostic relevance of thymidilate metabolism enzymes activities and their association with tumor growth. METHODS: Enzymes activities were determined by the radioisotope method and spectrophotometrically in tumor tissues and blood serum of 74 patients. RESULTS: It was demonstrated that thymidine phosphorylase activity in gastric tumors is lower by 2.6 times as compared to non-neoplastic mucosa of resection margin. This being accompanied by decrease of its activity in the blood serum (from 47.9 ± 2.6 to 14.65 ± 2.4 nmol/min·mg, p < 0.001). An increase of thymidine kinase activity was revealed both in tumor tissues (more than 3.5 times) and in blood serum (from 3.9 ± 0,7 nmol/mg·h, to 6.8 ± 1.0 nmol/mg·h, p < 0.01). Changes in their activity in the postoperative period depended on the type of surgical procedure and tumor eradication. CONCLUSION: It could be suggested that control of individual dynamics of the enzymes activities in blood serum may be used as informative tool for monitoring of patients and treatment optimization.

Assessment of thymidine phosphorylase function: measurement of plasma thymidine (and deoxyuridine) and thymidine phosphorylase activity.

We describe detailed methods to measure thymidine (dThd) and deoxyuridine (dUrd) concentrations and thymidine phosphorylase (TP) activity in biological samples. These protocols allow the detection of TP dysfunction in patients with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). Since the identification of mutations in TYMP, the gene encoding TP, as the cause of MNGIE (Nishino et al. Science 283:689-692, 1999), the assessment of TP dysfunction has become the best screening method to rule out or confirm MNGIE in patients. TYMP sequencing, to find the causative mutations, is only needed when TP dysfunction is detected. dThd and dUrd are measured by resolving these compounds with high-performance liquid chromatography (HPLC) followed by the spectrophotometric monitoring of the eluate absorbance at 267 nm (HPLC-UV). TP activity can be measured by an endpoint determination of the thymine formed after 1 h incubation of the buffy coat homogenate in the presence of a large excess of its substrate dThd, either spectrophotometrically or by HPLC-UV.

Platelet-stored angiogenesis factors: clinical monitoring is prone to artifacts.

BACKGROUND: The analysis of angiogenesis factors in the blood of tumor patients has given diverse results on their prognostic or predictive value. Since mediators of angiogenesis are stored in platelets, their measurement in plasma is sensitive to inadvertent platelet activation during blood processing. METHODS: Variants of blood withdrawal and plasma preparation were evaluated by ELISA for the detection of TSP-1, PF-4, VEGF and PD-ECGF. A total of 22 pancreatic cancer patients and 29 healthy volunteers were evaluated. RESULTS: Plasma preparation with the anticoagulant mix of citrate, theophylline, adenosine, dipyridamole (CTAD) and immediate blood processing at 4°C was required for reproducible measurements of TSP-1, PF-4 and VEGF. Blood collection by venflon or inadvertent hemolysis during blood withdrawal caused significantly elevated TSP-1 and PF-4 values. When optimized plasma preparation was applied, a significant increase of TSP-1 and VEGF in cancer patients was detected (P=0.006; P< 0.001). CONCLUSION: The reliable plasma analysis of circulating platelet-stored angiogenesis factors requires preparation with CTAD at 4°C and blood collection by butterfly needle. Suboptimal procedures of plasma preparation are commonly applied in clinical monitoring of angiogenesis parameters which may account for the differences in reported plasma values and may have masked their predictive or prognostic marker potential.

Vasculopathy in type 2 diabetes mellitus: role of specific angiogenic modulators.

