[Characteristics of PD-L1 expression in tumor cells and tumor microenvironment of DLBCL with MYD88 L265P mutation].

Objective: To study the effect of MYD88 L265P mutation on the expression of PD-L1 in tumor cells and tumor microenvironment in diffuse large B-cell lymphoma (DLBCL), and to provide theoretical basis for immunotherapy for patients. Methods: Multiplex ligation-dependent probe amplification (MLPA) was used to detect the frequency of MYD88 L265P mutation in 72 cases of DLBCL diagnosed by pathologists in Cancer Hospital of Chinese Academy of Medical Sciences from August 2008 to May 2010. Expression of PD-L1 in tumor cells and tumor microenvironment in all samples was evaluated using PD-L1 (22C3) and PD-L1 (SP142) with Ventana automatic immunohistochemical (IHC) platform. The relationship between MYD88 L265P mutation and the expression of PD-L1 in DLBCL tumor cells and tumor microenvironment was assessed. Results: Of the 72 cases of DLBCL, MYD88 L265P mutation was detected in 15 (20.8%) cases. Nine cases with JAK2 amplification were excluded, and the remaining 63 cases of DLBCL were divided into MYD88 L265P mutant group (n=14) and MYD88 L265P wild-type group (n=49). IHC results showed that among the 14 cases of MYD88 L265P mutant groups, PD-L1 (22C3) was positive in 7 cases (7/14) of tumor cells and PD-L1 (SP142) was positive in 4 cases (4/14) of tumor microenvironment. Among the 49 cases of MYD88 L265P wild-type group, 9 cases (18.4%) were positive for PD-L1 (22C3) in tumor cells, and 38 cases (77.6%) were positive for PD-L1(SP142) in tumor microenvironment. In addition, among the 16 cases with PD-L1(22C3) expression in tumor cells, only 2 of the 7 cases with MYD88 L265P mutation were positive for PD-L1 (SP142) in tumor microenvironment. All 9 cases with wild-type MYD88 L265P were positive for PD-L1 (SP142) in tumor microenvironment. Statistical analysis showed that the expression level of PD-L1 (22C3) in tumor cells in the MYD88 L265P mutant group was significantly higher than that in the MYD88 L265P wild-type group (P=0.017). The expression level of PD-L1 (SP142) in tumor microenvironment in the MYD88 L265P mutant group was significantly lower than that in the MYD88 L265P wild-type group (P=0.001). Conclusions: MYD88 L265P mutation may play an important role in the regulation of PD-L1 expression in DLBCL tumor cells and tumor microenvironment. Further studies will provide a theoretical basis for immunotherapy of DLBCL patients with MYD88 L265P mutation.

Effects of long non-coding RNA URHC on proliferation, apoptosis and invasion of colorectal cancer cells.

The article "Effects of long non-coding RNA URHC on proliferation, apoptosis and invasion of colorectal cancer cells, by Z.-G. Gu, G.-H. Shen, J.-H. Lang, W.-X. Huang, Z.-H. Qian, J. Qiu, published in Eur Rev Med Pharmacol Sci 2018; 22 (6): 1658-1664-DOI: 10.26355/eurrev_201803_14577-PMID: 29630109" has been withdrawn from the authors. The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/14577.

Effects of long non-coding RNA URHC on proliferation, apoptosis and invasion of colorectal cancer cells.

OBJECTIVE: To investigate the effect of long non-coding RNA URHC on the proliferation, apoptosis and invasion of colorectal cancer cells. PATIENTS AND METHODS: The expression of lncRNA-URHC in tissues and cells was tested by Real-time quantitative PCR. The expression of lncRNA-URHC was down-regulated by RNA interference (siRNA). The Real-time quantitative polymerase chain reaction (PCR) method was used to detect the interference efficiency. Cell counting kit-8 (CCK-8), flow cytometry, and transwell were used to detect the effect of lncRNA-URHC on the proliferation, apoptosis and invasion of colorectal cancer cells. The effect of lncRNA-URHC on epithelial-mesenchymal transition (EMT)-related markers was detected by Western blot. RESULTS: LncRNA-URHC expression was significantly increased in colorectal cancer cells compared with normal cells, and the expression of lncRNA-URHC in colorectal cancer cells was higher than that in the normal cell. After down-regulated the expression of lncRNA-URHC, the proliferation and invasion of colorectal cancer cells were decreased, while cells apoptosis was promoted. Down-regulation of lncRNA-URHC could enhance the expression of E-cadherin and reduce the expression of N-cadherin, vimentin and snail. CONCLUSIONS: Down-regulation of lncRNA-URHC can inhibit the progression of colorectal cancer.

