The association of peripheral immune markers with brain cortical thickness and surface area in South African people living with HIV.

A spectrum of cognitive impairments known as HIV-associated neurocognitive disorders (HAND) are consequences of the effects of HIV-1 within the central nervous system. Regardless of treatment status, an aberrant chronic neuro-immune regulation is a crucial contributor to the development of HAND. However, the extent to which inflammation affects brain structures critical for cognitive status remains unclear. The present study aimed to determine associations of peripheral immune markers with cortical thickness and surface area. Participants included 65 treatment-naïve HIV-positive individuals and 26 HIV-negative controls. Thickness and surface area of all cortical regions were derived using automated parcellation of T1-weighted images acquired at 3 T. Peripheral immune markers included C-C motif ligand 2 (CCL2), matrix metalloproteinase 9 (MMP9), neutrophil gelatinase-associated lipocalin (NGAL), thymidine phosphorylase (TYMP), transforming growth factor (TGF)-ß1, and vascular endothelial growth factor (VEGF), which were measured using enzyme-linked immunosorbent assays. Associations of these markers with thickness and surface area of cortical regions were evaluated. A mediation analysis examined whether associations of inflammatory markers with cognitive functioning were mediated by brain cortical thickness and surface area. After controlling for multiple comparisons, higher NGAL was associated with reduced thickness of the bilateral orbitofrontal cortex in HIV-positive participants. The association of NGAL with worse motor function was mediated by cortical thickness of the bilateral orbitofrontal region. Taken together, this study suggests that NGAL plays a potential role in the neuropathophysiology of neurocognitive impairments of HIV.

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.

Seroprevalencia y detección de infección primaria por citomegalovirus mediante prueba de avidez IgG en el primer trimestre de embarazo / Seroprevalence and detection of primary infection by cytomegalovirus with IgG avidity test during the first quarter of pregnancy

Objetivo. Conocer la seroprevalencia y detección de infección primaria por citomegalovirus (CMV) mediante prueba de avidez de inmunoglobulina G (IgG) durante el primer trimestre del embarazo en el Hospital General de Morelia, Michoacán. Material y métodos. Se estudiaron 177 pacientes mediante prueba de Elisa modificada, la cual utiliza inmunoanálisis quimioluminiscente de micropartículas (CMIA) para detección de anti-CMV (IgG e inmunoglobulina M [IgM]) e IgG avidez. Resultados. Del total de pruebas, 90.4% resultaron positivas para IgG; de éstas, 2.3% resultaron reactivas a IgM. En este segundo grupo, la prueba de IgG avidez reportó avidez baja en 1.1% y alta en el mismo porcentaje; 9.6% fueron seronegativas. Conclusiones. Se encontró similitud con lo publicado en México. Los profesionales de la salud deben conocer los algoritmos para el diagnóstico y manejo oportuno de la infección por CMV mediante la prueba de avidez de IgG.

Objective. To determine the seroprevalence and detection of primary infection by cytomegalovirus (CMV) with immunoglobulin G (IgG) avidity test during the first quarter of pregnancy in the General Hospital in Morelia, Michoacan. Materials and methods. A total of 177 patients were studied employing a modified Elisa test using a chemiluminescent microparticle immunoassay (CMIA) for the detection of CMV antibodies (IgG and immunoglobulin M [IgM]), and IgG avidity. Results. 90.4% were positive for IgG, and of these, 2.3% were also reactive for IgM, and in this group the IgG avidity test reported low avidity for 1.1% and higher avidity in the same percentage. 9.6% were seronegative. Conclusions. Similarity was found with published studies in Mexico. Health professionals should know the clinical algorithms for diagnosis and proper management of CMV infection using the IgG avidity test.

Elevated levels of IL-23 in a subset of patients with post-lyme disease symptoms following erythema migrans.

