Effects of CpG ODN on dendritic cells in anti-hepatocarcinoma action

AIM: To study the effect of oligonucleotides containing unmethylated CpG motif (CpG ODN) on mouse bone marrow-derived dendritic cells (BMDCs) in anti-HcaF cytotoxicity. METHODS: BMDCs were stimulated by CpG ODN in combination with tumor antigen (TAg). The expression of CD80 on BMDC surface was analyzed by FCAS. Level of IL-12 (p70) in supernatants of BMDC culture was detected by ELISA. The proliferation of T cells was examined by MTT assay. Cytotoxicity of CTL induced by CpG ODN combining with TAg was detected by MTT assay. RESULTS: CpG ODN combining or not combining with TAg up-regulated the expression of CD80 on BMDC surface and stimulated BMDCs to produce a high level of IL-12. CpG ODN-activated BMDC promoted the proliferation of T cells. CTL induced by CpG ODN in combination with TAg appeared strong specific cytotoxicity on Hca-F cells. CONCLUSION: CpG ODN may effectively induce the functional maturation of mouse BMDC in vitro. CpG ODN in combination with TAg can enhance the anti-HcaF cytotoxicity of CTL.     

微量乳酸脱氢酶释放法检测NK细胞活性的几个主要影响因素

对乳酸脱氢酶释放法测定小鼠脾脏 NK细胞活性中的酶促反应时间、效靶细胞比例、检测波长、细胞毒试验孵育时间、维持液中小牛血清 ( FCS)浓度等 5个重要的影响因素进行了探索。结果显示 ,酶促反应时间以 1 0 min最佳 ;效靶细胞比例以 1 5∶ 1为适宜 ;最适检测波长为 570 nm,其结果线性关系好 ;细胞毒试验孵育时间以 2 h为宜 ;维持液中小牛血清以低浓度为宜 ,其结果更接近自然杀伤百分率计算公式 ,并选用 2 %为本试验小牛血清的体积分数     

