The kinetics of cytokine production and CD25 expression by porcine lymphocyte subpopulations follow

When recommending books at a bookstore, its essential to consider the interests and preferences of the reader. Here are a few suggestions spanning various genres that are sure to cater to a diverse audience:1. Fiction: For those who enjoy a good story, To Kill a Mockingbird by Harper Lee is a timeless classic that explores themes of racial injustice and moral growth. Its rich narrative and memorable characters make it a mustread.2. Science Fiction: Fans of the genre might appreciate Dune by Frank Herbert. This epic tale of political intrigue, environmental challenges, and human evolution is a masterpiece that has captivated readers for decades.3. Mystery/Thriller: For those who cant put down a pageturner, Gone Girl by Gillian Flynn offers a gripping story of deceit, obsession, and the dark side of relationships.4. NonFiction: Sapiens: A Brief History of Humankind by Yuval Noah Harari provides an engaging and thoughtprovoking exploration of the history of our species, from the emergence of Homo sapiens in Africa to the present day.5. Biography/Autobiography: Becoming by Michelle Obama is an inspiring memoir that offers insight into the life of the former First Lady, her journey, and her reflections on the experiences that have shaped her.6. Selfhelp: The 7 Habits of Highly Effective People by Stephen R. Covey is a perennial favorite that offers practical advice on personal development and effectiveness.7. Young Adult: The Hunger Games by Suzanne Collins is a dystopian novel that has resonated with young readers for its themes of survival, rebellion, and the human spirit.8. Fantasy: Harry Potter series by J.K. Rowling is a staple for fantasy lovers, offering a magical world of witches and wizards that has captured the imagination of readers of all ages.9. Historical Fiction: All the Light We Cannot See by Anthony Doerr is a beautifully written novel that tells the parallel stories of a blind French girl and a German boy during World War II.10. Poetry: For those who appreciate the power of verse, The Waste Land by T.S. Eliot isa modernist masterpiece that explores themes of disillusionment and cultural collapse.When recommending books, its also important to consider the readers current mood or what they hope to gain from their reading experience. Whether theyre looking for escapism, knowledge, or inspiration, theres a book out there to suit every need.。

BD Biosciences1-2900 Argentia RoadMississauga, ON • L5N 7X9 • CANADALocal Tel: (905) 542-8028 • Toll-Free Tel: (888) 259-0187Local Fax: (905) 542-9391 • Toll-Free Fax: (888) 229-9918Cytokine ELISAIntroductionDue to the amplifying potential of enzyme labels, immunoassays which utilize enzyme-conjugated antibodies have become increasingly popular because of their high specificity and sensitivity.1 In 1971, Engvall and Perlmann2 coined the term “enzyme-linked immunosorbent assay” which is perhaps better known by the acronym, “ELISA”, to describe an enzyme-based immunoassay method which is useful for measuring antigen concentrations.Cytokine sandwich ELISA are sensitive enzyme immunoassays that can specifically detect and quantitate the concentration of soluble cytokine and chemokine proteins. The basic cytokine sandwich ELISA method makes use of highly-purified anti-cytokine antibodies (capture antibodies) which are noncovalently adsorbed (“coated” – primarily as a result of hydrophobic interactions) onto plastic microwell plates. After plate washings, the immobilized antibodies serve to specifically capture soluble cytokine proteins present in samples which were applied to the plate. After washing away unbound material, the captured cytokine proteins are detected by biotin-conjugated anti-cytokine antibodies (detection antibodies) followed by an enzyme-labeled avidin or streptavidin stage. Following the addition of a chromogenic substrate-containing solution, the level of coloured product generated by the bound, enzyme-linked detection reagents can be conveniently measured spectrophotometrically using an ELISA-plate reader at an appropriate optical density (OD). Data storage and reanalysis are greatly simplified when the plate reader is connected to a computer.By including serial dilutions of a standard cytokine protein solution of known concentration, the sandwich ELISA supports the development of standard curves. Standard curves (aka“calibration curves”) are generally plotted as the standard cytokine protein concentration (typically ng or pg of cytokine/ml) versus the corresponding mean OD value of replicates. The concentrations of the putative cytokine-containing samples can be interpolated from the standard curve. This process is made easier by using an ELISA computer software program.3 Generally, it is useful to perform a dilution series of the unknown samples to be assured that the OD will fall within the linear portion of the standard curve. Depending on the nature of the ELISA reagents used, investigators may choose to apply different curve fit analysis to their data, including either linear-log, log-log, or four-parameter transformations.1,4,5Although opinions differ, one convention for determining the ELISA sensitivity isto choose the lowest cytokine concentration that gives a signal which is at leasttwo or three standard deviations above the mean background signal value.6,7Because of the enzyme-mediated amplification of the detection antibody signal,the sandwich ELISA can measure physiologically relevant (i.e., > 5-10 pg/ml)concentrations of specific cytokine and chemokine proteins, which are present inmixed cytokine milieus, e.g., from stimulated lymphocyte culture supernatants.Although many different types of enzymes have been used, horseradishperoxidase (HRP) and alkaline phosphatase (AKP) are the enzymes that are oftenemployed in ELISA methods.1,8Application NotesCytokine sandwich ELISA are exquisitely specific because antibodies directedagainst two or more distinct epitopes are required.9 Therefore, sandwich ELISAcan discriminate between cytokines that can have overlapping biologicalfunctions which are not resolvable in a bioassay. Although cytokine sandwichELISA are very useful for cytokine detection and measurement, several limitationsfor the interpretation of ELISA data must be mentioned.9 For example, becausetest samples often come from tissue culture supernatants or biological fluidswhich are conditioned with cytokines produced by mixed cell populations, theELISA data does not provide direct information on the identities and frequenciesof individual cytokine producing cells. Techniques such as the"Immunofluorescent Staining of Intracellular Cytokines" are required for thislatter type of analysis.Several key issues need to be considered when designing experiments that involvecytokine and chemokine protein measurements using sandwich ELISA. Forinstance, it is well known that cytokine production by stimulated cell populationsis transient and that the kinetics of expression of different cytokine genes canvary. For these reasons, it may therefore be necessary to collect test samples atseveral time points to better characterize cytokine-production by an experimentalanimal or by a cultured cell population. As an example, in the case of stimulated mouse CD4+ T cell populations, the levels of IL-2 produced are detected relativelyearly after stimulation whereas the accumulated levels of IL-5 protein rise later inculture.10It should also be noted that cytokine production can be stimulus- and cell subset-dependent. For example, in the case of T cells, it is well known that naive T cellshave a limited cytokine production capability (i.e., primarily can produce IL-2)whereas memory T cells can produce high levels and different types of cytokineproteins including IFN-g and IL-4, as well as IL-2. 11,12 Moreover, T cell subsetshave been found to produce cytokines differentially in response to differentstimuli.12,13 Another consideration is that cytokine protein concentrations,measured at any one time point, may reflect the concurrent processes of cytokinesecretion, cytokine uptake by cells and cytokine protein degradation. Because ofthese processes, the measured level of cytokine protein may significantlyunderestimate the actual cytokine-producing potential of cells. In these cases, itmay be necessary to use complementary techniques such as multi-proberibonuclease protection assay analysis or immunofluorescent intracellular cytokinestaining with flow cytometric analysis to gauge the relative levels of cytokineexpression by various test cell populations.The levels of immunoreactive cytokine proteins detected by ELISA may or may notcorrelate directly with the levels of bioactive cytokine protein.9,14 For example, anELISA may utilize anti-cytokine antibodies that cannot discriminate between theprecursor (inactive) and mature (bioactive) forms of a cytokine protein such as TGFb1. Moreover, an ELISA may detect partially-degraded cytokine proteins which have retained their immunoreactive properties (i.e., at least two recognizable epitopes) but may have lost their bioactivity. In conclusion, cytokine sandwich ELISA are useful indicators of the presence and levels of cytokine and chemokine proteins but they do not actually provide information concerning the biological potency of the detected proteins.With these caveats in mind, one can infer from the presence and amount of cytokine protein detected, the potential mechanisms by which particular effector cell populations perform their functions. Moreover, sandwich ELISAs can detect soluble cytokine receptors which may be important for cytokine regulation. Soluble cytokine receptors may act as antagonists or as carrier proteins in vivo and may serve as disease markers in in vitro tests.15 It should be noted that in addition to providing a rich source of information for clinical and basic science research studies, sandwich ELISA for measuring cytokines and their receptors have become increasingly important as diagnostic tools and for monitoring therapeutic regimens,16 e.g., biological response modification regimens utilizing recombinant cytokine proteins. In the latter cases, highly optimized sandwich ELISA kits designed to minimize interference or nonspecific reactivities presented by patient samples is highly desirable.ELISA Protocol General ProcedureCapture antibody:1.Dilute the purified anti-cytokine capture antibody to 1-4 µg/ml a in BindingSolution. Add 50 µl of diluted antibody to the wells of an enhanced protein-binding ELISA plate (e.g.,Nunc Maxisorb; cat. no. 446469).2.Seal plate to prevent evaporation. Incubate overnight at 4°C.Blocking:3.Bring the plate to RT, remove the capture antibody solution, and block non-specific binding by adding 200 µl of Blocking Buffer per well.4.Seal plate and incubate at RT for 1-2 hr.5.Wash ≥3 times d with PBS/Tween ® .Standards and Samples:6.Add standards b,c and samples (diluted in Blocking Buffer/Tween ®h at 100 µl perwell.7.Seal the plate and incubate it for 2-4 hr at RT or overnight at 4°C.e8.Wash ≥4 times d with PBS/Tween® .Detection antibody:9.Dilute the biotinylated anti-cytokine detection antibody to 0.5-2 µg/ml inBlocking Buffer/Tween®.h Add 100 µl of diluted antibody to each well.10.Seal the plate and incubate it for 1 hr at RT.11.Wash ≥4 times d with PBS/Tween® .Avidin-Horseradish Peroxidase (Av-HRP):12. Dilute the Av-HRP conjugate (cat. no. 13007E) or other enzyme conjugate c,f toits pre-titered optimal concen-tration in Blocking Buffer/Tween ®. Add 100 µl per well.13. Seal the plate and incubate it at RT for 30 min.14. Wash ≥5 times d with PBS/Tween ® .Substrate:15. Thaw ABTS Substrate Solution c,f within 20 min of use. Add 100 µl of 3% H2O2per 11 ml of substrate and vor-tex. Immediately dispense 100 µl into each well. Incubate at RT (5-80 min) for colour development. The colour reaction can be stopped by adding 50 µl of Stopping Solution.16. Read the optical density (OD) for each well with a microplate reader set to 405nm.gSOLUTIONS:§ Binding Solution: 0.1 M Na 2HPO 4 , adjust pH to 9.0 with 0.1 M NaH 2PO 4.§ PBS Solution: 80.0 g NaCl, 11.6 g Na 2HPO 4 , 2.0 g KH2PO4 , 2.0 g KCl; q.s. to 10L; pH to 7.0.§ PBS/Tween ®: 0.5 ml of Tween ® -20 in 1 L PBS.§ Blocking Buffer: Prepare 10% fetal bovine serum (FBS), 10% newborn calf serum (NBCS ) or 1% BSA (immunoassay grade) in PBS. The Blocking Buffershould be filtered to remove particulates before use.§ Blocking Buffer/Tween ®: Add 0.5 ml Tween ® -20 to 1 L Blocking Buffer.§ ABTS Substrate Solution: Add 150 mg 2,2’-Azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (e.g., Sigma, cat. no. A-1888) to 500 ml of 0.1 M anhydrous citricacid (e.g., Fisher; cat. no. A-940) in dd H 20; pH to 4.35 with NaOH. Aliquot 11ml per vial and store at -20°C. Add 100 µl 3% H 2O 2 prior to use.§ 3% H 2O 2 Solution: Add 10 ml of 30% H 2O 2 to 90 ml of H 2O 2. Protect from prolonged exposure to light.§ Stopping Solution: (20% SDS/50% DMF): Add 50 ml of dimethylformamide (DMF) (Pierce, cat. no. 20672) to 50 ml dd H 20, then add 20.0 g sodium dodecylsulfate (SDS) (CMS, cat. no. 424-749).Cytokine ELISA Helpful Hintsa. To determine the optimal signal and lowest background for the ELISA, thecapture antibody (1-4 µg/ml) and detection antibody (0.25-2 µg/ml should be titrated against each other in a preliminary experiment. An appropriate range of serial dilutions for the cytokine standard should be included. A suggested range is generally provided on the Technical Data Sheet (TDS) for ELISAreagents. Generally, use of the capture antibody at 2 µg/ml and the detecting antibody at 1 µg/ml provides strong ELISA signals with low back-ground.b. CYTOKINE STANDARD HANDLING: Please read the TDS for each recombinantcytokine carefully. Handling instructions are lot-specific. For maximumrecovery of cytokine, the vial of cytokine should be quick-spun beforeopening. Lyophilized cytokines should be reconstituted as indicated in the lot-specific TDS. BD Biosciences recommends keeping the cytokine solution in a concentrated form (e.g., ≥1 µg/ml) and in the presence of a protein carrier for long-term storage.c. The linear region of cytokine ELISA standard curves are generally obtainable ina series of eight two-fold dilutions of the cytokine standard, from 2000 pg/ml to 15 pg/ml. To increase sensitivity beyond that obtainable with the standardELISA protocol, amplification kits, tertiary reagents, or alternateenzyme/substrate systems can be used.d.High backgrounds in blank wells (i.e., OD > 0.20) or poor consistency ofreplicates can be overcome by increasing the stringency of washes andoptimizing the concentration of capture and detection antibodies. Forexample, during washes, the wells can be soaked for ~ 1 minute intervals.Moreover, lower concentrations of detecting antibody or more washes after the detecting antibody stage can reduce background.e.For optimal sensitivity, overnight incubation of standards and samplesis recommended.f.If using peroxidase as the enzyme for colour development, avoid sodium azidein wash buffers and diluents, as this is an inhibitor of peroxidase activity.g.If no signal is observed, check the following: a) verify that appropriateantibody clones were used; b) check the activity of the enzyme/substratesystem: e.g.,coat 1 µg/ml of biotinylated detecting antibody in several wells in binding buffer for a few hr. After blocking, wash several times then proceed with the cytokine ELISA protocol from Step 13. If the enzyme/substrate system is active, then a strong signal should be seen; c) verify the activity of cytokine standard or try a new sample of standard.h.When measuring cytokines in complex fluids, such as serum, samplediluents which include irrelevant Ig are suggested.17References:1.Crowther, J. R. 1995. ELISA. Theory and Practice. Methods Mol. Biol. 42:1-223.2.Engvall, E., and P. Perlmann. 1971. Enzyme-linked immunosorbent assay (ELISA). Quantitativeassay of immunoglobulin G. I mmunochem.8:871-874.3.Davies, C. 1994. Principles. In The Immunoassay Handbook. D. Wild, ed. Stockton Press, NewYork, p. 3-47.4.Rogers, R. P. C. 1984. Data Analysis and Quality Control of Assays: A Practical Primer. InPractical Immuno Assay. W. R. Butt, ed. Marcel Dekker, Inc., New York.5.Davies, C. 1994. Calibration curve fitting. In The Immunoassay Handbook. D. Wild, ed, NewYork, p. 118-123.6.Davies, C. 1994. Concepts. In The Immunoassay Handbook. D. Wild, ed. Stockton Press, NewYork, p. 83-115.7.Pathak, S. S., A. van Oudenaren, and H. F. J. Savelkoul. 1997. Quantification ofimmunoglobulin concentration by ELISA. In Immunology Methods Manual, vol. 2. I. Lefkovitz, ed. Academic Press Inc., San Diego, p. 1056-1075.8.Wild, D., and C. Davies. 1994. Components. In The Immunoassay Handbook. D. Wild, ed. Press,New York, p. 49-82.9.Mosmann, T. R., and T. A. T. Fong. 1989. Specific assays for cytokine production by T cells. J.Immunol. Meth.116:151-158.10.Hobbs, M. V., W. O. Weigle, D. J. Noonan, B. E. Torbett, R. J. McEvilly, R. J. Koch, G. J. Cardenas,and D. N. Ernst. 1993. Patterns of cytokine gene expression by CD4 + T cells from young and old mice. J. Immunol. 150:3602-3614.11.Ehlers, S., and K. A. Smith. 1991. Differentiation of T cell lymphokine gene expression: The invitro acquisition of T cell memory. J. Exp. Med.173:25-36.12.Cerottini, J.C., and H. R. MacDonald. 1989. The cellular basis of T-cell memory. Annu. Rev.Immunol. 7:77-89.13.Farber, D. L., M. Luqman, O. Acuto, and K. Bottomly. 1995. Control of memory CD4 T cellactivation: MHC class II molecules on APCs and CD4 ligation inhibit memory but not naive CD4 T cells. Immunity 2:249-259.14.Carter, L. L., and S. L. Swain. 1997. Single cell analyses of cytokine production. Curr. Opin.Immunol.9:177-182.15.Callard, R. E., and A. J. H. Gearing. 1994. Cytokine receptor superfamilies. In The Cytokine FactsBook. Academic Press Inc., San Diego, p. 18-27.16.Rossio, J. L. 1997. Cytokines and immune cell products. In Weir's Handbook of ExperimentalImmunology. Fifth Edition. D. M. Weir, L. A. Herzenberg, L. A. Herzenberg, and C. Blackwell, eds. Blackwell Science, Inc., Cambridge, MA.17.Abrams, J.S. 1995. Immunoenzymetric assay of mouse and human cytokines using NIP-labeledanti-cytokine antibodies. Current Protocols in Immunology (J. Coligan, A. Kruisbeek, D.Margulies, E. Shevach, W. Strober, eds). John Wiley and Sons, New York. Unit 6.20.。