BACKGROUND: The causes of post-Lyme disease symptoms are unclear. Herein, we investigated whether specific immune responses were correlated with such symptoms. METHODS: The levels of 23 cytokines and chemokines, representative of innate and adaptive immune responses, were assessed in sera from 86 antibiotic-treated European patients with erythema migrans, 45 with post-Lyme symptoms and 41 without symptoms, who were evaluated prior to treatment and 2, 6, and 12 months thereafter. RESULTS: At study entry, significant differences between groups were observed for the type 1 helper T cell (TH1)-associated chemokines CXCL9 and CXCL10, which were associated with negative Borrelia cultures, and the type 17 helper T cell (TH17)-associated cytokine interleukin 23 (IL-23), which was associated with positive cultures and the development of post-Lyme symptoms (P ≤ .02). Moreover, of the 41 patients with detectable IL-23 levels, 25 (61%) developed post-Lyme symptoms, and all 7 with IL-23 levels ≥ 230 ng/mL had such symptoms. Furthermore, antibody responses to the ECGF autoantigen were more common in patients with post-Lyme symptoms (P = .07) and were correlated directly with IL-23 levels (P = .02). Despite the presence of post-Lyme symptoms, all posttreatment culture results were negative, antiborrelial antibody responses declined, and there were no objective signs of disseminated disease, suggesting that spirochetal eradication had occurred with treatment in all patients. CONCLUSIONS: High TH1-associated responses correlated with more effective immune-mediated spirochetal killing, whereas high TH17-associated immune responses, often accompanied by autoantibodies, correlated with post-Lyme symptoms, providing a new paradigm for the study of postinfectious symptoms in a subset of patients with Lyme disease.

The development and validation of an immunoassay for the measurement of anti-thymidine phosphorylase antibodies in mouse and dog sera.

Erythrocyte encapsulated thymidine phosphorylase (EE-TP) is under development as an enzyme replacement therapy for mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a fatal metabolic disorder resulting from an inherited deficiency of the enzyme thymidine phosphorylase. We report here the development and validation of a sensitive electrochemiluminescent (ECL) bridging immunoassay to support Good Laboratory Practice (GLP)-compliant preclinical safety studies of EE-TP in the mouse and dog. Affinity-purified rabbit anti-E. coli thymidine phosphorylase (TP) antibody was used as a calibrator standard with an effective working range of 2.5-7500 ng/mL. The minimum required dilution (MRD) for both mouse and dog sera was 1:10. The mean analytical recoveries for anti-TP antibodies spiked into serum at 70 ng/mL and 7000 ng/mL were 117.9% and 93.2%, respectively for mouse, and 112.0% and 104.3%, respectively for dog. The intra-assay precision (coefficient of variation, CV) ranged between 1.1% and 8.0% in mouse serum, and 1.9% and 2.5% in dog serum. Inter-assay precision ranged between -1.6% and 6.7% in mouse serum, and -13.0% and -2.5% in dog serum. Assay cut-point/screening cut-point correction factors were 201.37 and 44.4, respectively for mouse and dog sera. For future analysis of positive test samples, less than 37.12% (mouse) and 31.41% (dog) inhibition of the assay signal in the confirmation assay will confer anti-TP antibody specificity. Assay drift and hook effects (prozone) were not observed. The intra-assay and inter-assay accuracy for robustness were within ±20%.

An evaluation of thymidine phosphorylase as a means of preventing thymidine rescue from the thymidylate synthase inhibitor raltitrexed.

The antitumour effect of thymidylate synthase inhibitors such as raltitrexed (RTX) may be reversed by salvage of thymidine (Thd). Since thymidine phosphorylase (TP) depletes Thd, the potential for tumour-selective depletion of Thd using antibody-mediated delivery of TP to tumours was investigated. In vitro studies demonstrated that 25 x 10(-3) units/ml TP depleted extracellular Thd (3 microM) and restored sensitivity to the growth inhibitory effects of RTX in Lovo and HT29 cell lines. Thymidine concentrations in xenograft tumours were inversely proportional to the activity of TP in the tumour, and the presence of a subcutaneous Lovo xenograft reduced plasma Thd concentrations from 0.92 +/- 0.07 to 0.37 +/- 0.04 microM. Intravenous administration of native TP enzyme depleted plasma Thd to 5 nM, but following rapid elimination of TP, plasma Thd returned to pretreatment values. There was no effect on tumour TP or Thd. Conjugation of TP to the A5B7 F(ab)2 antibody fragment, which targets carcinoembryonic antigen (CEA) expressed on colorectal cell-lines such as Lovo, did result in selective accumulation of TP in the tumour. However, there was no tumour-selective depletion of Thd and there did not appear to be any potential benefit of combining antibody-targeted TP with RTX.