Antifollicular cell-mediated and humoral immunity in canine alopecia areata

Abstract A combination of cellular and humoral immunological assaults directed against follicular matrical cells is suspected to cause alopecia areata (AA) in humans. The specific aims of this study were to determine whether cytotoxic T lymphocytes (CTL) were increased in the blood and skin lesions of canine AA. Additionally, we wished to determine if circulating antifollicular antibodies were present in the dog. Finally, we aimed to investigate whether bulbar inflammation was associated with decreased matrical cell proliferation and keratinocyte apoptosis. Canine AA lesions were infiltrated by intrabulbar CTL, perifollicular helper T cells and dendritic cells. A higher percentage of CTL was present in canine AA peripheral blood compared with that of normal dogs. Tissue-fixed and circulating antifollicular IgG antibodies were detected. Reduced matrical proliferation and increased keratinocyte apoptosis was observed in inflamed hair bulbs. This study demonstrates the existence of antifollicular cell-mediated and humoral immunological responses in canine AA. Résumé— L'association d'agressions immunologiques cellulaires et humorales dirigées contre les cellules matricielles folliculaires est suspectée d'être à l'origine de l'Alopécie Areata chez l'homme. Le but de cette étude était de montrer si le nombre de lymphocytes T cytotoxiques est augmenté dans le sang et dans les lésions cutanées lors d'Alopécie Areata chez le chien. En plus, nous souhaitions montrer la présence d'anticorps circulants antifolliculaire chez le chien. Enfin, nous voulions voir si l'inflammation du bulbe pileux était associée à une diminution de la prolifération des cellules matricielles et une apoptose des kératinocytes. Lors d'Alopécie Areata chez le chien, existe une infiltration lésionnelle par des lymphocytes T cytotoxiques intrabulbaires, des lymphocytes T auxiliaires périfolliculaires et des cellules dentritiques. Le pourcentage de lymphocytes T cytotoxiques était plus important dans le sang périphérique provenant de sujets à Alopécie Areata que dans le sang d'animaux sains. Par ailleurs, des anticorps antifolliculaire (IgG) circulants et fixés dans les tissus sont détectés. Une réduction de la prolifération des cellules matricielles et une augmentation de l'apoptose des kératinocytes sont observées dans les bulbes pileux atteints. Cette étude démontre l'existence de réponses immunologiques cellulaires et humorales contre le follicule pileux dans l'Alopécie Areata du chien. [Olivry, T., Moore, P.F., Naydan, D.K., Puget, B.J., Affolter, V.K., Kline, A.E. Antifollicular cell-mediated and humoral immunity in canine alopecia areata (Immunité contre le follicule pileux dans l'Alopécie Areata du chien). Veterinary Dermatology 1996; 7 : 67–79.] Resumen Se cree que la alopecia areata (AA) en la especie humana resulta de la combinación de la acción inmunológica celular y humoral contra células de la matriz folicular. Los objetivos especificos de este estudio fueron determinar si los linfocitos T citotóxicos (CTL) se encontraban aumentados en sangre y en las lesiones cutáneas de animales con AA. Además, descamos investigar la posible presencia de anticuerpos antifoliculares circulantes en el perro. Finalmente, nos propusimos investigar si la inflamación bulbar se encontraba asociada a una disminución en la proliferación de células de la matriz y a la apoptosis de queratinocitos. Las lesiones de la AA canina mostraban infiltración por CTL intrabulbares, células T colaboradoras y células dendríticas. Se encontró un porcentaje mayor de CTL en la sangre periférica de perros con AA respecto a perros normales. Se detectaron anticuerpos IgG antifoliculares circulantes y en tejido fijado. Se observó una disminución en la proliferación matrical y un aumento en queratinocitos apoptóticos en los bulbos foliculares inflamados. Este estudio demuestra la existencia de respuestas inmunitarias antifoliculares de tipo celular y humoral en la AA canina. [Olivry, T., Moore, P.F., Naydan, D.K., Puget, B.J., Affolter, V.K., Kline, A.E. Antifollicular cell-mediated and humoral immunity in canine alopecia areata (Immunidad antifolicular en la alopecia areata canina). Veterinary Dermatology 1996; 7 : 67–79.] Zusammenfassung— Eine Kombinaton von zellulären und humoralen immunologischen Angriffen, die gegen die follikulären Matrixzellen gerichtet sind, wird als Ursache bei der Alopecia areata (AA) des Menschen vermutet. Die besondere Absicht dieser Studie war, festzustellen, ob die zytotoxischen T-Lymphozyten (CTL) im Blut und in den Hautveränderungen bei kaniner AA erhöht sind. Zusätzlich wollten wir feststellen, ob beim Hund zirkulierende antifollikuläre Antikörper vorkommen. Schließlich beabsichtigten wir, zu untersuchen, ob die bulbäre Entzündung mit verminderter Matrixzellproliferation und Keratinozytenapoptose einhergeht. Die Veränderungen bei kaniner AA wurden von intrabulbären CTL, perifollikulären T-Helferzellen und dendritischen Zellen infiltriert. Verglichen mit dem peripheren Blut gesunder Hunde kam bei kaniner AA eine höherer Prozentsatz an CTL vor. Es wurden gewebsfixierte und zirkulierende antifollikuläre IgG-Antikörper festgestellt. In entzündeten Haarbulbi wurde eine reduzierte Matrixproliferation und eine erhöhte Keratinozytenapoptose beobachtet. Diese Studie zeigt die Existenz von antifollikulären zellvermittelten und humoralen immunologischen Reaktionen bei kaniner AA. [Olivry, T., Moore, P. F., Naydan, D. K., Puget, B. J., Affolter, V. K., Kline, A. E. Antifollicular immunity in canine alopecia areata (Antifollikuläre Immunität bei der Alopecia areata des Hundes). Veterinary Dermatology 1996; 7 : 67–79.]     

4种因素对NK细胞杀伤活性的影响

为了探讨效应细胞与靶细胞的比例(效靶比)、孵育时间、显色时间以及小牛血清浓度对NK细胞杀伤活性的影响,采用乳酸脱氢酶释放法来测定不同因素对NK细胞杀伤活性的影响,结果表明,最佳效靶比和孵育时间分别为50∶1和2 h。在最优效靶比及孵育时间的基础上,显色0.5 h,小牛血清浓度为50 mL/L时NK细胞活性最高,证明4种因素对NK细胞杀伤活性均有不同程序的影响。     

Cloning and Sequencing of cnf1 from Escherichia coli Incriminated in Mink and Bovine Colibacillosis