・２４６・虫国塞旦匡蕴！！！Ｑ生！旦箜！鲞筮！塑￡！ｉ塑№丛鲤：坠垫！Ｑ：！堂：§，丛！：２ＣＸＣ趋化因子配体１６与冠心病关系的研究进展陈静文严激在冠状动脉粥样硬化性心脏病的发病机制学说中，除了已被人们接受的脂质浸润学说和损伤反应学说外，炎症反应的作用也逐渐被人们所认识。

炎症反应贯穿于粥样硬化斑块的形成、演变以及破裂的整个病理过程。

炎症细胞与血管内环境中的细胞、细胞因子、炎症介质等的相互作用极其复杂，而趋化因子则是联系炎症与动脉粥样硬化（ａｔｈｅｒｏｓｃｌｅｒｏｓｉｓ，ＡＳ）发生的枢纽。

ＣＸＣ趋化因子配体１６（ｃｘＣＬｌ６／ＳＲ－ＰＳＯＸ）是一种具有结合磷脂酰丝氨酸和氧化型低密度脂蛋白（ＯＸ－ＬＤＬ）作用的趋化因子，同时也是清道夫受体家族成员之一。

近年来，国内外学者在其与冠心病关系方面研究甚多，争议也较多，现就其目前的研究进展作一综述。

ｌＣＸＣＬｌ６概述血管壁上的几乎所有细胞如平滑肌细胞、内皮细胞和巨噬细胞都有表达ＣＸＣＬｌ６，尤其是在已存在ＡＳ斑块内膜上的脂质负荷的巨噬细胞中表达极为丰富…。

ＣＸＣ趋化因子受体６（ＣＸＣＲ６）是其唯一受体，主要在ＣＤ４＋Ｔ细胞、ＣＤ８＋Ｔ细胞、Ｂ细胞、ＮＫＴ细胞、树突状细胞中表达口１。

ＣＸＣＬｌ６在人体内有两种存在形式，即跨膜结合型和分泌型。

不同形式的ＣＸＣＬｌ６生物学作用不同而又相互关联：以分泌型存在时，主要作为ＣＤ８＋Ｔ细胞的趋化诱导剂；以跨膜结合型存在时，通过它的趋化因子活性区参与活化Ｔ淋巴细胞与血管内皮细胞之问的识别和粘附，促进大量的特异性炎症细胞浸润。

金属蛋白水解酶１０（ａｄｉｓｉｎｔｅｇｒｉｎａｎｄｍｅｔａｌｌｏｐｒｏｔｅａｓｅ１０，ＡＤＡＭ一１０）是两种形式转化的关键因素，跨膜结合型ＣＸＣＬｌ６被ＡＤＡＭ－１０水解后脱落形成分泌型ＣＸＣＬｌ６，分布于外周血中。