Identification of the angiogenic endothelial-cell growth factor-1/thymidine phosphorylase as a potential target for immunotherapy of cancer.

Characterization of the antigens recognized by tumor-reactive T cells isolated from patients successfully treated with allogeneic HLA-matched hematopoietic stem cell transplantation (SCT) can lead to the identification of clinically relevant target molecules. We isolated tumor-reactive cytotoxic CD8(+) T-cell (CTL) clones from a patient successfully treated with donor lymphocyte infusion for relapsed multiple myeloma after allogeneic HLA-matched SCT. Using cDNA expression cloning, the target molecule of an HLA-B7-restricted CTL clone was identified. The CTL clone recognized a minor histocompatibility antigen produced by a single nucleotide polymorphism (SNP) in the angiogenic endothelial-cell growth factor-1 (ECGF1) gene also known as thymidine phosphorylase. The SNP leads to an Arg-to-His substitution in an alternatively translated peptide that is recognized by the CTL. The ECGF1 gene is predominantly expressed in hematopoietic cells, although low expression can also be detected in other tissues. The patient from whom this CTL clone was isolated had mild graft-versus-host disease despite high numbers of circulating ECGF-1-specific T cells as detected by tetramer staining. Because solid tumors expressing ECGF-1 could also be lysed by the CTL, ECGF-1 is an interesting target for immunotherapy of both hematologic and solid tumors.

Platelet endothelial cell adhesion molecule-1 and angiogenic factor expression in idiopathic membranous nephropathy.

BACKGROUND: Idiopathic membranous nephropathy (IMN), a principal disease of glomerular capillaries, was investigated for some aspects of glomerular capillary injury and repair (angiogenesis). METHODS: Fifteen cases of IMN were studied immunohistochemically for expression of the endothelial cell antigen platelet endothelial cell adhesion molecule-1 (PECAM-1[CD31]) and the angiogenesis-stimulating factors vascular endothelial growth factor (VEGF) and thymidine phosphorylase (TP). An equal number of normal control kidneys of fetal and mature origin were tested for the same antigens. RESULTS: Normal tissues expressed PECAM-1 in both glomerular and interstitial endothelial cells, whereas VEGF and TP were expressed in the tubular epithelium. IMN was characterized by complete or partial loss of PECAM-1 expression from glomerular capillaries and a parallel gain/expression of this antigen by the tubular epithelium. In addition, VEGF and TP expression was lost or considerably reduced from tubular cells of IMN. CONCLUSION: We hypothesize that PECAM-1 expression by tubular epithelial cells represents uptake of CD31(+) cell-surface fragments released by glomerular endothelial cells after glomerular damage. The damage is confounded by the failure of angiogenic mechanisms to promote glomerular angiogenesis (repair) because both VEGF and TP stimulation by the tubular epithelium is eliminated. It is suggested that immunohistochemical detection of VEGF or TP in the tubular epithelium may be useful in understanding the pathogenesis of IMN.

Development and characterization of 1C6-203, a new monoclonal antibody specific to human thymidine phosphorylase.

Thymidine phosphorylase (dThdPase) is an essential enzyme for activation of the oral cytostatic drug capecitabine and its intermediate metabolite, doxifluridine, to 5-fluorouracil in tumors. Methods to estimate dThdPase expression in tumor tissue might be useful to predict the efficacy of capecitabine and doxifluridine in cancer patients. We established a new monoclonal antibody (MAb), 1C6-203, applicable for dThdPase immunohistochemistry and compared its staining characteristics with those of a previously established MAb, 654-1. In 4% paraformaldehyde-fixed colorectal carcinoma, 1C6-203 and 654-1 stained cancer cells in 19/30 and 9/30 patients, respectively. In 10% formalin-fixed colorectal carcinoma, 1C6-203 and 654-1 stained cancer cells in 18/30 and 6/30 patients, respectively. In negative 10% formalin-fixed tissues, microwave treatment improved the positivity of 654-1-stained cancer cells. These results suggest that an epitope recognized by 1C6-203 is resistant to epitope masking by formaldehyde fixation, whereas that for MAb 654-1 is sensitive. Therefore, MAb 1C6-203 might be more suitable than MAb 654-1 for evaluating dThdPase expression in colorectal carcinoma.