[Progress in diagnosis and treatment of radioactive iodine-refractory differentiated thyroid carcinoma].

The radioactive iodine-refractory differentiated thyroid carcinoma (RIR-DTC) is a complex process that involves multiple genetic changes and multiple signaling pathways.Radionuclide imaging, genomics and proteomics are effective to clarify the mechanism and helpful in clinical diagnosis and therapy.The treatment of RIR-DTC includes the removal of distant metastases, drug therapy, external radiotherapy and radiofrequency ablation.This review mainly focuses on the pathogenesis, diagnosis and treatment of RIR-DTC.

[Expressions and clinical significances of paired box gene 2 and cyclin D1 in advanced ovarian serous carcinoma].

Objective: To investigate the expressions and clinical significances of paired box gene 2 (Pax2) and cyclin D1 protein in advanced ovarian serous carcinoma. Methods: From January 2003 to December 2013, the pathologic tissues of 202 patients with advanced ovarian serous cancer (Ⅲ-Ⅳ) who underwent initial cytoreductive surgery were collected. The expressions of Pax2 and cyclin D1 protein were detected by immunohistochemistry in tissue microarray. The relationships of their expressions with the clinicopathological features and prognosis of the patients were analyzed. Results: The positive rate of Pax2 protein expression of the 202 patients with ovarian serous adenocarcinoma was 24.8% (50/202) and that of cyclin D1 was 25.2% (51/202). The expressions of Pax2 and cyclin D1 were not significantly related with age, clinical stage and pathological grade of ovarian serous adenocarcinoma patients (P>0.05). The median overall survival (OS) time of Pax2-negative patients was 53 months and the progression-free survival (PFS) time was 29 months. The median OS time of Pax2-positive patients was 66 months and PFS time was 33 months, the OS of Pax2-negative patients was significant different from that of Pax2-positive patients (χ(2)=4.06, P=0.04). The median PFS time of Pax2-negative patients was not significant different from that of Pax2-positive patients (χ(2)=2.43, P=0.11). The median OS time of cyclin D1-negative patients was 62 months and PFS time was 30 months. The median OS time of cyclin D1-positive patients was 48 months and PFS time was 22 months. The median OS time of cyclin D1-negative patients was significantly different from that of cyclin D1-positive patients (χ(2)=4.71, P=0.03), while the median PFS time of cyclin D1-negative patients was marginally different from that of cyclin D1-positive patients (χ(2)=0.59, P=0.41). Multivariate analysis showed that the expression of Pax2 was an independent factor of the prognosis for patients with ovarian serous adenocarcinoma (RR=0.597, 95% CI 0.371-0.962, P<0.034). Conclusion: The expressions of Pax2 and cyclin D1 are associated with the prognosis of patients with advanced ovarian serous adenocarcinoma while Pax2 is an independent prognostic factor.

[Comparison of paired box genes 8 and 2 expression in epithelium tissues and the related tumors].