而作为清道夫受体，发挥着从血管壁清除ＯＸ－ＬＤＬ等有害物质的作用。

ＣＸＣＬｌ６作为一种具有多种功能的蛋白，其在冠心病炎症反应中具有不可忽视的地位。

The TLR2-MyD88-NOD2-RIPK2signalling axis regulates a balanced pro-inflammatory and IL-10-mediatedanti-inflammatory cytokine response to Gram-positive cell wallsLilian O.Moreira,1,2‡Karim C.El Kasmi,1†‡Amber M.Smith,1,2David Finkelstein,3Sophie Fillon,1†Yun-Gi Kim,4Gabriel Núñez,4Elaine Tuomanen 1and Peter J.Murray 1,2*1Department of Infectious Diseases,St.Jude Children’s Research Hospital,332North Lauderdale,Memphis,TN 38105,USA.2Department of Immunology,St.Jude Children’sResearch Hospital,332North Lauderdale,Memphis,TN 38105,USA.3Hartwell Center for Biotechnology and Bioinformatics,St.Jude Children’s Research Hospital,332North Lauderdale,Memphis,TN 38105,USA.4Department of Pathology,University of Michigan,1500East Medical Center Drive,Ann Arbor,MI 48109,USA.SummarySystemic infection with Streptococcus pneumoniae is associated with a vigorous pro-inflammatory response to structurally complex cell wall fragments (PnCW)that are shed during cell growth and antibiotic-induced autolysis.Consistent with previ-ous studies,inflammatory cytokine production induced by PnCW was dependent on TLR2but independent of NOD2,a cytoplasmic NLR protein.However,in parallel with the pro-inflammatory response,we found that PnCW also induced pro-digious secretion of anti-inflammatory IL-10from macrophages.This response was dependent on TLR2,but also involved NOD2as absence of NOD2-reduced IL-10secretion in response to cell wall and translated into diminished downstream effects on IL-10-regulated target gene expression.PnCW-mediated production of IL-10via TLR2required RIPK2a kinase required for NOD2function,and MyD88but differed from that known for zymosan in that ERK pathway activation was not detected.As mutations inNOD2are linked to aberrant immune responses,the temporal and quantitative effects of activation of the TLR2-NOD2-RIPK2pathway on IL-10secretion may affect the balance between pro-and anti-inflammatory responses to Gram-positive bacteria.IntroductionIL-10secretion from T cells,macrophages and dendritic cells is an essential regulatory mechanism to temper excessive inflammatory responses (Murray,2006).Mice lacking IL-10are extremely sensitive to a wide variety of pro-inflammatory stimuli including LPS,and systemic infections with bacteria and parasites that provoke a strong inflammatory response (Murray,2006).In all cases,IL-10is required to restrain the inflammatory response and protect the host against the damaging effects of pro-inflammatory cytokines.Systemic infection with S.pneumoniae provokes a generalized inflammatory response linked with devastating neurological sequelae and a fatality rate of ~15%(Tuomanen et al .,1995).Administration of purified pneumococcal cell wall (PnCW)recapitulates the majority of the inflammatory responses observed in systemic S.pneumoniae infection,suggest-ing that the pro-inflammatory properties of PnCW are a primary driver of inflammatory pathology (Moreillon and Majcherczyk,2003;Fillon et al.,2006;Orihuela et al .,2006).Because PnCW is mainly composed of peptidogly-can,teichoic acid,cell wall linked proteins and lipoteichoic acids,TLR2is considered especially important in the process of PnCW detection by macrophages and den-dritic cells that are then stimulated to produce inflam-matory cytokines (Yoshimura et al .,1999).IL-10can modulate PnCW induced inflammation (Orihuela et al .,2006),but the mechanism leading to IL-10production is entirely unclear.Modulation of inflammation has been suggested to involve NOD2,a cytoplasmic protein of the larger NLR family (Inohara and Nunez,2003;Inohara et al .,2005;Murray,2005;Strober et al .,2006;Kanneganti et al .,2007).For example,single nucleotide polymorphisms in NOD2have been observed at an increased frequency in Crohn’s disease (Lesage et al .,2002).However,severalReceived 2May,2008;revised 31May,2008;accepted 2June,2008.*For correspondence.E-mail peter.murray@;Tel.(+1)9014953219;Fax (+1)9014953099.†Present address:University of Colorado Health Sciences Center,Denver,CO,USA;‡Contributed equally to this work.Cellular Microbiology (2008)10(10),2067–2077doi:10.1111/j.1462-5822.2008.01189.xFirst published online 8July 2008©2008The AuthorsJournal compilation ©2008Blackwell Publishing Ltdloss-of-function mice in the Nod2gene do not have gut immunopathology when maintained under normal condi-tions(Pauleau and Murray,2003;Kobayashi et al.,2005; Maeda et al.,2005;Mariathasan et al.,2006;Barreau et al.,2007).These data are consistent with the fact that some people have mutations in one or both NOD2 alleles but have no clinical inflammatory bowel disease (Hugot et al.,2007).Thus NOD2likely plays a role in more complex pathways modulating pro-inflammatory responses(Strober et al.,2007;Xavier and Podolsky, 2007).NOD2-deficient mice,their immune cells and human cells bearing predicted loss-of-function NOD2alleles,are non-responsive to muramyl dipeptide(MDP),a‘minimal’component of bacterial peptidoglycan able to induce pro-inflammatory responses.A second pathway of MDP sensing appears to be involved in IL-1b processing and is mediated by NLR family members Cryopyrin(NLRP3)and Nalp1(NLRP1)(Faustin et al.,2007;Pan et al.,2007; Marina-Garcia et al.,2008).At this stage it is unclear whether human cells bearing two copies of the NOD2 frame-shift mutation are completely non-responsive to MDP because the truncated mutant protein should still be pared with other bacterial components such as LPS,peptidoglycan or CpG DNA,MDP is a very weak agonist of myeloid-derived cells,especially when considered by molar comparison.However,MDP syner-gizes with other TLR agonists to stimulate cytokine, chemokine and nitric oxide production.This effect is the basis of variants of the‘MDP synergy’assay,a common ‘readout’of NOD2function.In contrast to the lack of MDP responsiveness,macrophages from all NOD2-deficient mice have been generally reported to have‘normal’responses to highly defined TLR agonists.At present,the specific roles of MDP,the origin of MDP in mammalian infection systems and the link between NOD2and MDP remain unresolved.In the studies described here,we observed that TLR2 and NOD2were together responsible for IL-10production when macrophages were exposed to PnCW.By contrast, inflammatory cytokine production in response to PnCW was TLR2dependent and NOD2-independent.Our studies reveal an unexpected pathway of signal integra-tion in response to a complex microbial product that stimulates multiple signalling pathways in macrophages.ResultsWhen S.pneumoniae grow in vivo,cell wall components are released into the host environment by several pro-cesses:cell wall turnover that occurs during normal growth,antibiotics that induce autolysis,and subpopula-tions that undergo spontaneous autolysis as a mecha-nism of interbacterial gene transfer.The latter process lead in part to the discovery of DNA as the source of genetic information.Released cell wall fragments stimu-late a complex pathway of local and systemic inflamma-tory responses including neuronal damage and memory loss(Tuomanen et al.,1995).Because cell wall fragments derived from yeast(zymosan)induce robust IL-10secre-tion from myeloid-derived cells(Dillon et al.,2004;2006; Rogers et al.,2005;Slack et al.,2007),we tested if puri-fied PnCW(Fillon et al.,2006)also induced IL-10produc-tion from bone marrow-derived macrophages(BMDMs). PnCW induced previously unsuspected high amounts of IL-10(Fig.1).As purified PnCW is composed primarily of peptidoglycan,a TLR2agonist,this response was predict-ably TLR2dependent(Fig.1).In parallel experiments we also observed that IL-10and IL-10mRNA production in response to PnCW was also dependent on NOD2 (Figs1B,D and E).The reduction in IL-10production from PnCW-stimulated NOD-deficient macrophages was not to the same extent as Tlr2-/-BMDMs but nevertheless highly reproducible.We also observed that BMDMs derived from Tlr2-/-;Nod2-/-mice secreted undetectable IL-10,demonstrating that TLR2and NOD2might function in the same or related pathways for IL-10production (Figs1B,D and E).By contrast,IL-10production in response to LPS was indistinguishable in Tlr2-/-,Nod2-/-or Tlr2-/-;Nod2-/-cells(Fig.1C).We tested if NOD2was required for IL-10production in response to Pam3CSK4,a synthetic lipid that signals via exclusively TLR2.We observed that Nod2-/-BMDMs produced IL-10in response to Pam3CSK4similar to control cells(data not shown).In contrast,Pam3CSK4did not induce IL-10in Tlr2-/-or Tlr2-/-;Nod2-/-cells as expected(data not shown).NOD2was therefore not required for the‘canoni-cal’TLR2activation pathway stimulated by Pam3CSK4 but was important to the physiological PnCW stimulus. Because PnCW is a complex macromolecule,we tested its cellular fate after exposure to BMDMs.We visu-alized PnCW fragments in macrophages through the use of FITC-labelled PnCW(Fig.1F and G).Macroph-ages consumed all the PnCW over a period of~24h and thereafter,the PnCW remained internalized and appar-ently unchanged over7days when the experiments were paring these data with the kinetics of cytokine secretion,we suspect that macrophages are stimulated with early,external contact with PnCW.By contrast,phagocytosed PnCW appeared to persist within macrophages.Further experiments will be necessary to determine if macrophages can generate MDP from the PnCW and any signalling connections between NOD2 and in-dwelling PnCW.We next asked how broadly NOD2influenced overall macrophage transcriptional responses to PnCW.We per-formed transcriptome analysis of BMDMs derived from background-matched C57BL/6Nod2+/+or Nod2-/-mice2068L.O.Moreira et al.©2008The AuthorsJournal compilation©2008Blackwell Publishing Ltd,Cellular Microbiology,10,2067–2077stimulated for 0,1or 4h with PnCW at a concentration and conditions identical to that used for the experiments in Fig.1.We analysed the data first by principal component analysis (data not shown)and then by ordering gene expression according to degree of induction relative to the untreated (0h)time point data (Table 1).We observed that the absence of NOD2had limited effects on the overall inflammatory response to PnCW (Table 1,Fig.2A)consistent with the notion that other receptors,especially TLR2,mediate a strong cellular activation by PnCW.Of the mRNAs whose expression was altered,we noted that IL-10mRNA expression was reduced in NOD2-deficient cells relative to controls consistent with the ELISA and mRNA data shown in Fig.1.Because IL-10has essential autocrine-paracrine inhibitory effects on activated mac-rophages,we next asked if a panel of known downstream target genes of IL-10signalling (Lang et al .,2002;El Kasmi et al .,2007)were affected by the reduction of IL-10after PnCW stimulation of NOD2-deficient macrophages.We observed that expression of multiple IL-10target genes was lowered relative to controls in NOD2-deficient macrophages at 4h post stimulation (Fig.2B).This is consistent with the data that PnCW stimulates IL-10production in a NOD2-dependent way that leads,in part,to autocrine-paracrine downstream effects on IL-10-responsive gene expression.By contrast to the requirement for both TLR2and NOD2in IL-10production,we observed that alltestedbined signals from TLR2and NOD2are required for PnCW-induced IL-10production.A.Schematic representation of the composition of PnCW obtained by biochemical purification from whole lysed pneumococci.The upper part of the figure is cartoon of the bacteria cell membrane and wall indicating that the wall composed primarily of peptidoglycan,to which multiple proteins,lipopeptides and lipids are tethered,surrounds the cell membrane of pneumococci.Following purification,PnCW is predominantly peptidoglycan and teichoic acids.This fraction is particulate is nature,analogous to zymosan fractions from yeast cell walls.B.IL-10production from BMDMs stimulated with PnCW measured over time by ELISA.Results are combined from independent experiments (n =7).C.IL-10production from LPS stimulation of BMDMs from the same genotypes used in (B).D.Northern blotting analysis of IL-10mRNA over time (h)following PnCW stimulation of BMDMs from the indicated genotypes.Data from Nod2-/-;Tlr2-/-BMDMs were obtained from a different part of the gel as shown the separation from the other part of the gel.E.qRT-PCR analysis of IL-10mRNA production following PnCW stimulation.Data points are averages of duplicate samples.Data are representative of three independent experiments.F.Confocal microscopy to follow the fate of PnCW after contact with BMDMs.FITC-labelled PnCW was added to C57BL/6BMDMs andfollowed over time.Representative images at times 0,5,24and 48h are shown.At each time,cultures were fixed and labelled with phalloidin to visualize actin.Note that the FITC signal is not visible until sufficient PnCW particle have been phagocytosed and concentrated inside macrophages (24h).All images were collected using a 10¥objective,except the 48h time point (20¥).G.Higher power image of PnCW inside BMDMs at 48h (40¥objective).The TLR2-MyD88-NOD2-RIPK2signalling axis 2069©2008The AuthorsJournal compilation ©2008Blackwell Publishing Ltd,Cellular Microbiology ,10,2067–2077pro-inflammatory genes induced by PnCW were depen-dent on TLR2(Fig.3),but independent of NOD2as shown by the microarray data(Table1).These results are consistent with the notion that TLR2is the primary pathway by which macrophages detect PnCW and that NOD2plays a downstream role infine tuning the signal-ling response,in this case by regulating IL-10production. NOD2has been reported to form heterotypic com-plexes with another CARD domain protein,RIPK2(Kobayashi et al.,2002;Park et al.,2007;Hasegawa et al.,2008).Like NOD2-deficient cells,macrophages lacking RIPK2cannot respond to MDP,suggesting that NOD2and RIPK2function in the same pathway for MDP responsiveness(Park et al.,2007).Furthermore,RIPK2 is also required for NOD1function(which does not‘sense’MDP),suggesting that RIPK2is genetically and biochemi-cally linked to both NOD1and NOD2activity(Park et al., 2007).We therefore used macrophages derived from twoTable1.Gene expression increased by PnCW treatment.Gene Rank(WT)Fold(4h)Rank(Nod2-/-)Fold(4h)P(time)P(genotype) Gm19601222.141205.56 1.41E-130.5314Irg12124.622145.62 4.32E-110.8515Cxcl13102.854111.82 2.17E-110.3880Tnf482.043124.60 1.02E-110.8967Cxcl1578.065102.79 4.75E-110.3036Ccl3677.61676.38 4.39E-110.9913Saa3776.27854.01 2.37E-140.1997Ptgs2857.721629.27 4.13E-090.9574Ccl4954.471432.23 3.61E-120.6201Il1b1051.931532.008.30E-070.7485Gpr109a1151.731037.097.80E-140.2307Slc7a111251.721924.34 4.11E-140.6030Ptgs21351.634616.038.02E-080.1154Il1a1445.045015.37 6.39E-070.7571Il61544.031135.24 1.02E-060.3773Socs31643.811332.65 2.46E-110.2271Socs31740.921726.16 1.79E-110.4451Fpr11838.412621.24 2.66E-090.8110 Tnfaip31936.912024.25 6.30E-090.4238Cxcl22036.81760.84 6.79E-140.9650BB5485872136.547111.55 1.85E-060.7213Tnfsf92235.176412.63 5.17E-080.9962Socs32335.101825.02 1.24E-120.2296Ccl22434.012123.20 1.41E-090.5926Ccl72533.98942.028.91E-100.3433Fpr-rs22631.152721.21 3.19E-120.6983Il1rn2729.496911.71 2.74E-100.0624Ets22829.254915.50 3.77E-090.5326Ccrl22929.133019.91 2.75E-080.7250LOC6229763028.831233.45 6.43E-120.1185Il1rn3128.083218.99 4.52E-110.1198 Tnfaip33227.382421.82 1.42E-100.8902Zc3h12c3325.122322.497.12E-100.3216Ccl123425.125913.15 1.25E-080.2671Cdc42ep23524.164416.31 1.03E-080.7966Gpr843623.463119.54 3.15E-100.2886Il1rn3723.357311.359.55E-120.1113Nr4a33822.464815.73 2.80E-080.7223Ccl53922.153618.02 1.01E-070.2819Ch25h4021.836512.599.66E-110.2134Cd404121.426612.25 2.74E-110.8623Cd404221.315413.89 6.89E-120.3615Zc3h12c4321.072821.05 3.52E-100.7250Clec4e4420.752920.40 2.51E-090.9522Malt14520.223318.56 5.46E-120.8940 Hivep34619.932222.60 3.95E-150.4636 Ptges4719.705314.23 2.23E-100.2554Cd694819.603418.55 3.57E-070.5920Cd404919.486013.08 2.69E-120.7930Traf15019.184316.55 5.61E-080.2824 Probe sets from the top50induced targets in PnCW-stimulated control cells are ordered1through50based on fold induction relative to the untreated control cells at4h.The correlative rank of genes expressed in PnCW-stimulated NOD2-deficient macrophages relative to the control ranking is listed along with the P-values(ANOVA)by time relative to unstimulated cells and by genotype.2070L.O.Moreira et al.