The role of thymidine phosphorylase expression in the invasiveness of gastric carcinoma.

BACKGROUND: Thymidine phosphorylase (TP) has angiogenic activity in various cancer tissues. Gastric carcinomas are classified into two histologic groups: differentiated and undifferentiated adenocarcinomas. There are differences in the modes of development and the extent of infiltration between the two groups. The purpose of the current study was to determine whether TP is involved in the invasiveness and progression of these two types of gastric carcinoma. METHODS: To investigate the expression and localization of TP and the microvessel counts, the authors examined specimens from 149 gastric carcinoma patients. The specimens were stained using monoclonal antibody against TP and polyclonal antibody against factor VIII. To determine the cell type expressing TP, immunohistochemical staining using a monoclonal antibody against CD68 that is specific for macrophages and double staining using antibodies to both TP and CD68 were performed. RESULTS: The proportion of TP positive tumors in differentiated adenocarcinomas was higher than that in undifferentiated adenocarcinomas. The TP positive differentiated adenocarcinomas invaded more deeply than the TP negative ones, but this was not the case with undifferentiated adenocarcinomas. TP was expressed mainly in the invasive edges of tumors and was expressed more frequently in macrophages than in tumor cells. TP expression was correlated with microvessel count and CD68 expression. Patients with TP positive carcinomas had a poorer prognosis than those with TP negative differentiated adenocarcinomas. CONCLUSIONS: TP expressed in macrophages may be correlated with microvessel count and play an important role in tumor invasiveness and progression in differentiated gastric adenocarcinoma.

Expression of the angiogenic protein, platelet-derived endothelial cell growth factor, in coronary atherosclerotic plaques: In vivo correlation of lesional microvessel density and constrictive vascular remodeling.

Recent information indicates that platelet-derived endothelial cell growth factor (PD-ECGF), a 45-kDa angiogenic protein, is expressed in the endothelium of various tissues and that its level of expression is correlated with the number of microvessels in human tumors. Because the formation of neovessels is also thought to play a role in atherosclerotic vascular remodeling, we analyzed PD-ECGF expression in fresh, coronary plaque tissues obtained by directional coronary atherectomy. Specimens from 31 patients were collected and analyzed by reverse transcription-polymerase chain reaction, histochemical staining, immunohistochemistry, and in situ hybridization with the use of PD-ECGF-specific primers and probes. Lesional vascular remodeling was assessed by intravascular ultrasound. PD-ECGF immunoreactivity and mRNA were found in plaque macrophages, endothelial cells of plaque neovessels, and stellate smooth muscle cells of 20 atherectomy specimens (64.5%). PD-ECGF immunoreactivity was correlated with the number of lesional microvessels and mast cells. Double-staining experiments revealed a close spatial proximity of PD-ECGF-positive cells and mast cells. Furthermore, the numbers of microvessels and mast cells were significantly higher in lesions lacking compensatory enlargement. The data indicate that PD-ECGF is expressed within cells of the atherosclerotic plaque and may be involved in driving angiogenesis in concert with mast cells.

Platelet-derived endothelial cell growth factor inhibits DNA synthesis in vascular smooth muscle cells.