Objective: To explore the expressional differences between paired box genes 2(Pax2) and 8 (Pax8) protein in different kinds of epitheliums and tumors, and to investigate the clinicopathologic significance. Methods: Expression levels of Pax2 and Pax8 protein were detected in 75 cases of different human epithelium tissues and 255 cases of different tumors on tissue microarray by immunohistochemistry. Results: Pax2 and Pax8 selectively expressed in different tissues. The positive rates of Pax8 protein expressed in the normal epithelium of the thyroid, urinary system and female reproductive system were 100% (2/2), 60.0% (3/5) and 76.9% (10/13), respectively. The positive rates of Pax2 expressed in the epithelium tissues of urinary system and the female reproductive system were 40.0% (2/5) and 38.5% (5/13) respectively. However, the expression of Pax2 protein was not detected in the normal thyroid epithelium. The positive rate of Pax8 protein expressing in the epithelium of reproductive system was significantly higher than that of Pax2 protein (P<0.05). The tumors derived from different tissues also expressed different levels of protein Pax2 and Pax8. The positive rates of Pax8 in renal cell carcinoma, thyroid carcinoma and endometrial adenocarcinoma were 65.2% (15/23), 66.7% (10/15) and 80.0% (4/5), respectively. The positive rates of Pax2 in renal cell carcinoma, thyroid carcinoma and endometrial adenocarcinoma were 34.8% (8/23), 13.3% (2/15) and 20.0% (1/5), respectively. The positive rates of Pax8 protein expressed in renal cell carcinoma, thyroid carcinoma and endometrial adenocarcinoma were significantly higher than those of Pax2 protein (P<0.05). The positive rates of Pax8 in ovarian serous carcinoma, endometrial carcinoma and clear cell carcinoma were 92.9% (26/28), 81.8% (9/11) and 82.4% (14/17), respectively. The positive rates of Pax2 in ovarian serous carcinoma, endometrial carcinoma and clear cell carcinoma were 28.6% (8/28), 9.1% (1/11) and 17.6% (3/17), respectively. The positive rates of Pax8 protein expressed in ovarian serous carcinoma, endometrial carcinoma and clear cell carcinomawere significantly higher than those of Pax2 protein (P<0.05). Conclusions: Pax2 and Pax8 are specifically expressed in female reproductive system and uritany system. However, the positive expression of Pax8 is superior to that of Pax2. The combined expression of Pax8 and Pax2 can be used in the differential diagnosis of epithelial tumors derived from different origins.

Clinical value of fluid bolus contrast flow meter during hysterosalpingography.

The purpose of this study was to investigate the clinical value of the fluid bolus contrast flow meter during hysterosalpingography. Hysterosalpingography information of 342 cases, which included a manual handset group of 213 cases and a bolus instrument group of 129 cases were reviewed. Comparative analysis was used to compare the two groups in order to assess the clinical adverse reactions, contrast agent reflux, and image quality. In the instrument bolus group compared with the manual handset group, the clinical adverse reactions decreased from 75.12 to 31.78% (P < 0.001); the backflow phenomenon of the contrast agent decreased from 13.62 to 3.10% (P < 0.01); and image quality significantly improved, with the A class film rate increasing from 54.46 to 68.99% (P < 0.01) and the C class film rate decreasing from 8.92 to 2.33% (P < 0.05). The use of a contrast bolus through the liquid inlet of the hysterosalpingography instrument can provide fully dynamic observation, reducing the contrast agent reflux and adverse reactions as well as improving the image quality and diagnostic accuracy. In addition, the medical staff is not subjected to radiographic radiation. Therefore, it is a safe and reliable imaging method.

Protein-protein interaction network and significant gene analysis of osteoporosis.

This study used DNA microarray data to identify differentially expressed genes of osteoporosis and provide useful information for treatments of the disease. We downloaded gene expression data of Osteoporosis GSE35956 from the Gene Expression Omnibus database, which included five normal and five osteoporosis samples. We then identified the differentially expressed genes between normal and disease samples using the R language software, and constructed the protein interaction network. DAVID was used to perform the biological process enrichment and KEGG pathway cluster analyses. We used the Cytoscape plug-in unit, Cluster ONE, to perform cluster module analysis to find hub proteins of the network module and to analyze their Gene Ontology (GO) functions. A total of 294 genes were found to be differentially expressed between normal and disease samples, which were used to construct the differential gene-protein interaction network. GO function analysis revealed that the genes' functions were mainly involved in the intracellular signaling cascade. KEGG pathway analysis suggested that the main metabolic pathways of these genes were those of cancer: the neurotrophin/T cell/Fc epsilon RI/B cell/ ErbB/p53 signaling pathway, the cell cycle pathway, and the chronic myeloid leukemia pathway. Screening analysis of hub proteins revealed that KRT18 had the highest hub degree. In conclusion, we found differentially expressed genes related to osteoporosis. GO biological process enrichment and KEGG pathway enrichment analyses identified significant osteoporosis genes and their molecular functions. Finally, module analysis of hub proteins in interaction networks showed that cell death was one of the main biological processes of osteoporosis genes.

Microwave irradiation is a useful tool for improving isolation of actinomycetes from soil.