©2008The AuthorsJournal compilation©2008Blackwell Publishing Ltd,Cellular Microbiology,10,2067–2077strains of Ripk2-/-mice and mice lacking both NOD1and NOD2(Nod1-/-;Nod2-/-)to probe if IL-10production was inhibited.We observed by ELISA and qRT-PCR analysis that RIPK2-deficient cells had a similar phenotype to NOD2-deficient BMDMs(Fig.4)in terms of reduced IL-10 production in response to PnCW.Furthermore,macroph-ages from Nod1-/-;Nod2-/-mice,which have been reported to have similar phenotypes to Ripk2-/-macroph-ages(Park et al.,2007),also had a deficiency in IL-10production when stimulated with PnCW.Finally, when introduced into Nod2-/-macrophages by retroviral-mediated transduction,human NOD2rescued IL-10in response to PnCW and MDP responsiveness (Fig.4E–G).Analogous studies with NOD1were not pos-sible because human NOD1producer lines do not make virus at sufficiently high titre to infect stem cells.Collec-tively,these data provide evidence that NOD2and RIPK2 function in the same pathway that mediated IL-10produc-tion in response to PnCW.Zymosan,a complex cell wall fraction from yeast,has been shown to induce IL-10production by the TLR2-DECTIN1-SYK pathway and the TLR2-DECTIN1-ERK pathway(Dillon et al.,2004;2006;Rogers et al.,2005;Slack et al.,2007).We therefore asked if PnCW also stimulated ERK activation with the idea that ERK could be a common downstream mediator of multiple TLR2-driven pathways,including the TLR2-NOD2pathway.However, we observed that PnCW induced low or negligible ERK phosphorylation(Fig.5).Therefore,PnCW seems to stimulate IL-10production via pathways distinct from those induced by zymosan,even though both the DECTIN1-SYK and NOD2-RIPK2pathways depend on TLR2.Finally,IL-10production in response to PnCW was dependent on MyD88(Fig.5E),suggesting that the TLR2-MyD88pathway is absolutely necessary for PnCW-induced IL-10production.MyD88was not,however, required for MDP sensing(Fig.5F)as expected as shown by the use of a sensitive MDP stimulation assay where nitric oxide production is measured in response to MDP and IFN-g(Totemeyer et al.,2006).By contrast,MDP sensing was dependent on NOD2.DiscussionOur data suggest that NOD2is a component of a signalling module that regulates IL-10production in aFig.2.Absence of NOD2has a limited effecton PnCW-induced inflammatory transcription.A.Select inflammatory targets induced byPnCW in control or NOD2-deficient BMDMs.Data for each target mRNA is plotted as theaverage difference signal intensity.Data areaverages from independent(n=4)Affymetrixarrays performed per genotype and per time.No significant differences(paired t-test)weredetected between plete datasets for this experiment have been depositedin the GEO database.B.Expression of IL-10target genes isaffected in the absence of NOD2function.Signal intensity for IL-10(left most in thepanel)and selected genes whose expressionis dependent,in part,on autocrine-paracrineIL-10production.*P<0.05,paired t-testcomparing4h time points between controland Nod2-/-BMDMs(n=4).The TLR2-MyD88-NOD2-RIPK2signalling axis2071©2008The AuthorsJournal compilation©2008Blackwell Publishing Ltd,Cellular Microbiology,10,2067–2077stimulus-specific and cell type-specific way(Fig.6).Acti-vation of TLR2-MyD88by Pam3CSK4leads to ERK acti-vation and IL-10induction.In contrast,activation of TLR2-MyD88by PnCW invokes NOD2and RIPK2and bypasses ERK activation to produce IL-10.Activation of TLR2and DECTIN by zymosan leads to ERK activation and IL-10induction.It is well accepted that IL-10is essen-tial for regulating the amplitude of most,if not all inflam-matory responses in vivo.Therefore,mutant forms of NOD2may be a component of homeostatic regulation of IL-10in myeloid-derived cells.Notably,Netea and col-leagues have demonstrated that human monocyte-derived macrophages and dendritic cells isolated from people bearing NOD2mutations have reduced IL-10pro-duction in response to peptidoglycan and MDP(Netea et al.,2004;2005;Kullberg et al.,2008).We found parallels between PnCW stimulation of IL-10 production and the activation of dendritic cells by zymosan,a large fragmentary remnant of yeast cell walls. In both cases,immune cells are exposed to particulate fractions of cell walls of varying sizes and displaying a variety of different sugars,lipids and structural biopolymers.Zymosan stimulates IL-10production by a MyD88-independent pathway involving TLR2,DECTIN1, SYK and ERK(Dillon et al.,2004;2006;Rogers et al., 2005;Slack et al.,2007).PnCW stimulation of IL-10also requires TLR2,but does not seem to activate substantial ERK phosphorylation(Fig.5).Instead,MyD88,NOD2 and RIPK2are required.The PnCW and zymosan recog-nition pathways present a considerable contrast to the activation of immune cells with Pam3CSK4through TLR2, a pathway that has an absolute requirement for MyD88 but not NOD2.Therefore,there are different signalling routes to IL-10production following TLR2activation (Fig.6).Similarly,additional diversity of signalling for IL-10production has been observed for receptors such as the LPS-mediated pathway that requires TLR4and p38 (Park et al.,2005),and the immune complex pathway that enhances IL-10production via FcR ligation(Edwards et al.,2006).BTK also plays a key role in stimulus-specific IL-10production:the absence of BTK leads to a decrease in IL-10produced from macrophages and a correspond-ing increase in inflammatory mediators normally blocked by IL-10(e.g.IL-12,IL-6)(Kawakami et al.,2006;SchmidtFig.3.Pro-inflammatory cytokinetranscription in response to PnCW isdependent on TLR2.BMDMs from theindicated genotypes were stimulated withPnCW for the times shown.Cytokine mRNAswere measured by qRT-PCR.Data arerepresentative of three independentexperiments.2072L.O.Moreira et al.©2008The AuthorsJournal compilation©2008Blackwell Publishing Ltd,Cellular Microbiology,10,2067–2077et al .,2006).However,the reduced amounts of IL-10made in the absence of BTK,like the absence of NOD2,is insufficient to initiate the severe inflammatory bowel disease observed in the complete absence of IL-10(Schmidt et al .,2006)(L.M.and P .J.M.,unpubl.data).Therefore,NOD2and BTK both seem to function to tune stimulus-specific IL-10production.We found that neither TLR2nor NOD2were absolutely required for PnCW stimulation of IL-10production.IL-10was made,although in much lower amounts,in the absence of either TLR2or NOD2.However,cells lacking both TLR2and NOD2had a complete abrogation of IL-10production.These data suggest that TLR2and NOD2have a genetic interaction.A similar finding has been made in the ECOVA gut inflammation system where pathology observed in the absence of NOD2was reversed in the combined deficiency of both NOD2and TLR2(Watanabe et al .,2006).It is however,prematuretoFig.4.IL-10expression in response to PnCW is dependent on the RIPK2pathway.A.BMDMs from Ripk2-/-or Nod1-/-;Nod2-/-mice were stimulated with PnCW and IL-10measured over time by ELISA.B.As in (A),but following LPS stimulation.C and D.Analysis of BMDMs from an independent Ripk2mutant strain showing IL-10and IL-10mRNA produced in response to PnCW requires RIPK2.E and F.Reconstitution of Nod2-/-hematopoietic stem cells with human or mouse NOD2cDNAs rescues IL-10production in response to PnCW.Stem cells from Nod2-/-mice were infected with retroviruses containing hNOD2or mNOD2cDNAs and selected by cell sorting for YFP and plated into media containing CSF-1to drive differentiation into macrophages.After differentiation,macrophages were stimulated with LPS or LPS and MDP to measure the MDP synergy with TLR4signalling (E)or IFN-g signalling (F)or IL-10production after PnCW treatment (G).Data are representation of two independent infection studies.In the case of mNOD2,sufficient cells were obtained to perform the MDP-IFN-g synergy assay only.The TLR2-MyD88-NOD2-RIPK2signalling axis 2073©2008The AuthorsJournal compilation ©2008Blackwell Publishing Ltd,Cellular Microbiology ,10,2067–2077suggest that TLR2and NOD2function in a linear pathway where NOD2is downstream of TLR2.Instead,we suggest that cross-talk between the TLR2and NOD2pathways occurs in a stimulus-and cell-specific context.Cross-talk is not revealed when TLR2is stimulated with Pam 3CSK4but instead is exposed by agents such as PnCW and peptidoglycan,at least in the BMDMs used here.This idea may help resolve previous reports that suggested that NOD2was not involved in TLR2signalling (Kobayashi et al .,2005):dependence on both stimulus and cell type is needed to expose differences among pathways.Recent data from human monocytes bearing NOD2mutations has also revealed a complex interplay between NOD2and TLR2(Borm et al .,2008).Using transcriptome profiling,we also found that the absence of NOD2affected a surprisingly low number of mRNAs when BMDMs were stimulated with PnCW.These data are in keeping with published studies suggestingthatFig.6.Model of IL-10production myeloid lineage cells(macrophages and DCs)stimulated by agents that activate TLR2,alone or in combination with other cell surface molecules.Fig.5.PnCW activation of BMDMs does not activate the ERK or p38pathways but requires MyD88signalling.A.C57BL/6BMDMs were stimulated with Pam 3CSK4,LPS or PnCW over time and ERK phosphorylation measured byimmunoblotting for phosphorylated forms of ERK or reprobed for total ERK amounts.B.A similar experiment to (A)was performed using BMDMs from the genotypes indicated on the right.C.The same lysates from (B)were probed for phospho-p38.Note that PnCW does not activate ERK or p38in any of these experiments.D.BMDMs from control,Nod2-/-,Tlr2-/-or MyD88-/-mice were stimulated with PnCW for 8h and IL-10measured by ELISA.Data are average ϮSD from independent samples (n =3).E.BMDMs were stimulated with MDP orMDP +IFN-g overnight and nitrites measured by the Griess assay.Stimulation with IFN-g alone did not produce detectable nitrites (data notshown).2074L.O.Moreira et al.©2008The AuthorsJournal compilation ©2008Blackwell Publishing Ltd,Cellular Microbiology ,10,2067–2077loss of NOD2has minimal effects on pro-inflammatory TLR signalling(Pauleau and Murray,2003;Kobayashi et al.,2005;Park et al.,2007).However,PnCW is an example of a pathogen‘signal’that can activate multiple pathways including the TLR2pathway.It is not surprising that NOD2plays limited roles in the overall transcriptional response as the mammalian immune system most likely has developed multiple,overlapping ways to recognize and respond to such a complex material.Nevertheless, thefinding that IL-10was one of the most affected mRNAs,along with a cohort of IL-10-regulated genes, suggests that further work is warranted to determine the role of NOD2in IL-10production in humans bearing muta-tions in NOD2.It is tempting to speculate that IL-10regulation has key functional significance in chronic inflammatory states such as the inflammatory bowel diseases.Most research-ers agree that common pathways of inflammatory media-tor production,arrived at by a multitude of genetic and environmental mechanisms,drive disease and are the central focus of therapeutic intervention through anticy-tokine antagonists.IL-10plays an essential regulatory role in controlling the products of the common inflamma-tory pathway:IL-12,TNF-a,IL-6,etc.Therefore,small disturbances in temporal,anatomic or quantitative IL-10 amounts may translate into greater inflammation.Experimental proceduresMiceNod2-/-mice have been previously described(Pauleau and Murray,2003).Nod2-/-;Tlr2-/-mice on a C57BL/6background (n=5generations)have been described(Watanabe et al., 2006).Tlr2-/-mice were purchased from the Jackson Laboratories.Mice were bred and genotyped according to pub-lished protocols.For some experiments,Nod2-/-mice were backcrossed10generations to the C57BL/6background.All animal experiments were performed in accordance with protocols governed by the St Jude Animal Care and Use Committee(P.J. M.,Principal Investigator).Nod1-/-;Nod2-/-and Rip2k-/-mice have been described previously(Chin et al.,2002;Kobayashi et al.,2002;Park et al.,2007).Cell preparation and stimulus conditionsBone marrow-derived macrophages were isolated and cultured as described(Lang et al.,2002).Macrophages were stimulated with highly purified PnCW prepared as described previously (Tuomanen et al.,1985a,b).Briefly,S.pneumoniae unencapsu-lated strain R6were grown in C+Y medium,bacteria were boiled in SDS,mechanically broken by shaking with acid-washed glass beads,and treated sequentially with DNase,RNase,trypsin,LiCl, EDTA and acetone.PnCW was confirmed to be free of protein by analysis for non-cell wall amino acids using mass spectrometry. The absence of contaminating endotoxin was confirmedfirst by the Limulus test(Associates of Cape Cod)and then by ELISA measurement of hIL-8production by HEK293cells transfected with plasmids designed to express TLR4and MD2(a generous gift of Dr Doug Golenbock,UMass)in the presence of purified PnCW.Forfluorescence microscopy experiments,BMDMs were labelled with Phalloidin and4′,6-diamidino-3-phenylindole (DAPI),a nuclear stain.PnCW was directly labelled with 1mg ml-1FITC(Sigma-Aldrich)solution in carbonate buffer (pH9.2)for1h at room temperature in the dark and washed twice with PBS containing calcium and magnesium(Gosink et al., 2000).ELISAELISA was performed as previously described(El Kasmi et al., 2006).Capture and detection antibodies used were purchased from BD Pharmingen.Detection limits for the ELISAs were 50pg ml-1(IL-10)and10pg ml-1(IL-12p40).Northern blotting,RT-PCR and immunoblottingTotal RNA was isolated from primary macrophages using Trizol. Reverse transcription(qRT-PCR)was performed as described previously using primers and probes(Applied Biosciences)spe-cific for each cytokine mRNA(Lang et al.,2002).Immunoblotting was performed using the following rabbit polyclonal antibodies: antiphospho-p42,p44ERK and anti-phospho-p38from Cell Sig-naling Technology used at1:500final dilution.Anti-p42,p44ERK and anti-p38were from the same source and were used at afinal concentration of1:1000.Transcriptome analysisRNA samples from BMDM cultures representing four PnCW preparations,two genotypes(wild-type and the NOD2deficient) and three time points(0,1and4h)were arrayed on Affymetrix GeneChip analysis murine430v2GeneChip arrays.Signal was acquired with MAS5.0software and natural log transformed [ln(signal+20)]to stabilize variance and better conform the data to the normal distribution.Principal components analysis(Partek 6.1)revealed a batch effect which,being orthogonal to hour and genotype,was removed by capturing and mean adjusting residu-als of a one-way ANOVA-based on PnCW batch(STATA/SE9.2). The batch-corrected transformed signal was tested for exposure time and genotype effects in a two-way ANOVA using Partek.The P-values for the time effect were corrected for multiple compari-sons using the false discovery rate(Benjamini et al.,2001).Fold change calculations were based on the geometric means of the batch corrected data between the4h and0h time points for each genotype.Array data has been submitted to the GEO data-base(accession GSE8960).Retroviral-mediated transduction of stem cellsRetroviral transduction of murine bone marrow cells was per-formed as described previously(El Kasmi et al.,2006;Holst et al.,2006a,b).For the transduction experiments,we used fetal liver stem cells isolated from E14pregnant Nod2-/-female mice. Fetal liver cells were cultured for48h in complete DMEM with 20%FBS,20ng ml-1murine IL-3,50ng ml-1human IL-6and The TLR2-MyD88-NOD2-RIPK2signalling axis2075©2008The AuthorsJournal compilation©2008Blackwell Publishing Ltd,Cellular Microbiology,10,2067–2077。