Platelet-derived endothelial cell growth factor (PD-ECGF) is linked to angiogenesis in human cancer. Direct studies have demonstrated that PD-ECGF is a potent mitogen for endothelial cells in vivo. Because endothelial repair and smooth muscle cell proliferation are two processes that affect arterial wall structure and tone, we analyzed the effects of PD-ECGF on DNA synthesis and creatine kinase BB-specific activity (CK) in human umbilical artery smooth muscle cells (SMC) and in a human umbilical endothelial cell line (E304). In SMC, PD-ECGF (0.001 to 10 U/mL) inhibited DNA synthesis dose dependently (-24% + 6% to -63% + 15%) assessed by 3[H]thymidine incorporation into DNA, whereas in E304 it stimulated DNA synthesis dose dependently (30% + 4% to 100% + 4%). In both SMC and E304, however, PD-ECGF elicited an increase in CK-specific activity by 54% to 130% and 79% to 163%, respectively. These effects were reversed by a specific anti-PD-ECGF antibody. In E304 cells PD-ECGF enhanced 17beta-estradiol (E2) or dihydrotestosterone (DHT)-induced DNA synthesis from 56% to 122% and from 127% to 359%, and CK activity from 70% to 180% and from 90% to 190%, respectively. In SMC PD-ECGF, an inhibitor of 3[H]thymidine incorporation by itself, markedly enhanced the stimulatory effect of low concentrations of E2 and DHT on 3[H]thymidine incorporation. It also increased E2 and DHT CK induction from 40% to 140% and from 52% to 120%, respectively. In both E304 and SMC, PD-ECGF inhibited the proliferative and the CK-inducing effects of platelet-derived growth factor (PDGF) and immunoglobulin F1 (IGF1). Thus, PD-ECGF, an established growth promoter for endothelial cells, is a potent inhibitor of DNA synthesis in human arterial SMC. However, in both E304 endothelial cells and SMC, PD-ECGF enhances the stimulatory effect of low concentrations of gonadal steroids on 3[H]thymidine incorporation. PD-ECGF antagonizes PDGF- and IGF1-induced DNA synthesis in both E304 and SMC cells. By inhibiting arterial SMC proliferation and accelerating endothelial cell replication, PD-ECGF may buffer the effect of PDGF and favorably modulate arterial wall response to injury.

Prediction of response to 5'-deoxy-5-fluorouridine (5'-DFUR) in patients with inoperable advanced gastric cancer by immunostaining of thymidine phosphorylase/platelet-derived endothelial cell growth factor.

BACKGROUND: No reliable method is available for predicting the response to chemotherapy in patients with gastric cancer. The anticancer drug 5'-deoxy-5-fluorouridine (5'-DFUR) is converted into 5-fluorouracil (5-FU) by thymidine phosphorylase (dThdPase). We studied the relation between the expression of dThdPase in tumor tissue and the response to treatment with 5'-DFUR to determine if this enzyme can be used to predict the response to chemotherapy. METHODS AND MATERIALS: We performed endoscopic biopsy and studied the expression of dThdPase by immunostaining with anti-dThdPase monoclonal antibody in 41 patients with inoperable advanced gastric carcinomas before they received multiple-drug chemotherapy, including 5'-DFUR. The relation between the expression of dThdPase and the response to chemotherapy was studied. We also studied the characteristics of positive cells against anti-dThdPase monoclonal antibody. RESULTS: The response rate among patients whose tumors were positive for dThdPase expression (56.8%, 21/37) was higher than that among patients whose tumors were negative for dThdPase expression (0%, 0/4; p = 0.048). Spindle-shaped cells darkly expressing dThdPase were sporadically seen surrounding cancer nests in some patients. Multiple-antibody immunostaining suggested that these spindle-shaped cells were perivascular mesenchymal cells. The response rate was 82.4% (14/17) among patients with spindle-shaped cells strongly positive for dThdPase, as compared with 29.2% (7/24) among those with spindle-shaped cells weakly positive or negative for dThdPase (p = 0.00131). The expression of spindle-shaped cells darkly stained with dThdPase was the factor most strongly related to the response to chemotherapy (odds ratio = 35.513, p = 0.0027). CONCLUSIONS: The expression of dThdPase in stromal spindle-shaped cells may be a useful index in predicting the response to 5'-DFUR in patients with gastric cancer.

Immunohistochemical expression of thymidine phosphorylase in human endometrial cancer.