Actinomycetes are an important source of novel, biologically active compounds. New methods need to be developed for isolating previously unknown actinomycetes from soil. The objective of this experiment was to study microwave irradiation of soil as a means for isolating previously unknown actinomycetes. Soil samples were collected at ten elevations between 800 and 3670 m on Taibai Mountain, Shaanxi Province, China. Moistened soil samples were irradiated at 120 W heating power (2450 MHz) for 3 min using a household microwave oven. Irradiation increased total actinomycete, streptomycete, and antagonistic actinomycete counts on three types of culture media. Irradiation also increased the number of culturable actinomycete isolates. Some actinomycete isolates were culturable only after the soil was irradiated, whereas other isolates could not be cultured after irradiation. Irradiation of soil from elevations > 3000 m increased actinomycete counts significantly but had little effect on the number of culturable actinomycete isolates. In contrast, irradiation of samples from elevations < 3000 m had relatively little effect on actinomycete counts, but significantly increased the number of culturable actinomycete isolates. We used 16S rDNA sequence analysis to identify 14 actinomycete isolates that were only culturable after irradiation. Microwave irradiation of soil was helpful for isolating Streptomyces spp., Nocardia spp., Streptosporangium spp., and Lentzea spp. Slightly more than 90% of the identified actinomycete species were biologically active. In conclusion, microwave irradiation is a useful tool for isolating biologically active actinomycetes from soil.

Interferon-inducible protein-10 as a marker to detect latent and active tuberculosis in rheumatoid arthritis.

SETTING: effective tuberculosis (TB) screening should be performed before anti-tumour necrosis factor alpha (TNF-α) treatment in rheumatoid arthritis (RA). The usefulness of the tuberculin skin test (TST) and QuantiFERON®-TB Gold (QFT-G) for detecting latent tuberculosis infection (LTBI) is limited. OBJECTIVE: we tested the diagnostic performance of interferon-gamma (IFN-γ) inducible protein 10 (IP-10) and IFN-γ for detecting LTBI in RA patients receiving anti-TNF-α treatment. DESIGN: IP-10 levels were determined by enzyme-linked immunosorbent assay in 56 RA patients and 18 active TB patients. TST was performed using the Mantoux method and QFT-G was performed by measuring IFN-γ levels in whole blood treated with TB-specific antigens. RESULTS: twenty-four (42.9%) TST-positive patients were defined as having LTBI. Significantly higher levels of baseline, early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) stimulated IP-10 were observed in active TB patients (median 209.9 pg/ml, 899.0 pg/ml and 880.2 pg/ml, respectively) and RA patients with LTBI (165.3 pg/ml, 904.4 pg/ml and 747.5 pg/ml, respectively), compared to those without LTBI (89.3 pg/ml, 579.4 pg/ml and 515.0 pg/ml, respectively). Baseline IP-10 has high sensitivity (83.3% and 100%) and medium specificity (67.9% and 59.6%), while ESAT-6-stimulated IP-10 has high sensitivity (87.5% and 100%) and specificity (85.7% and 71.2%) for detecting LTBI and TB. The performance of IP-10 is superior to IFN-γ for detecting LTBI (TST+) and active TB. CONCLUSION: IP-10 may be used for detecting LTBI and as a potential biomarker to identify active TB in RA patients receiving anti-TNF-α treatment.

Latent tuberculosis infection among close contacts of multidrug-resistant tuberculosis patients in central Taiwan.

SETTING: Both the tuberculin skin test (TST) and the QuantiFERON®-TB Gold In-Tube test (QFT-GIT) may be used to detect Mycobacterium tuberculosis infection. A positive reaction to either test can indicate latent tuberculosis infection (LTBI). These tests can be used to study the rate of infection in contacts of multidrug-resistant tuberculosis (MDR-TB) patients. OBJECTIVE: To evaluate the transmission status of MDR-TB patients in Taiwan by examining their close contacts and to compare the efficiency of TST and QFT-GIT. DESIGN: Chest radiographs, TST and QFT-GIT were performed in household contacts of confirmed MDR-TB patients to determine their infection status. RESULTS: A total of 78 close contacts of confirmed MDR-TB patients were included in the study. The majority of the MDR-TB patients were parents of the close contacts and lived in the same building; 46% of the subjects were TST-positive and 19% were QFT-GIT-positive, indicating LTBI that was likely to develop into active MDR-TB. There was a lack of consistency between TST and QFT-GIT results in subjects with previous bacille Calmette-Guérin vaccination. CONCLUSION: Household contacts of MDR-TB patients are likely to develop LTBI; thus, follow-up and monitoring are mandatory to provide treatment and reduce the occurrence of active infection.