Colibacillosis is responsible for significant losses to the mink and cattle industries. Previous work in our laboratory and by others has suggested that possession of cnf1, the gene encoding cytotoxic necrotizing factor (CNF1), may contribute to the virulence of isolates of E. coli from mink and cattle. The cnf1 gene from E. coli isolated from a mink with colisepticaemia and a bovid with scours was amplified and cloned as a 3.5 kb fragment, and the fragment was sequenced. The cnf1 sequences from the mink and bovine isolates of E. coli were compared to each other and to cnf1 sequences of E. coli from urinary tract and diarrhoea-associated infections of humans. The difference was only 7 nucleotides between the cnf1 sequences of the mink and bovine isolates of E. coli, which translated into 7 differences in amino acids. The cnf1 sequence of the mink isolate of E. coli had 15 nucleotide differences from the cnf1 sequences of the human isolate of E. coli (GenBank X70670), which translated into 11 differences in amino acids between these proteins. The cnf1 sequence of the bovine isolate of E. coli had 14 nucleotide differences from the cnf1 sequence of the human isolate of E. coli (GenBank X70670), which translated into 10 differences in amino acids between these proteins. The highly conserved sequences of the amino acids of CNF1 proteins make them a promising target for detection and control of the CNF1-producing E. coli involved in disease among various host species.     

Zorrimidazolone, a bioactive alkaloid from the non-indigenous mediterranean stolidobranch Polyandrocarpa zorritensis

Chemical analysis of the Mediterranean ascidian Polyandrocarpa zorritensis (Van Name 1931) resulted in the isolation of a series of molecules including two monoindole alkaloids, 3-indolylglyoxylic acid (3) and its methyl ester (4), 4-hydroxy-3-methoxyphenylglyoxylic acid methyl ester (1) and a new alkaloid we named zorrimidazolone (2). The structure of the novel compound 2 has been elucidated by spectroscopic analysis and bioactivity of all compounds has been investigated. Zorrimidazolone (2) showed a modest cytotoxic activity against C6 rat glioma cell line.     

Bioactive cembranoids from the dongsha atoll soft coral Sarcophyton crassocaule

Seven new cembranoids, sarcocrassocolides F-L (1-7), have been isolated from a soft coral Sarcophyton crassocaule. Their structures were determined by extensive spectroscopic analysis. Most new compounds exhibited significant cytotoxic activity against a limited panel of cancer cell lines, and the structure-activity relationship was studied. Compounds 1-7 were found to display significant in vitro anti-inflammatory activity in LPS-stimulated RAW264.7 macrophage cells by inhibiting the expression of the iNOS protein. Compound 4 was also found to effectively reduce the level of COX-2 protein.     

Differential cytotoxicity of the isolated protein fraction of Escumite bean (Phaseolus acutifolius)

The Escumite bean (Phaseolus acutifolius) is an edible legume which in raw form is highly toxic to rats. Proteins were separated by DEAE cellulose and affinity chromatography. Hemagglutinating activity, trypsin inhibitory effect, cytotoxicity on human peripheral lymphocytes and on intestinal epithelial cells of rats, and mitogenic activity were assayed with each protein fraction. Hemagglutinins and trypsin inhibitory fractions showed differential toxicity with lymphocytes as compared to intestinal epithelial cells. A protein fraction without the previous activities was cytotoxic mainly to intestinal epithelial cells.     

Polyoxygenated Steroids from the Octocoral Leptogorgia punicea and in Vitro Evaluation of Their Cytotoxic Activity

Five new polyoxygenated marine steroids—punicinols A–E (1–5)—were isolated from the gorgonian Leptogorgia punicea and characterized by spectroscopic methods (IR, MS, ¹H, ¹³C and 2-D NMR). The five compounds induced in vitro cytotoxic effects against lung cancer A549 cells, while punicinols A and B were the most active, with IC₅₀ values of 9.7 μM and 9.6 μM, respectively. The synergistic effects of these compounds with paclitaxel, as well as their effects on cell cycle distribution and their performance in the clonogenic assay, were also evaluated. Both compounds demonstrated significant synergistic effects with paclitaxel.     

Bioactive Cembranoids,Sarcocrassocolides P–R,from the Dongsha Atoll Soft Coral Sarcophyton crassocaule

New cembranoids, sarcocrassocolides P–R (1–3) and four known compounds (4–7) were isolated from the soft coral Sarcophyton crassocaule. The structures of the metabolites were determined by extensive spectroscopic analysis. Compounds 3–5 and 7 were shown to exhibit cytotoxicity toward a limited panel of cancer cell lines and all compounds 1–7 displayed potent in vitro anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells by inhibiting the expression of inducible nitric oxide synthase (iNOS) protein. Compound 7 also showed significant activity in reducing the accumulation of cyclooxygenase-2 (COX-2) protein in the same macrophage cells.