Effects of endothelial cells in apical periodontitis on nitric oxide production by polymorphonuclear leukocytes

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1) Samples:Surgically removed periapical lesions were examined by hematoxylin-eosin stains. Sixteen of 30 samples were apical cyst, whereas 14 were apical glanuloma.2) iNOS immunohistochemistry:Human specific iNOS anti-sera were purified from rabbits immunized with iNOS specific synthesized peptide. Immunohistochemistry using iNOS anti-sera was performed for cryostat sections of apical cyst. Endothelial cells, epithelial cells, fibroblasts and macrophages were iNOS positive. Lymphocytes and polymorphonuclear leukocytes also showed iNOS positive.3) Double immunohistochemistry of iNOS and IL-1β:Some iNOS-positive cells showed IL-1β positive, and some did not. On the other hand, some IL-1β-positive cells showed iNOS positive, and some did not. The data revealed that autocrine and paracrine effects of IL-1β on iNOS production. In addition, IL-1β-positive cells were present adjacent to iNOS-positive cells.4) Detection of iNOS mRNA:Human umbilical vein endothelial cells (HUVEC) were co-cultured with peripheral blood polymorphonuclear leukocytes (PB-PMNs) stimulated with lipopolysaccharide and IL-1β. HUVEC were harvested, and RNA was extracted using TRIsol. The RNA was reverse-transcribed into complementary DNA (cDNA). Human iNOS mRNA expression was then amplified by polymerase chain reaction using cDNA, human iNOS specific PCR primers and DNA polymerase. Amplified PCR products were fractionated using 1.7% agarose gel with ethidium bromide and were visualized on UV light.5) Southern hybridization:After the PCR products were loaded on agarose gels, the fractionated message expression was transferred to nylon membrane. Digoxigenin-labeled iNOS-specific probes were hybridized to the DNA. Autoradiography was developed on Kodak x-ray analysis films. The intensity was recorded using a densitometer. Specificity of RT-PCR was then confirmed.