Type 2 diabetes mellitus (T2DM) is largely defined by hyperglycemia that promotes vascular complications. Abnormal angiogenesis has been claimed to have a role in this disease. This study aimed to investigate serum levels of both conventional and other markers of angiogenesis not well studied before in diabetes, and to correlate findings with age of the patients, glycemic control, presence of microvascular complications, and oxidative stress. Thirty-eight patients with T2DM and 13 age- and sex-matched healthy persons representing controls were recruited. Serum levels of basic fibroblast growth factor (b-FGF) was measured by immunosorbent assay kit; advanced glycosylation end products, platelet-derived endothelial cell growth factor (PD-ECGF), cathepsin-D (CD), gangliosides, hyaluronic acid (HA), nitric oxide (NO), lipid peroxides (LPER), superoxide dismutase, and total thiols by chemical methods; and copper (Cu) by atomic absorption flame photometry. Advanced glycosylation end products and angiogenic factors (b-FGF, PD-ECGF, CD, gangliosides, HA, and Cu) were significantly higher in patients than controls. Oxidative stress markers, NO, and LPER were significantly higher while total thiols were significantly lower in patients than controls. These changes were more pronounced with age, poor glycemic control, and presence of microvascular complications. Angiogenesis dysfunction coinciding with elevated levels of many angiogenic growth factors may point to their malfunctioning due to oxidative stress and/or protein glycation at the factor and the receptor levels. This necessitates further investigations.

Angiogenic biomarkers in children with congenital heart disease: possible implications.

BACKGROUND: Vascular endothelial growth factor (VEGF), platelet derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) and leptin are known as potent angiogenic factors The objective of the study was to evaluate these angiogenic factors VEGF, PD-ECGF/TP and leptin in children with congenital heart disease (CHD) and the factors that lead to angiogenesis in such cases. METHODS: Sixty CHD children were studied and divided into two groups (n=30); cyanotic-CHD (C-CHD) and acyanotic-CHD (A-CHD). Twenty five healthy children were included as controls. RESULTS: Significantly higher serum levels of VEGF, PD-ECGF/TP activity and leptin were detected in patients with CHD, particularly in patients with C-CHD. CHD patients with SpO2<90%, pulmonary hypertension (PH), severe pulmonary stenosis (PS), detectable collaterals, cardiomegaly and/or heart failure showed significantly higher levels of these factors than those with higher SpO2 or those without these findings. CONCLUSION: Hypoxia, PH and PS are important factors that lead to harmful angiogenesis. However, angiogenesis could be essential in some cases of CHD as coarctation of aorta to enhance renal perfusion. This may provide new ways for therapeutic strategies aiming at reducing or promoting angiogenesis in CHD to improve patient's outcome.

Oxidative stress and tumor progression in colorectal cancer.

BACKGROUND/AIMS: Elevated oxidative status has been found in many types of cancer cells. Recent studies have shown that the enzymatic product of thymidine phosphorylase (TP) generated reactive oxygen species (ROS) within cancer cells. The aim of this study was thus to evaluate the signal transduction pathway and the role of ROS in colorectal cancer. METHODOLOGY: Blood specimens were obtained from the drainage vein of the tumor during operation in 76 patients with colorectal cancer. Serum ROS levels were measured using the derivative-Reactive Oxygen Metabolites (d-ROM) test and serum TP levels were examined by a highly sensitive ELISA method. RESULTS: There was no significant correlation between serum levels of ROS and TP. Serum ROS levels were elevated in proportion to tumor invasion and had a significant positive correlation with tumor size (p < 0.05). However, they did not increase in patients with liver metastasis. CONCLUSIONS: These findings suggest that ROS are independent of TP-triggered signaling transduction and are associated with increased tumor invasion, but not liver metastasis in patients with colorectal cancer. From this point of view, new strategies related to ROS may provide improved therapeutic results as well as a preventative effect on carcinogenesis of the colorectum.

Frequency of mitochondrial defects in patients with chronic intestinal pseudo-obstruction.