-综述•骨源性细胞因子在激素性股骨头坏死发生发展中的研究进展蒋捷,黄林科，胡峰△(广西医科大学第二附属医院，广西南宁530007)［摘要］股骨头坏死是骨外科常见的难治性疾病,其机制仍有待研究。

目前为止,医源性糖皮质激素是非创伤性股骨头坏死的主要原因。

激素的长期使用可导致股骨头骨细胞凋亡、血液循环障碍所致缺血缺氧，最终导致股骨头塌陷。

激素性股骨头坏死的发生发展与骨组织中细胞直接接触和其间接分泌的细胞因子调控相关。

本文综述了骨组织中成骨细胞分泌的核因子k B受体结合配体，骨保护素,骨碱性磷酸酶，骨细胞表达硬化素，破骨细胞分泌骨形态发生蛋白2等因子在SONFH的研究进展，骨源性细胞因子在SONFH中扮演重要作用。

［关键词］骨源性细胞因子;激素性股骨头坏死;临床分型;病理表现;综述［中图分类号］R681［文献标识码］A［文章编号］961-5639(2621)61-482-04doi:16.3969/E R w x961-5639.402000025Resevrch prggress of bone derived cytokines in developmenS of steaid induced osteynecrgsis of the femorai hecdJIANG Jia,HUANG Lia-Ue,HU Feng'(Department of OrtUopebic trauma,the Second Affiliated Hospital of Guanypi Medical Univer­sity?Nanning Citp,Guangpi Zhuang Autonomous Repiox530007,China)J Abstrach]Avascular necrosis of the femoral heah is a common refracton disease in ortUopebic shraeru/whose mechanism remains to be studied.So far,iatngenic glucocorncoids are the main cause of nox-EaumaWe necrosis of the femoral heah.Long term treatment of glucocorncoids leafs to osteocyte apoptosis of femoral heah,ischemia and hypoxia caused bp blood cinulaWon OisorUer,and eventuaLy leafs to coXapse of femoral heah.The occurrence and Oevelopment of steroid induced osteonecrosis of the femoral heah are related to the direct contact of cells in bone tissue and the repulaLox of cypdines secreted indirectly.We reviewed the research progress of Nuclear factor kaypa B receptor binding ligand,RANKL,OsPoproPpeEn；OPG and Bone aldaLne phosphamse；BAP secreted bp osteoblasts, eceeeohnt(SOST)peoducedbsoeheocsheeatdBotemoepeogetehncpeohent-2BMP-2eeceehedbsoeheoceaehenteheeondntducedoeheote-eeoeneoeheeeemoeaeeead0Botedeeneedeshoenteepeasatnmpoehatheoeenteheeondntdueedoeheoteeeoeneoeheeeemoeaeeead0J Key woras]Bone PeEved cytodines;2proid induced necrosis of the femoral heah；Clinical classification;PatUologicai manifestation；Reenew股骨头坏死(OWewecrwis of femoral heah,ONFH)是骨外科常见的难治性疾病,严重影响患者的生活质量。

大鼠弥漫性脑损伤后血清TNF-α和IL-10的含量变化栗浩【摘要】目的探讨急性颅脑损伤后血清肿瘤坏死因子α(TNF-α)和白细胞介素10(IL-10)的含量变化及其对临床的指导意义.方法采用双抗体夹心ABC-ELISA方法 .检测60例急性颅脑损伤患者伤后血清TNF-α和IL-10含量的变化.结果 TNF-α、IL-10在伤后早期即明显升高.结论 TNF-α和IL-10参与了急性颅脑损伤后的炎性反应过程,血清中TNF-α和IL-10含量在颅脑损伤后明显升高,可能在继发性脑损害中起重要作用.【期刊名称】《泰山医学院学报》【年(卷),期】2008(029)009【总页数】2页(P663-664)【关键词】急性颅脑损伤;肿瘤坏死因子α;白细胞介素10【作者】栗浩【作者单位】新汶矿业集团华丰医院,山东,宁阳,271413【正文语种】中文【中图分类】R651脑损伤可分为局限性脑损伤和弥漫性脑损伤(diffuse brain injury, DBI)两大类。

后者主要包括弥漫性轴索损伤(diffuse axonal injury, DAI)、弥漫性脑肿胀(diffuse brain swelling, DBS)、弥漫性微血管损伤和缺氧性脑损害等四种类型[1]。

目前认为炎症反应在急性颅脑损伤后的继发性脑损害中发挥重要作用[2]。

为了探讨炎性细胞因子TNF-α和IL-10在急性颅脑损伤发病过程中的作用，我们通过建立弥漫性脑损伤大鼠模型检测伤后大鼠不同时期血清TNF-α和IL-10含量探讨血清中TNF-α和IL-10含量变化以及对预后的影响。

1 资料和方法1.1 材料采用成年健康SD雄性大鼠，体重325～375 g，共42只。

实验动物由山东大学实验动物中心提供。

双抗体夹心ABC-ELISA试剂购自上海麦莎生物科技有限公司。

1.2 动物模型的制备根据Marmarous'弥漫性脑损伤动物模型有改进[3]， SD大鼠用乙醚吸入麻醉，麻醉前30 min肌肉注射阿托品0.5 mg，先进行颈部气管插管准备，颈部剃毛后用碘伏进行消毒，纵性切开颈部皮肤，锐性分离皮下筋膜，向头部翻起甲状腺，纵性锐性分离气管部肌肉，暴露气管，应用无菌纱布覆盖切口，再腹卧位固定大鼠，沿大鼠头部中线切开皮肤，分离皮下组织及筋膜暴露前至冠状缝后至人字缝之间的颅骨，用磷酸锌水泥将直径为10 mm，厚度为3 mm的圆形钢板镶在上述部位，待水泥固定后打击。