Combining polymerase chain reaction restriction enzyme analysis with phenotypic characters for mycobacteria identification in Taiwan.

SETTING: Many hospitals use the fully-automated BACTEC 960 Mycobacteria Growth Indicator Tube (MGIT) system and acid-fast staining to detect acid-fast bacilli (AFB) in clinical specimens; however, labour-intensive biochemical methods are used for further mycobacterial species identification. OBJECTIVE: To develop a user-friendly algorithm for mycobacterial species identification from AFB smear-positive BACTEC tubes. DESIGN: AFB smear-positive BACTEC tubes were collected and mycobacteria were isolated and identified by biochemical methods. The tubes were subgrouped by rpoB duplex polymerase chain reaction restriction enzyme analysis (rpoB DPRA). The results were combined with key phenotypic characters of mycobacteria isolated from the tubes to develop a species identification algorithm with 16S rDNA sequencing of the isolate being used as the gold standard method. RESULTS: By rpoB DPRA, 441 AFB smear-positive BACTEC tubes were correctly subgrouped into 100 tubes containing Mycobacterium tuberculosis complex, 335 tubes containing non-tuberculous mycobacteria and six tubes containing both. A species identification algorithm was developed by combining the rpoB DPRA results of the tubes with growth rate, photoreactivity and two biochemical results of mycobacteria recovered from the tubes. CONCLUSION: This user-friendly algorithm can be used for mycobacterial species identification from AFB smear-positive BACTEC tubes.

Combining the Capilia TB assay with smear morphology for the identification of Mycobacterium tuberculosis complex.

SETTING: Many hospitals in Taiwan use the fully automated BACTEC Mycobacterial Growth Indicator Tube (MGIT) 960 system to identify mycobacteria in clinical specimens, while the labour-intensive BD ProbeTec ET (CTB) system or biochemical methods are used to identify Mycobacterium tuberculosis complex (MTC) in mycobacterially positive BACTEC cultures. OBJECTIVE: To evaluate whether the Capilia TB assay can be used to replace the BD ProbeTec ET (CTB) system or biochemical methods for identifying MTC in BACTEC cultures. DESIGN: Mycobacterially positive BACTEC cultures were collected and MTC in the cultures was identified using biochemical methods. MTC was identified using serpentine cording in smears, the Capilia TB assay or the BD ProbeTec ET (CTB) system, and the results were compared. RESULTS: Using 233 mycobacterially positive BACTEC cultures, the sensitivity and specificity of identification of the Capilia TB assay were respectively 96.9% and 98.6%, while those of the BD ProbeTec ET (CTB) system were respectively 99.4% and 97.3%. Combining the Capilia TB assay with serpentine cording in smears led to 100% specificity for intersected results and 100% sensitivity for combined results. CONCLUSION: The Capilia TB assay can be used to identify MTC in BACTEC cultures. By combining the assay with serpentine cording in smears, false-positives and -negatives may be reduced.

Autoantibodies against tumour suppressor protein p53 in pleural effusions of patients with tuberculosis pleurisy.

BACKGROUND: Autoantibodies against the p53 proteins (p53 Abs) can be detected in the serum, ascites, saliva and pleural effusions of various malignant patients. It is suggested that p53 Abs in pleural effusions might have some value for tumour diagnosis, prognosis or monitoring. The present study investigated the prevalence of p53 Abs in the pleural effusions of 90 patients with various diseases. METHODS: Patients with suspicious pleural effusions in chest film received thoracocentesis and their pleural effusions were collected. The presence of p53 Abs in effusion was detected by immunoblotting. Differences of p53 Abs with respect to the patient's age, gender, white blood cell count, lactate dehydrogenase, total proteins and adenosine deaminase scores were calculated by chi2-test. RESULTS: p53 Abs were detected in 14.4% (13/90) of our patients, with prevalences of 10.5% (6/57) and 21.2% (7/33) among patients with benign and malignant diseases, respectively. Notably, 16.1% (5/31) of patients with tuberculosis pleurisy were positive for p53 Abs. These five patients had no history of cancer and, so far, have had no manifestations related to tumorigenesis. CONCLUSIONS: As far as we know, this is the first report regarding the detection of p53 Abs in pleural effusions from patients with tuberculosis pleurisy.