OBJECTIVE: To investigate correlations between the expression of thymidine phosphorylase (TP) by endometrial cancer cells and the density of microvessels within the tumor, the clinicopathologic features, and the prognosis. METHODS: We examined tumor specimens obtained from 46 patients with endometrial cancer (9 FIGO stage IA, 16 stage IB, 8 stage IC, 1 stage IIA, 6 stage IIB, and 6 stage IIIC). The cellular expression of TP and the intratumoral density of microvessels were determined by immunohistochemistry using monoclonal antibodies to TP and factor VIII-related antigen, respectively. We investigated the relationship between the cellular expression of TP and the following factors: clinicopathologic features (menopausal status, histologic type, tumor size, histologic grade, myometrial invasion, cervical invasion, and metastasis), the microvessel count, and the disease-free survival period. RESULTS: Of the 46 tumors, 19 (41%) were TP-positive. The microvessel count was significantly higher in TP-positive tumors than in TP-negative tumors (P = 0.01, Mann-Whitney U test). There was no significant correlation between TP expression and clinicopathologic features, and there was no significant difference in the disease-free survival period between patients with TP-positive tumors and patients with TP-negative tumors. CONCLUSION: TP expression was not correlated with clinicopathologic features or prognosis, but was associated with an increased density of microvessels in endometrial cancer. These findings suggest that TP may play an important role in angiogenesis and may be involved in the tumorigenesis of endometrial cancer.

Different angiogenic pathways in human cervical cancers.

OBJECTIVE: The objective of this study is to clarify the association between the expression of two types angiogenic factors, vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF)/thymidine phosphorylase(dThdPase) and clinicopathological features, including tumor angiogenesis, in cervical cancers. METHODS: The expression of VEGF and PD-ECGF was evaluated by immunohistochemical staining of tumor specimens from 73 patients with stage Ib-IIb cervical cancer (51, squamous cell carcinoma; 19, adenocarcinoma; 3, adenosquamous carcinoma) who underwent radical hysterectomy. The microvessel density was assessed by immunostaining for factor VIII-related antigen in the most neovascularized area. RESULTS: The microvessel density in adenocarcinomas was significantly higher than that in squamous cell carcinomas (P < 0.01). The intensity of VEGF expression in adenocarcinomas was significantly stronger than that in squamous cell carcinomas (P < 0.05). In contrast, the expression of PD-ECGF in squamous cell carcinomas was significantly higher than that in adenocarcinomas (P < 0.0001) and adenosquamous carcinomas (P < 0.01). There was an inverse relationship between VEGF expression and PD-ECGF expression among all patients studied (P < 0.001). The microvessel density was significantly correlated with the intensity of VEGF expression (P < 0.05). In contrast, there was no correlation between the microvessel density and the expression of PD-ECGF. CONCLUSIONS: The expression of VEGF appears to be involved in the promotion of angiogenesis in cervical cancers. Furthermore, we propose that angiogenic pathways may be different in different types of cervical cancers.

[Successful treatment of hepatic metastasis of gastric cancer with 5'-DFUR and Lentinan].

Satisfactory therapeutic effects are rarely obtained with oral chemotherapy for gastric cancer. We have experienced successful treatment for synchronous hepatic metastasis of gastric cancer with 5'-DFUR and Lentinan. The patient was a 78-year-old female, diagnosed as having gastric cancer with multiple hepatic metastases, who underwent gastrectomy. Immunohistochemistry of the resected specimens with anti-thymidine phosphorylase (dThdPase) antibody yielded positive results for dThdPase in the primary tumor as well as the hepatic metastases. Two months after surgery, administration of 400 mg of 5'-DFUR per day and 2 mg i.v. of Lentinan every other week was started. Four months after discharge, carcinoembryonic antigen (CEA) in plasma showed an abrupt logarithmic decline. Furthermore, a 99% reduction in hepatic metastases was demonstrated by abdominal CT. At present, 22 months after surgery, the patient is managed on an outpatient basis with no complaints of any side effects. Immunochemotherapy using 5'-DFUR and Lentinan may be effective against gastric malignancies expressing dThdPase activity.

Preparation of anti-human thymidine phosphorylase monoclonal antibodies useful for detecting the enzyme levels in tumor tissues.

The antitumor activity of cytostatic 5'-deoxy-5-fluorouridine (5'-dFUrd) depends on its being converted to 5-fluorouracil (5-FUra) by the enzyme thymidine phosphorylase (dThdPase, EC 2.42.4). We prepared mouse anti-human dThdPase monoclonal antibodies to serve as tools for clinical studies with this drug. Partially purified dThdPase obtained form HCT116 human colon cancer cells grown in athymic mice was used as and antigen for the immunization of mice. Six hybridomas were cloned which produced anti-human dThdPase antibodies, as detected by Western blot analysis with human dThdPase. With these antibodies, we developed an ELISA method sensitive enough to measure dThdPase levels, even in tumor tissue samples weighing as little as 10 mg. In addition, one monoclonal antibody was suitable for immunologically staining the enzyme in tumor tissues. Thus, these anti-human dThdPase monoclonal antibodies could be used to measure levels of the enzyme in tumor cells, which is essential for the activation of 5'-dFUrd.