BACKGROUND & AIMS: Chronic intestinal pseudo-obstruction (CIPO) is a rare disorder caused by intestinal dysmotility and characterized by chronic symptoms suggesting bowel obstruction in the absence of fixed, occluding lesions. CIPO has been associated with primary defects of the mitochondrial oxidative phosphorylation pathway, although the frequency of mitochondrial disorders in patients with CIPO is unknown. This study evaluates mitochondrial function in patients with CIPO. METHODS: A retrospective study was performed of data collected from 80 CIPO patients at a tertiary centre over a 25-year period. Mitochondrial disorders were detected by analysis of serum lactate and thymidine phosphorylase activities, brain magnetic resonance images, and muscle biopsies. Genes encoding thymidine phosphorylase, mitochondrial DNA tRNA(leu(UUR)) or tRNA(lys), and DNA polymerase-gamma were analyzed for mutations. RESULTS: Mitochondrial defects were identified in 15 patients (10 women; median age at diagnosis 32 years), representing 19% of the study cohort. All 15 patients had extra-digestive symptoms, 5 had mutations in the thymidine phosphorylase gene, 2 had mutations in tRNA(leu(UUR)), and 5 had mutations in the DNA polymerase-gamma gene. No genetic defect was detected in 3 of the patients with mitochondrial disorders. Patients with mitochondrial CIPO differed from patients without mitochondrial defects in their very severe nutritional status (frequent and long-term requirement for parenteral nutrition) and poor prognosis (frequent digestive and neurologic complications that led to a high incidence of premature death). CONCLUSION: Mitochondrial disorders seem to be an important cause of CIPO. Patients with CIPO, especially severe cases with associated neurologic symptoms, should be tested for mitochondrial defects.

[Lactate dehydrogenase, adenosine deaminase and thymidine phosphorylase activity of blood and tissues in breast cancer].

Lactate dehydrogenase (LDH), adenosine deaminase and thymidine phosphorylase activity was analyzed in the blood serum, primary tumor and adjacent uninvolved breast tissues from 49 women with adenocarcinoma and from 10 ones with benign adenofibroma. The LDH activity was increased in both cancerous and adjacent tissues. Serum LDH level reflects cell membrane alterations not only in the tumor node cells but also to a greater extent--in the surrounding unmalignant tissues. The discovered changes in nucleosides catabolic enzyme's activity in patients with breast cancer are correlated with LDH activity and its level in the blood serum.

Prognostic significance of the serum thymidine phosphorylase levels in venous blood drainage specimens in patients with colorectal cancer.

BACKGROUND/AIMS: We investigated whether the serum Thymidine phosphorylase (TP) levels in venous blood drainage specimens were associated with the prognosis and risk of liver metastasis in patients with resectable colorectal cancer. METHODOLOGY: From 88 patients with colorectal cancer, specimens of venous blood drainage were obtained during operation. The serum TP levels were measured by a highly sensitive Enzyme-Linked Immunosorbent Assay (ELISA) method. RESULTS: Subsequently, 88 patients were divided into two groups based on the levels of TP. The dividing line was determined to be 55ng/mL. The TP-high group (> 55ng/mL) had a significantly shorter overall survival than the TP-low group (< 55ng/mL). A multivariate analysis indicated that the serum TP level in venous blood drainage specimens to be a better prognostic factor independent of the traditional pathologic parameters. The serum TP levels of 3 patients with metachronous liver metastasis were high (> 55ng/mL). CONCLUSIONS: These findings suggest that the serum TP levels in venous blood drainage specimens reflect the prognosis of patients with colorectal cancer undergoing curative resection, particularly the risk of liver metastasis.

Neoadjuvant treatment of colorectal cancer with bevacizumab: the perioperative angiogenic balance is sensitive to systemic thrombospondin-1 levels.

PURPOSE: Colorectal cancer patients receiving neoadjuvant treatment with bevacizumab, a monoclonal antibody neutralizing vascular endothelial growth factor (VEGF), may suffer from wound healing complications after surgery as the antibody persists in patient blood. We characterized the systemic angiogenic balance in the perioperative period to evaluate its effect on physiologic angiogenesis. EXPERIMENTAL DESIGN: Nineteen patients receiving combination chemotherapy and bevacizumab for six neoadjuvant cycles were compared with 14 patients receiving chemotherapy without bevacizumab. Plasma from perioperative days -1, +1, +7, and +21 was analyzed for VEGF, thrombospondin-1 (TSP-1), and PD-ECGF concentrations. The angiogenic capacity was further tested in an in vitro assay of endothelial cell proliferation and migration. RESULTS: On day +1, the onset of wound healing was reflected in a change of balance, i.e., an increase of proangiogenic factors VEGF and platelet-derived endothelial cell growth factor compared with low TSP-1 inhibitor levels in both treatment groups. Patients with bevacizumab therapy showed significantly higher blood levels of total VEGF throughout the evaluation period. However, most VEGF molecules were inactive, i.e., complexed with antibody. Nevertheless, the capacity to stimulate endothelial growth was higher for these plasma samples and was reflected in low TSP-1 levels and an altered TSP-1 sensitivity. When purified TSP-1 protein was added, plasma samples of the bevacizumab but not the chemotherapy group showed reduced endothelial growth. CONCLUSIONS: Feedback mechanisms of bevacizumab therapy are not restricted to VEGF expression but seem to involve additional factors, such as TSP-1, which influences the systemic angiogenic balance and permits endothelial growth.