Mycobacterium avium complex-associated hemophagocytic syndrome in systemic lupus erythematosus patient: report of one case.

Hemophagocytic syndrome (HPS) in systemic lupus erythematosus(SLE) patients has not commonly been reported. In this case study, we report the first case of Mycobacterium avium complex (MAC)-associated hemophagocytic syndrome in a patient with systemic lupus erythematosus (SLE). This SLE patient, a 15-year-old girl, had been on a high dose of prednisolone (> 0.5mg/kg/day) for more than 3 years. She presented with a spiking fever, hepatosplenomegaly, pancytopenia, hyperferritinemia and adult respiratory distress syndrome. Bone marrow examination revealed hemophagocytosis as well as non-caseating granulomatosis. There was no indication of SLE fare-up. She responded poorly to initial treatment with methyl-prednisolone, intravenous immumoglobulin, etoposide, and drugs for Mycobacterium tuberculosis including rifampin, ethambutol, isoniazid and pyramide. However, gastric lavage culture revealed MAC. Following treatment with clarithromycin, ciprofloxacin and amikacin, her condition gradually improved and she was discharged 3 months after admission. In SLE patients with pancytopenia and hyperferritinemia, MAC-associated HPS should be considered in the differential diagnosis.

Prognostic significance of vascular endothelial growth factor expression in human ovarian carcinoma.

The influence of vascular endothelial growth factor (VEGF) expression and microvessel density (MVD) on prognosis and the relationship between VEGF expression and MVD in ovarian carcinoma are not well defined. We studied VEGF expression in parallel with MVD by immunohistochemistry in 94 ovarian tumours (64 malignant, 13 borderline, and 17 benign) and correlated the results with the clinicopathologic prognostic factors of the disease to clarify their significance in this disease. Assessment of VEGF mRNA isoforms by RT-PCR was also performed. Of the malignant, borderline, and benign ovarian tumours respectively, two (3%), four (31%) and 16 (94%) were negative, 31 (48%), seven (54%) and one (6%) had low expressions, and 31 (48%), two (15%) and none (0%) had high expressions of VEGF. There were significant associations between the VEGF expression and disease stage (P= 0.002), histologic grade (P= 0.0004), and patient outcome (P= 0.0002). MVD did not correlate significantly with the clinicopathologic parameters. Likewise, no correlation was found between MVD and VEGF expression. The survival of patients with high VEGF expression was significantly worse than that of patients with low and negative VEGF expression (P = 0.0004). Multivariate analysis revealed that disease stage and VEGF expression were significant and independent prognostic indicators of overall survival time (P = 0.008 and P = 0.006 respectively). These findings suggest that in conjunction with the established clinicopathologic prognostic parameters of ovarian carcinoma, VEGF expression may enhance the predictability of patients at high risk for tumour progression who are potential candidates for further aggressive therapy.

B7.1 expression by the weakly immunogenic F98 rat glioma does not enhance immunogenicity.

Enhanced immunogenicity has been reported following transfection of a variety of immunogenic tumors with the B7.1 co-stimulatory molecule. The purpose of the present study was to determine if transfection of a weakly immunogenic rat brain tumor, the F98 glioma, with the gene encoding B7.1 could enhance its immunogenicity. F98 cells were transfected with a plasmid containing the B7.1 gene, and stable transfectants (F98/B7.1) were obtained. Flow cytometric analysis confirmed the expression of B7.1 and MHC class I antigens on the cell surface. To investigate the effects of B7.1 expression on the tumorigenicity of the F98 glioma, Fischer rats were implanted intracerebrally with either F98 (wild-type) or F98/B7.1 transfected cells. No significant differences in survival times were noted. Mean survival times of 21.8 and 24.0 days were observed for the respective groups at a challenge dose of 103 cells. These differences in survival time were not significant. To determine if expression of B7.1 enhanced the immunogenicity of the F98 glioma, rats were vaccinated weekly for 3 weeks with 107 mitomycin C-treated F98 or F98/B7.1 cells injected subcutaneously and then challenged intracerebrally with F98 cells 1 week later. Unvaccinated animals or those that received wild-type F98 cells as a vaccine had a survival time (mean +/- s.d.) of 22.3 +/- 1.5 days following tumor challenge versus 20.0 +/- 1.7 days for rats that had been vaccinated with F98/B7.1. Although we recognize that it might be possible to design more effective vaccination regimes, nevertheless, our data indicate that transfection of the B7.1 gene into the F98 rat glioma did not enhance its immunogenicity, and that other approaches will be required.