国P小免疫学杂忐202丨彳I:丨月第44卷第1期1丨丨丨J Immunol J训.2021，V〇1.44,!\\). 1• 14 •16734394.2012.03.004.Liu LL,Fang F. Tht* research progress of pattern-recognition recep­tor recognizing cytomegalovirus[ J . International Journal of Immu­nology ,2012 ,35 (3 ) ：180- 182. D()I：10.3760/cma. j. issn. 1673-4394.2012.03.004.[3 J Zeleznjak J，l)«povi(. B，Krmpotic A，et al. Mouse cytomegalovirusencoded immunoevasins and evolution of Ly49 receptors - sidekicksor enemies? [ J]. Immunol l^tt ,2017,189 ：40-47. D()I：10. 1016/j. imlet.2017.04.007.[4] Hammer Q,Kuckert T,Romagnani C. Natural killer cell specificityfor viral infections! J .Nat Immunol ,2018,19( 8) ：8()0-808. DOI ：10. 1038/s41590-018-0163-6.[5 ]Schluh TE,Sun JC, Walton SM,et al. Comparing the kinetics ofNK cells,CI>4,and CD8 T cells in murine cytomegalovirus infec-tion: J]. J Immunol，2011，187 (3 ) : 1385-1392. DOh l〇. 4049/jimmunol. 1100416.[6 !Beaulieu AM,Sun JC. Tracking effet；lor and memory NK cells dur­ing MCMV infection f J .Methods M ol Biol, 2016, 1441 ：1-12.DOI： 10. 1007/978-1 4939-3684-7_l.[7]李莎，朱艳，张在利.MCMV早期感染对小鼠CD8 + T，7S T和NK细胞功能的影响[J].免疫学杂志，2018,34(8) :645457.DOI ： 10. 13431/j. cnki. immunol. j. 20180101.Li S,Zhu Y,Zhang ZL. The impact of early c-ytomegaloviru.s infec-tion on CD8 + T,*y8T and NK cells in mice. [ J]. ImmunologicalJournal,2018,34(8) ：645-657. DOI ： 10. 13431/j. cnki. immunol.j. 20180101._ 8 Robbins SH,Tessmer MS,Mikayama T,et al. Expansion and (*on- tradion of tlie NK cell compailment in response to murine cytomeg­alovirus infection[ J . J Immunol, 2004,173 ( 1) ： 259-266. DOI ：10.4049/j immunol. 173. 1.259.[9 ]Sumaria N,van Dommelen SL, Aiuloniou CK,et al. The roles of in-teiferon-gamma and perforin in antiviral immunity in mice that dif­fer in genetically detemiined NK-cell-mediated antiviral activity[J]. Immunol Cell Biol, 2009,87 ( 7 )： 559-566. DOI：10. 1038/icb. 2009.41.[10 ：Parikh BA,Piersma SJ, Pak-Wittel MA,et al. Dual requirement ofcytokine and activation receptor triggering for cytotoxic control ofmurine cytomegalovirus by NK cells J . 1M^)S1^111^,2015,11(12) ：el005323. DOI： 10. 1371/journal, ppat. 1005323.[11]卢元元.BALB/(•和C57BL/6小鼠抗MCMV免疫应答的差异与其感染结局的相关性[丨)].武汉:华中科技大学，2019.Lu YY. The Relationship between the difference of immune reac­tions against MCMV and the infections outcomes in BALB/c andC57BL/6 m ice[I)'. Wuhan ： Hua/liong L iiiversity ,2019.[12: Bahic M, Krmpotic A.Jonjic S. All is fair in virus-host interac­tions：NK cells and cytomegalovims J . Trends M ol Med,2011 ,17(11) ：677-685. DOI ： 10. 1016/j. molmed. 2011.07.003.[13 _Adams NM,Lau CM, Fan X,et al. Transcription factor IRF8 or­chestrates the adaptive natural killer cell response[ J . Immunity ,2018,48 (6)：1172-1182. c6. DOI：10. 1016/j. imniuni. 2018.04.018.!14 ] Nal>ekura T, Lanier LL. Activating receptors for self-MHC class I enhance effector functions and memor>' differentiation of NK cellsduring mouse cytomegalovirus inlcction [J j. Immunity, 2016,45(1) ：74-82. DOI ： 10. 1016/j. imniuni. 2016.06. 024.(收稿 H 期:20204)7-31)读者•作者•编者关于参考文献著录格式要求参考文献著录格式基本参照此行GB/T7714-2005《文后参考文献著录规则》，采用顺序编码制著录，依照文献在文中出现的先后顺序用阿拉伯数字加方括号标出将参考文献按引用先后顺序（用阿拉伯数字标出）全部排列于文末参考文献中的作者，丨~3名全部列出，3名以上只列前3名，后加“，等或其他与之相应的文字，如“，e ta l.”_著录作者姓名时将姓放在前，名缩写放在姓后面：外文期刊名称用缩写，以《Index Medicus》中的格式为准；中文期刊用全名每条参考文献均须著录起止页码，文献题名项后需标注文献类型标志项目作者必项将参考文献与其原文核对无误举例如下[1] You CH, Lee KY , Chey WY,et al. Electrogastrographic study of patients with unexplained nausea, bloating and vomiting[J].Gastroenterology, 1980,79(2) :311-315.(期刊格式)[2]汪敏刚.支气管哮喘[A]//戴自英.实用内科学.8版.北京：人民卫生出版社，1991:833-840.(专著中析出文献格式）[3 ] Sodeman WA Jr,Sodenmn WA. Pathologic physiology ： mechanisms of disease [ M]. 8tli ed. I^iladelphia ： Saunders, 1974： 457-472.(书籍格式）。

纳米二氧化硅对呼吸系统毒性及NF-κB在其中的作用摘要：纳米二氧化硅（SiNPs）作为纳米二氧化硅外观为白色无定形粉末，粒子尺寸在1~100nm之间，比表面积大且化学性质稳定，作为纳米技术消费产品中最常用的五种纳米材料之一，接触纳米SiO2的机会不断增加，有关暴露于其的健康危害的证据也在不断增加[1]，因此其安全性问题也逐步被大众所关注。

相关工矿职业人群通过呼吸道、皮肤、消化道等多种途径摄入体内，一般人群也可通过生物医疗、食品添加等途径将其摄入体内[2]。

SiNPs在体内、外均可诱导炎症和氧化应激反应，而长期接触SiNPs粉尘后，会引起以肺组织慢性持续性炎症与进行性纤维化为主要病理特点的全身性疾病[3]。

肺部由于其特殊的解剖结构及生理功能，使得呼吸系统成为重要的入侵途径之一。

核因子κB（nuclear factor kappa B, NF-κB）作为一种重要的转录因子，对于研究细胞功能、机体对疾病的反应和机体健康发展十分重要，有证据表明，NF-κB参与SiO2诱导的矽肺鼠模型的炎症纤维化发生发展过程，且抑制NF-κB后肺部炎症和纤维化均有所缓解。

因此，从SiNPs毒作用机制和NF-κB在其中发挥的作用两部分整理相关的研究进展。

关键词：纳米二氧化硅；肺毒性；NF-κB与常规颗粒相比，纳米颗粒更容易进入肺间隙，并在肺间隙中滞留更长的时间，纳米SiO2颗粒可通过多种途径进入机体，由于肺部特殊的解剖结构及生理功能，使得呼吸系统成为重要的入侵途径之一。

由于纳米SiO2具有较小的粒径及较大的比表面积，可逃避呼吸系统中细胞的吞噬作用，在肺泡中沉积和传播，也可透过气血屏障进入循环系统，对肺部以外的器官产生毒性作用。

NF-κB存在于绝大多数动物细胞内，参与细胞对刺激的反应，它的错误调节会引发许多疾病。

在纳米SiO2诱导的肺纤维化中，NF-κB信号通路一方面可能通过刺激巨噬细胞产生TNF-α、转化生长因子-β（Transforming growth factor-β，TGF-β）等炎性因子引起肺部炎症，另一方面可能通过调控肺泡上皮细胞中肺表面活性物质的表达以及调控EMT过程来影响肺纤维化的发展，是纳米SiO2的肺毒性研究中的关键靶点。