Expression of thymidine phosphorylase in peripheral blood cells of breast cancer patients is not increased by paclitaxel.

BACKGROUND: A synergistic cytotoxic effect has been hypothesized for taxanes and capecitabine, a prodrug of 5-fluorouracil. Based on preclinical studies, this synergism has been attributed to an up-regulation of the enzyme thymidine phosphorylase (TP). Beside tumour tissue, TP is highly expressed in white blood cells, possibly causing increased hematotoxicity, when taxanes are combined with capecitabine. So far, this hypothesis has not been investigated in humans. METHODS: A total of 128 consecutive blood samples were collected from eight patients with advanced breast cancer receiving paclitaxel weekly at a dose of 80 mg/m2. To assess the expression of TP in blood cells, samples were collected prior to first therapy, at the end of infusion, and up to 15 days thereafter. This procedure was repeated during the sixth application of paclitaxel. After isolation of the peripheral mononuclear blood cells, the expression of TP was assessed by ELISA. In parallel, paclitaxel level in plasma was evaluated at three selected time points as pharmacokinetic control parameter. RESULTS: Paclitaxel concentrations at the end of infusion did not change significantly from week 1 to week 6. The expression of TP in peripheral mononuclear blood cells decreased significantly after infusion below pretherapeutic values (p = 0.023; n = 8). After the nadir on day 3, the expression of TP increased moderately returning to baseline levels within one week. The overall picture in week 6 was similar to week 1. Using a trend analysis, neither a short-term nor a long-term induction of TP was observed. CONCLUSION: TP in peripheral mononuclear blood cells was hardly regulated under therapy with paclitaxel. Therefore, no increased haematotoxicity due to TP upregulation is expected from the combination of taxanes and capecitabine.

Determination of thymidine phosphorylase activity in human blood cells and fibroblasts by a nonradiochemical assay using reversed-phase high-performance liquid chromatography.

In this study, we demonstrated that the highest activity of thymidine phosphorylase (TP) was found in peripheral blood mononuclear (PBM) cells followed by that of thrombocytes and granulocytes whereas no activity of TP could be detected in erythrocytes. The activity of TP in leukocytes proved to be intermediate compared to the TP activity observed in PBM cells and granulocytes. The activity of TP also was readily detectable in human fibroblasts.

Allogeneic stem cell transplantation corrects biochemical derangements in MNGIE.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a multisystemic autosomal recessive disease due to primary thymidine phosphorylase (TP) deficiency. To restore TP activity, we performed reduced intensity allogeneic stem cell transplantations (alloSCTs) in two patients. In the first, alloSCT failed to engraft, but the second achieved mixed donor chimerism, which partially restored buffy coat TP activity and lowered plasma nucleosides. Thus, alloSCT can correct biochemical abnormalities in the blood of patients with MNGIE, but clinical efficacy remains unproven.

Infusion of platelets transiently reduces nucleoside overload in MNGIE.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is caused by thymidine phosphorylase (TP) deficiency, which leads to toxic accumulations of thymidine (dThd) and deoxyuridine (dUrd). In this work, we report that infusion of platelets from healthy donors to patients with MNGIE restored transiently circulating TP and reduced plasma dThd and dUrd levels, suggesting that treatments to achieve permanent restoration of circulating TP such as allogeneic stem cell transplantation or gene transfer might be therapeutic.