Neuropeptide Y-mediated pressor responses following high-frequency stimulation of the rat sympathetic nervous system.

Neuropeptide Y (NPY) is a potent pressor agent that is stored in the sympathetic nerves. In several species, NPY release is augmented when sympathetic impulse frequencies increase. We investigated the extent to which NPY contributes to the pressor response to high- and low-frequency electrical stimulation. Rats were pithed, and the sympathetic trunk was stimulated at either 20 or 3 Hz in the presence or absence of antagonists of NPY and alpha and beta adrenergic receptors. The 20-Hz stimulation raised plasma NPY levels, but the 3-Hz stimulation did not. The 20-Hz stimulation caused marked pressor responses that were maintained for several minutes after the end of stimulation regardless of whether rats were pretreated with adrenergic blockers. The NPY antagonists BIBP 3226 and 1229U91 reduced the size of the pressor response that followed 20 Hz stimulation by >50%. The rapid blood pressure spikes that occur during electrical stimulation are attenuated by alpha adrenergic but not by NPY antagonists. There is a prolonged pressor response after high-frequency stimulation of the sympathetic trunk in pithed rats that begins after 1 to 2 min of stimulation and lasts approximately 10 min after the end of stimulation. At least half of this pressor response is mediated by NPY.

Dual role of protein kinase C in the regulation of cPLA2-mediated arachidonic acid release by P2U receptors in MDCK-D1 cells: involvement of MAP kinase-dependent and -independent pathways.

Defining the mechanism for regulation of arachidonic acid (AA) release is important for understanding cellular production of AA metabolites, such as prostaglandins and leukotrienes. Here we have investigated the differential roles of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase in the regulation of cytosolic phospholipase A2 (cPLA2)-mediated AA release by P2U-purinergic receptors in MDCK-D1 cells. Treatment of cells with the P2U receptor agonists ATP and UTP increased PLA2 activity in subsequently prepared cell lysates. PLA2 activity was inhibited by the cPLA2 inhibitor AACOCF3, as was AA release in intact cells. Increased PLA2 activity was recovered in anti-cPLA2 immunoprecipitates of lysates derived from nucleotide-treated cells, and was lost from the immunodepleted lysates. Thus, cPLA2 is responsible for AA release by P2U receptors in MDCK-D1 cells. P2U receptors also activated MAP kinase. This activation was PKC-dependent since phorbol 12-myristate 13-acetate (PMA) promoted down-regulation of PKC-eliminated MAP kinase activation by ATP or UTP. Treatment of cells with the MAP kinase cascade inhibitor PD098059, the PKC inhibitor GF109203X, or down-regulation of PKC by PMA treatment, all suppressed AA release promoted by ATP or UTP, suggesting that both MAP kinase and PKC are involved in the regulation of cPLA2 by P2U receptors. Differential effects of GF109203X on cPLA2-mediated AA release and MAP kinase activation, however, were observed: at low concentrations, GF109203X inhibited AA release promoted by ATP, UTP, or PMA without affecting MAP kinase activation. Since GF109203X is more selective for PKCalpha, PKCalpha may act independently of MAP kinase to regulate cPLA2 in MDCK-D1 cells. This conclusion is further supported by data showing that PMA-promoted AA release, but not MAP kinase activation, was suppressed in cells in which PKCalpha expression was decreased by antisense transfection. Based on these data, we propose a model whereby both MAP kinase and PKC are required for cPLA2-mediated AA release by P2U receptors in MDCK-D1 cells. PKC plays a dual role in this process through the utilization of different isoforms: PKCalpha regulates cPLA2-mediated AA release independently of MAP kinase, while other PKC isoforms act through MAP kinase activation. This model contrasts with our recently demonstrated mechanism (J. Clin. Invest. 99:1302-1310.) whereby alpha1-adrenergic receptors in the same cell type regulate cPLA2-mediated AA release only through sequential activation of PKC and MAP kinase.