Animal(2012),6:10,pp1620–1626&The Animal Consortium2012doi:10.1017/S1751731112000481The effect of chitooligosaccharide supplementation on intestinal morphology,selected microbial populations,volatile fatty acid concentrations and immune gene expression in the weaned pig A.M.Walsh,T.Sweeney,B.Bahar,B.Flynn and J.V.O’Doherty-School of Agriculture,Food Science and Veterinary Medicine,University College Dublin,Lyons Research Farm,Newcastle,Co.Dublin,Ireland(Received24March2011;Accepted29January2012;First published online2March2012)An experiment(complete randomised design)was conducted to investigate the effects of supplementing different molecular weights (MW)of chitooligosaccharide(COS)on intestinal morphology,selected microbial populations,volatile fatty acid(VFA)concentrations and the immune status of the weaned pig.A total of28piglets(24days of age,9.1kg(6s.d.0.80)live weight)were assignedto one of four dietary treatments for8days and then sacrificed.The treatments were(1)control diet(0ppm COS),(2)control diet plus5to10kDa COS,(3)control diet plus10to50kDa COS and(4)control diet plus50to100kDa COS.The COS was included in dietary treatments at a rate of250mg/kg.Tissue samples were taken from the duodenum,jejunum and ileum for morphological measurements.Digesta samples were taken from the proximal colon to measure lactobacilli and Escherichia coli populations and digesta samples were taken from the caecum and proximal colon for VFA analysis.Gene expression levels for specific cytokines were investigated in colonic tissue of the pig.Supplementation of different MW of COS had no significant effect on pig performance during the post-weaning period(days0to8;P.0.05).The inclusion of COS at all MW in the diet significantly reduced faecal scores compared with the control treatment(P,0.01).Pigs fed the10to50kDa COS had a higher villous height(P,0.05)and villous height:crypt depth ratio(P,0.05)in the duodenum and the jejunum compared with the control treatment.Pigs fed the5to10kDa COS had a lower lactobacilli population(P,0.05)and E.coli population(P,0.05)in the colon compared with the control group.Pigs offered the5to10kDa COS had significantly lower levels of acetic acid and valeric acid compared with the control group(P,0.05). The inclusion of different MW of COS had no significant effect on the expression of the cytokines tumour necrosis factor-a,Interleukin (IL)-6,IL-8and IL-10in the gastro-intestinal tract of the weaned pig.The current results indicate that a lower MW of5to10kDa COS possessed an antibacterial activity,while the higher MW of10to50kDa was optimum for enhancing the intestinal structure. Keywords:chitooligosaccharide,pig,microbiology,intestinal morphologyImplicationOur results indicate that the inclusion of chitooligosaccharides (COSs)in piglet diets may moderate several gut health para-meters that contribute to some of the common problems that occur after weaning in the absence of in-feed antibiotics.It was observed that COSs with a molecular weight(MW)of5to 10kDa were more effective in reducing Escherichia coli populations while a MW of10to50kDa enhanced the intestinal structure.IntroductionThe weaning period imposes profound social and environ-mental stresses on the piglet such as removal from the sow,change in diet and mixing of piglets from different litters. Numerous studies have reported that there is a reduction in villous height(villous atrophy)and an increase in crypt depth (crypt hyperplasia)after weaning,which leads to increased susceptibility to intestinal gut dysfunction(Spreeuwenberg et al.,2001;Pierce et al.,2006).The post-weaning period is characterised by a reduction in feed intake,poor growth rates,diarrhoea and an increased risk of disease(Lalles et al., 2007).These negative effects on piglet growth during the weaning period were managed by growth-promoting anti-biotics.However,the European Union placed a total ban on the use of in-feed antibiotic growth promoters on the1st January2006due to public concerns regarding bacterial resistant and human health issues. Chitooligosaccharides(COS)may be a potential viable alternative to traditional antimicrobials in animal production.-E-mail:john.vodoherty@ucd.ie 1620Chitosan is a natural biopolymer derived by alkaline deacety-lation of chitin,which is the principal component of protective cuticles of crustaceans such as crabs,shrimps,prawns,lobsters and cell walls of some fungi such as aspergillus(Qin et al., 2006).Both chitin and chitosan are biopolymers composed of glucosamine and N-acetylated glucosamine(2-acetylamino-2-deoxy-D-glucopyranose)units linked by b(1to4)glycosidic bonds(Koide,1998).Low molecular weight(MW)COS is a water-soluble derivative of chitosan due to shorter chain lengths(Kim and Rajapakse,2005).Recently,both chitosan and its derivatives have generated considerable interest due to their biological activities,including antimicrobial,antitumour, immunoenhancing effects and the acceleration of wound healing(No et al.,2002;Liu et al.,2006)There is considerable variation in the literature on the biological properties of COS (Jeon et al.,2001;Liu et al.,2006).Most of this variation is partly due to the widely different MW used across studies.It is hypothesised that the biological properties of COS may be influenced by its MW and COS will enhance selected indices of health in weaned piglets.Material and methodsAll procedures described in this experiment were conducted under an experimental licence from the Irish Department of Health in accordance with the cruelty to Animals Act1876 and the European Communities(Amendments of the Cruelty to Animals Act1976)Regulations.Experimental dietsThe experiment was designed as a complete randomised block design and comprised four dietary treatments.Thedietary treatments were as follows:(1)control diet(0ppm COS),(2)control diet plus5to10kDa COS,(3)control diet plus10to50kDa COS and(4)control diet plus50to100kDa COS.The COS was sourced from Kitto Life Co.Ltd(Kyungki-do,Seoul,Korea)and was supplemented in the experimental diets at a concentration of250ppm.The diets were fed for 8days ad libitium,after which time the pigs were humanely sacrificed.The diets were formulated to have similar diges-tible energy(16MJ/kg)and standardised ileal digestible (SID)lysine(14g/kg)contents.All amino acids requirements were met relative to SID lysine(National Research Council, 1998).The ingredient composition and chemical analysis of the dietary treatments are presented in Table1.Animals and managementA total of28piglets(progeny of large white3(large white3landrace sows))were selected from a commercial pig unit at24days of age.The piglets had a weaning weight of9.1kg(s.d.50.80)and were blocked on the basis of litter of origin and live weight(n57).The piglets were individu-ally housed in fully slated pens(1.7m31.2m).They were individually fed and had ad libitum access to feed and water. The house temperature was thermostatically controlled at 308C throughout the experiment.This study was not a growth performance study but some performance data were recorded.The piglets were weighed at the beginning of the experiment(day0)and at the end of the experiment(day8). Food was available up to thefinal weighing and all remaining food was weighed back for the purpose of cal-culating feed efficiency.Pigs were observed for clinical signs of diarrhoea and a scoring system was applied to indicate the presence and severity of this as described by Pierce et al. (2006).Faeces scores were assigned daily for individual pigs from day0and continued until day8.The following faeces scoring system was used:15hard faeces,25slightly soft faeces in the pen,35soft,partially formed faeces,45loose, semi-liquid faeces and55watery,mucous-like faeces.Gut morphological analysisThe piglets were humanely sacrificed on day8by a lethal injection of Euthatal(pentobarbitone sodium BP–Merial Animal Ltd,Sandringham House,Essex,UK)at a rate of1ml/ 1.4kg BW.On removal of the digestive tract,sections of the duodenum(10cm from the stomach),the jejunum(60cm from stomach)and the ileum(15cm from caecum)were excised andfixed in10%phosphate-buffered formalin.The preserved segments were prepared using standard paraffin-embedding techniques.The samples were sectioned at5m m Table1Composition and chemical analysis of experimental diets (as-fed basis)Items Starter diet* Ingredient(g/kg)Whey permeate125.0 Wheat444.2 Soya bean meal142.5 Whey protein isolate130.0 Full-fat soybean80.0 Soya oil65.0 Vitamins and minerals 5.0 Lysine HCL 4.5 DL-methionine 1.6L-threonine 2.2 Analysis(g/kg,unless otherwise stated)DM892.5 CP(N36.25)224.2 GE(MJ/kg)18.2 Ash43.7 NDF110.3 Lysine-16.5 Methionine and cysteine-9.9 Threonine-10.7 Tryptophan- 2.5 Calcium-8.0 Phosphorous- 6.0 DM5dry matter;GE5gross energy.Starter diet provided(mg/kg completed diet):Cu,175;Fe,140;Mn,47;Zn, 120;I,0.6;Se,0.3;retinol,1.8;cholecalciferol,0.025;alpha-tocopherol,67; phytylmenaquinone,4;cyanocobalamin,0.01;riboflavin,2;nicotinic acid,12; pantothenic acid,10;choline chloride,250;thiamine,2;pyridoxine,0.015.*COS was included in dietary treatments T2–T4at a rate of250mg/kg.-Calculated for tabulated nutritional composition(Sauvant et al.,2004).Chitooligosaccharide in piglet diets1621thickness and stained with haemotoxylin and eosin(Pierce et al.,2006).Villous height and crypt depth were measured on the stained sections(43objective)using a light micro-scopefitted with an image analyser(Image Pro Plus,Media Cybernetics,Buckinghamshire,UK).Measurements of15well oriented and intact villi and crypts were taken for each seg-ment.Villous height was measured from the crypt–villous junction to the tip.Crypt depth was measured from the crypt–villous junction to the base.Results were expressed as the mean villous height or crypt depth in micrometres. Intestinal microfloraFor microbial analysis,digesta samples(,1061g)were aseptically recovered from the proximal colon of each pig immediately post slaughter.Digesta samples were stored in sterile containers(Sarstedt,Wexford,Ireland),placed on ice and transported to the laboratory within2h.A1.0g sample was removed from the digesta sample,serially diluted (1:10)in9.0ml aliquots of maximum recovery diluents (Oxoid,Basingstoke,UK)and spread plated(0.1ml aliquots) onto selective agars,as follows:Lactobacillus spp.were isolated on de Man,Rogosa and Sharp(MRS)agar(Oxoid) with an overnight(18to24h)incubation at378C in an atmosphere enriched with5%CO2,as recommended by the manufacturers(Oxoid).The Escherichia coli species were isolated on MacConkey agar(Oxoid)following aerobic incubation at378C for18to24h(O’Doherty et al.,2010). Target colonies of Lactobacilli and E.coli were identified by Gram stains and colony morphology(Salanitro et al.,1977). The API50CHL(BioMerieux,Biomerieux,Craponne,France) kit was used to confirm suspect Lactobacilli spp.Suspect E. coli colonies were confirmed with API20E(BioMerieux, France).This API system identifies the suspect colonies by measuring their ability to produce cytochrome oxidase. Typical colonies of each bacteria on each agar were counted, log transformed and the numbers of bacteria were expressed per gram of digesta after being serially diluted.Volatile fatty acid(VFA)analysisSamples of digesta from individual pigs were taken from the caecum and the proximal colon to measure the VFA concentration and molar proportions of VFAs.The VFA con-centrations in the digesta were determined using gas liquid chromatography according to the method described by Pierce et al.