Expression of thymidine phosphorylase in human gastric carcinoma.

The activity of thymidine phosphorylase (dThdPase) has been reported to increase in several types of malignant tumors. Experimental evidence has shown that dThdPase is identical to platelet-derived endothelial cell growth factor, and that dThdPase has angiogenic activity. We examined the expression of dThdPase to investigate whether the expression of dThdPase correlates with angiogenesis, clinicopathologic features and the prognosis of patients with human gastric carcinomas. Microvessels were assessed by immunostaining endothelial cells for factor VIII. We counted microvessels in the tumors of 158 patients whose tumors were completely removed surgically. Microvessels were counted in a x400 field in the most active areas of neovascularization. We purified a monoclonal antibody (TMA-1) against dThdPase and studied the expression of dThdPase using TMA-1 in the same serial sections as those used for the detection of factor VIII. The correlation between angiogenesis and dThdPase, and the clinicopathological significance of dThdPase, in patients with gastric carcinoma were examined. The positive expression of dThdPase was more frequent (P < 0.001) in gastric carcinomas (67/158, 43.4%) than that in normal tissues (12/158, 7.6%). The average microvessel count in dThdPase-positive gastric carcinomas was higher (P < 0.001) than that in dThdPase-negative carcinomas. The percentage of gastric carcinoma cells expressing dThdPase was significantly correlated with the microvessel count (P < 0.001). Further, the average size of dThdPase-positive carcinomas was significantly larger (P < 0.001) than that of negative carcinomas and the mean microvessel count in dThdPase-positive gastric carcinomas was also significantly higher (P < 0.001) than that in dThdPase-negative carcinomas. There was a significant correlation between the positive expression of dThdPase and microvessel count (P < 0.001) or lymph node metastasis (P = 0.013) by multivariate logistic analysis. Further, patients with dThdPase-positive carcinoma showed a significantly worse prognosis than those with dThdPase-negative carcinoma overall and in stage III. These findings indicate that the expression of dThdPase in gastric carcinomas is related to progression and metastasis, and this enzyme affects the prognosis of some patients with the disease.

Neurotrophic action of gliostatin on cortical neurons. Identity of gliostatin and platelet-derived endothelial cell growth factor.

Gliostatin is a polypeptide growth inhibitor of apparent M(r) = 100,000 with a homodimeric structure comprising two 50-kDa subunits, acting on astrocyte as well as astrocytoma cells (Asai, K., Hirano, T., Kaneko, S., Moriyama, A., Nakanishi, K., Isobe, I., Eksioglu, Y.Z., and Kato, T. (1992) J. Neurochem., 59, 307-317). The amino acid sequences of 13 tryptic peptides including the amino terminus were completely identical to those of platelet-derived endothelial cell growth factor (PD-ECGF) (Ishikawa, F., Miyazono, K., Hellman, U., Drexler, H., Wernstedt, C., Hagiwara, K., Usuki, K., Takaku, F., Risau, W., and Heldin, C.-H. (1989) Nature 338, 557-562). Gliostatin and PD-ECGF, purified from human placenta, shared growth inhibition on glial cells and growth promotion on endothelial cells, and exhibited similar values for half-maximal dose of glial growth inhibition (ID50 = 1.3 nM) and the half-maximal concentration of endothelial growth promotion (EC50 = 1.0 nM), suggesting that both factors evoke the biological actions through an identical receptor on each cell surface. We have further demonstrated evidence of a novel neurotrophic action of gliostatin/PD-ECGF toward embryonic rat cortical neurons in culture. The half-maximal concentration of gliostatin/PD-ECGF for neurotrophic action was 0.3 nM. All actions on glial, endothelial, and neuronal cells, were abolished by a monoclonal antibody against gliostatin. These data indicate that gliostatin/PD-ECGF may play important roles on development and regeneration of the central nervous system and may also involve the induction of angiogenesis for the formation of blood brain barrier.