(2007).A1-g sample was diluted with distilled water (2.53weight of sample)and centrifuged at14003g for4min(Sorvall GLC–2B laboratory centrifuge,Dupont, Wilmington,DE,USA).Then,1ml of the subsequent super-natant and1m l of internal standard(0.5g3-methyl-n-valeric acid in1l of0.15mol/l oxalic acid)were mixed with3ml of distilled water.Following centrifugation to remove the precipitate,the sample wasfiltered through Whatman 0.45m m polyethersulphone membranefilters into a chromato-graphic sample vial.A1-m l sample was injected into a model 3800Varian gas chromatograph with a25m30.53mm i.d. megabore column(coating CP-Wax58(FFAP)–CB(no. CP7614))(Varian,Middelburg,the Netherlands).RNA extraction and complementary DNA(cDNA)synthesis Tissue samples were collected from the mesenteric side of the colon,rinsed with ice-cold sterile phosphate-buffered saline(Oxoid)and stripped of overlying smooth muscle cells. Approximately1to2g of the porcine colon tissue was cut into small pieces and placed in tubes containing15ml of RNAlater(Applied Biosystems,Foster City,CA,USA)and immediately stored at2208C pending RNA extraction.Total RNA was extracted from colon tissue samples(25mg)using a GenElute Mammalian Total RNA Miniprep Kit(RTN70, Sigma-Aldrich,St Louis,MO,USA)according to the manu-facturer’s instructions.To eliminate possible genomic DNA contamination,total RNA samples were subjected to DNAse I(AMPD1,Sigma-Aldrich)treatment according to the man-ufacturer’s protocol.Then RNA purification was performed using a phenol–chloroform extraction method(Chomczynski and Sacchi,2006).The total RNA was quantified using a NanoDrop-ND1000Spectrophotometer(Thermo Fisher Scien-tific,Wilmington,DE,USA)and the purity was assessed by determining the ratio of the absorbance at260and280nm. All total RNA samples had260/280nm ratios above1.8.In addition,RNA integrity was verified by visualisation of the18 and28S ribosomal RNA bands stained with ethidium bromide after gel electrophoresis on1.2%agarose gels(Egel,Invitro-gen Inc.,Carlsbad,CA,USA).Total RNA(1m g)was reverse transcribed(RT)using the RevertAid H minusfirst strand cDNA synthesis kit(Fermentas GmbH,St Leon-Rot,Germany)with oligo dT primers.Thefinal RT product was adjusted to a volume of120m l using nuclease-free water.Real-time quantitative PCRAll primers for the selected cytokines,genes such as Inter-leukin-1a(IL-1a),IL-6,IL-10,tumour necrosis factor(TNF-a) and the reference genes b-actin(ACTB),b2-microglobin (B2M),glyceraldehyde-3-phosphate dehydrogenase(GAPDH) and peptidylprolyl isomerise A(PPIA)are presented in Table2. Amplification was carried out in a reaction volume of20m l containing10m l SYBR Green Fast PCR Mastermix(Applied Biosystem),forward and reverse primer mix(1m l),8m l DEPC treated water and1m l of template cDNA.Quantitative real-time PCR was carried out using an ABI PRISM7500Fast sequence detection system for96-well plates(Applied Biosys-tem).The thermal cycling conditions were as follows:an initial denaturation step at958C for10min,40cycles of958C for15s, followed by608C for1min.Dissociation analyses of the PCR product were performed to confirm the specificity of the resulting PCR products.All samples were run in triplicate.The cycle threshold value(C t)is defined as the fractional cycle number at whichfluorescence passes thefixed threshold.The mean C t values of triplicates of each sample were used for calculations.Normalisation of quantitative PCR dataNormalisation of the C t values obtained from real-time PCR was performed by(i)transforming the raw C t values into relative quantities using the formula,relative quantities5 (PCR efficiency)D C t,where D C t is the change in the C t valuesWalsh,Sweeney,Bahar,Flynn and O’Doherty 1622of the sample relative to the highest expression (minimum C t value),(ii)using geNorm,a normalisation factor was obtained from the relative quantities of four most stable housekeeping genes (GAPDH,B2M,ACTB and PPIA)and (iii)the normalised fold change or the relative abundance of each of the target genes was calculated by dividing their relative quantities by the normalisation factor.Statistical analysisThe experimental data were analysed as a randomised block design using the GLM procedure of SAS (2004).The individualpig served as the experimental unit.Food intake was inclu-ded as a covariate in the model for villous height,crypt depth and villous height to crypt depth ratio in the digestive tract.The microbial counts were log transformed.The data were checked for normality using the Proc Univariate function of SAS.The means were separated using the Tukey–Kramer Test.Probability values of ,0.05were used as the criterion of statistical significance.All results are presented in the tables as least square means 6standard error of the means (s.e.).ResultsPerformance and faecal scoringThe average faecal scores of the pigs are presented in Table 3.The supplementation of different MW of COS had no significant effect on the growth performance of the pig during the 8-day experimental period (P .0.05).However,the inclusion of COS at all MW in the diet significantly reduced faecal scores com-pared with the control treatment (P ,0.01).MicrobiologyThe effect of COS supplementation at different MW on selected microbial populations in the colon of the pig is shown in Table 3.Pigs offered diets containing 5to 10kDa COS had a lower E.coli number compared with the control (P ,0.05)and the 50to 100kDa COS (P ,0.05)treatments.The 10to 50kDa treatment had a neumerically lower E.coli number compared with the control group (P 50.09).Pigs offered diets containing 5to 10kDa COS had a significantly lower population of lacto-bacilli in the colon compared with the control group (P ,0.05)and the 50to 100kDa COS diet (P ,0.01).Pigs offered 50to 100kDa COS had a higher lactobacilli number than pigs offered 10to 50kDa COS (P ,0.05).The supplementation of different MW of COS had no significant dietary effect on the lacto-bacilli :E.coli ratio in the colon of the pig.Table 2Porcine-specific primers used for real-time PCR 1.Forward primer sequence (50-30)Gene 2.Reverse primer sequence (50-30)T m (8C)IL-6 1.AGACAAAGCCACCACCCCTAA59.82.CTCGTTCTGTGACTGCAGCAGCTTATC 62.7IL-8 1.TGCACTTACTCTTGCCAGAGAACTG 61.92.CAAACTGGCTGTTGCCTTCTT 61.7IL-10 1.GCCTTCGGCCCAGTGAA 57.62.AGAGACCCGGTCAGCAACAA 59.4TNF-a 1.TGGCCCCTTGAGCATCA55.22.CGGGCTTATCTGAGGTTTGAGA 60.3GAPDH 1.CAGCAATGCCTCCTGTACCA 62.22.ACGATGCCGAAGTTGTCATG 62.1B2M 1.CGGAAAGCCAAATTACCTGAAC 59.02.TCTCCCCGTTTTTCAGCAAAT 60.0ACTB 1.CAAATGCTTCTAGGCGGACTGT 59.02.TCTCATTTTCTGCGCAAGTTAGG 60.0PPIA1.CGGGTCCTGGCATCTTGT58.02.TGGCAGTGCAAATGAAAAACTG56.5IL 5interleukin;TNF 5tumour necrosis factor;GAPDH 5glyceraldehyde-3-phosphate dehydrogenase;B2M 5b 2-microglobin;ACTB 5genes b -actin;PPIA 5peptidylprolyl isomerise A.Primers were designed using Primer Express TM software and were synthesisedby MWG Biotech (Milton Keynes,UK).Table 3Effect of COS supplementation at different MW on faecal scoring,selected microbial populations in the proximal colon and the total VFA concentration and the proportions of VFAs in the caecum of the weaned pig (least square means and s.e.;n 57)Dietary treatmentsControl 5to 10kDa 10to 50kDa50to 100kDas.e.SignificanceFaeces scoring Days 0to 84.06b 3.31a 3.44a 3.38a 0.124**Proximal colonic bacterial population (log cfu/g of digesta)Escherichia coli5.94b 4.34a 4.71a 5.81b 0.477*Lactobacilli spp.7.39bc 6.24a 6.56ab 7.56c 0.347*VFA concentrations in the caecum Total VFA (mmol/g of digesta)95.8770.25103.00116.7913.006ns Acetic acid 67.36b 45.77a 70.30b 79.20b 8.543*Propionic acid 19.8416.5123.3927.05 3.616ns Isobutyric acid 0.770.490.810.790.157ns Butyric acid 6.45 6.14 6.817.51 1.575ns Isovaleric acid 0.67b 0.45a 0.68b 0.83b 0.092*Valeric acid0.790.881.011.410.282nsCOS 5chitooligosaccharide;MW 5molecular weight;VFA 5volatile fatty acid.Probability of significance:*P ,0.05;**P ,0.01;ns,P ,0.05.Means with the same superscript alphabets within rows are not significantly different (P .0.05).Chitooligosaccharide in piglet diets1623Volatile fatty acidsThe effects of COS supplementation at different MW on the VFA concentrations in the caecum are shown in Table3.The supplementation of different MW of COS had a significant effect on the concentrations of acetic acid(P,0.05)and isovaleric acid(P,0.05)in the caecum.Pigs fed5to10kDa COS had lower levels of acetic acid and isovaleric acid compared with the control(P,0.05),10to50kDa COS (P,0.05)and50to100kDa COS(P,005).There was no significant effect of MW on VFA concentrations(P.0.05)in the proximal colon(data not shown).Gut morphologyThe effects of varying COS MW on villous height,crypt depth and the villous height:crypt depth ratios in the gastro-intestinal tract are shown in Table4.Pigs fed the10to 50kDa COS had a higher villous height in the duodenum and the jejunum compared with the control group(P,0.05), 5to10kDa COS(P,0.01)and50to100kDa COS diets (P,0.05).There was no effect of dietary treatment on crypt depth in the duodenum(P.0.05).Pigs offered the10to 50kDa COS had a higher villous height:crypt ratio in the duodenum and the jejunum compared with the control group(P,0.05)and the5to10kDa COS diet(P,0.01).Cytokine gene expression analysisThe effects of COS supplementation on the immune response in colon tissues of the pig are shown in Table5.The supplementation of different MW of COS had no significant effect on the expression of the cytokines TNF-a,IL-6,IL-8 and IL-10(P.0.05)in the gastro-intestinal tract of the pig. DiscussionThe hypothesis of the current experiment is that the biolo-gical properties of COS may be influenced by its MW and COS will enhance selected indices of health in weaned pig-lets.It was demonstrated in the current study that the lower MW of5to10kDa possessed antibacterial activity while the higher MW of10to50kDa was optimum for enhancing intestinal structure.Dietary supplementation of COS at the low MW of5to 10kDa decreased both lactobacilli and E.coli counts,while the10to50kDa COS numerically decreased E.coli popula-tions in the colon of the pig.In a study by Liu et al.(2008), COS supplementation at different concentrations reduced E. coli concentrations in the caecum of the weanling pig.E.coli is considered to be one of the most important causes of post-weaning diarrhoea in weaned pigs;therefore,a reduction inTable4Effect of COS supplementation at different MW on villous height,crypt depth and the villous height:crypt depth ratio in the gastro-intestinal tract of the weaned pig(least square means and s.e.)Dietary treatments Control5to10kDa10to50kDa50to100kDa s.e.Significance Covariate(intake) Villous height(m m)Duodenum284.0a256.0a326.3b266.2a17.38*ns Jejunum271.6a270.7a316.5b260.8a16.15*ns Ileum239.8268.3251.5242.915.07ns ns Crypt depth(m m)Duodenum305.7330.2280.1311.718.98ns ns Jejunum294.1298.4281.6268.420.93ns ns Ileum207.5242.8228.2239.811.29ns ns Villous:crypt depth ratioDuodenum 1.0a0.8a 1.2b0.9a0.08*ns Jejunum0.9a0.9a 1.2b 1.0ab0.06*ns Ileum 1.2 1.1 1.1 1.00.06ns nsCOS5chitooligosaccharide;MW5molecular weight.Probability of significance:*P,0.05;**P,0.01;ns,P,0.05.Means with the same superscript alphabets within rows are not significantly different(P.0.05).Table5Effect of COS supplementation at different MW on the immune response in unchallenged proximal colon tissues(leastsquare means of fold change in normalised relative gene expression with their s.e.;n57animals)Dietary treatments Control5to10kDa10to50kDa50to100kDa s.e.SignificanceColonTNF-a0.3660.3530.3760.3620.0568nsIL-60.2480.3590.3180.3220.0645nsIL-80.3850.5440.3700.3580.0797nsIL-100.3640.3420.3110.3070.0614ns COS5chitooligosaccharide;MW5molecular weight;TNF5tumour necrosis factor;IL5interleukin.Probability of significance:*P,0.05;**P,0.01;ns,P,0.05.Walsh,Sweeney,Bahar,Flynn and O’Doherty1624E.coli populations may reduce the incidence of diarrhoea in post-weaned pigs(Fairbrother et al.,2005).Although many species of E.coli are commensal,high levels of specific E.coli (like ETEC)will increase the risk of disease.Unfortunately, ETEC numbers were not measured in the current study.In the current study,the faecal score was decreased in pigs fed the COS diets compared with the control.These results suggest that the supplementation of the5to10kDa and10 to50kDa COS reduces E.coli populations in the colon, resulting in a lower faecal score in the post-weaning period. The50to100kDa COS led to a reduced diarrhoea score but no reduction in E.coli populations;therefore,this MW of COS may be working as a bulking agent to affect the faecal score.The50to100kDa COS may retard the rate of passage through the intestine and may have the ability to absorb water.In the current study,it was demonstrated that supple-mentation of5to10kDa COS had the strongest antimicrobial effect against both lactobacilli and E.coli.This is in agreement with other studies in which low MW COS(5to10kDa)were shown to possess strong antibacterial properties compared with higher MW COS and the antibacterial properties of COS increased at a low MW of,5kDa against Gram-negative such as E.coli(Zheng and Zhu,2003;Kittur et al.,2005).In a study by Liu et al.(2010),COS supplementation decreased E.coli populations compared with the control in the caecum of weaned pigs,while Jeon et al.(2001)observed a anti-microbial effect of COS against Gram-positive bacteria such as Lactobacilli under in-vitro conditions.To explain COS antibacterial activity,two mechanisms have been proposed.Thefirst mechanism is that the posi-tively charged COS reacts with negatively charged molecules at the microbial cell surface,thereby altering cell perme-ability(Chung and Chen,2008).Therefore,COS may interact with the membrane of the cell to alter cell permeability. However,as evident from the current study,this activity may differ with varying MW as the50to100kDa group had no inhibitive effect on the selected microbial populations,while the MWs of5to10kDa and10to50kDa COS had the strongest inhibitive effect.The other antibacterial mechan-ism is the binding of COS with DNA to inhibit RNA synthesis (Liu et al.,2004).It has been proposed that COS penetrates the nuclei of the bacteria and interferes with RNA and pro-tein synthesis.It is noteworthy that all the COS samples used in the current study were soluble in aqueous solutions.Kim and Rajapakse(2005)found that COS with a MW of .30kDa were not effective as antibacterial agents due to their poor solubility in aqueous solutions at a neutral pH. Volatile fatty acids are the major end products of bacterial metabolism in the large intestine(Macfarlane and Macfarlane, 2003).Both protein and carbohydrate fermentation contribute to the production of acetic acid;however,branched-chain fatty acids such as isovaleric acid are produced from protein fermentation(Mackie et al.,1998).In the current study,the5 to10kDa group had the lowest selected microbial populations while also reducing isovaleric acid and acetic acid concen-trations in the caecum.The shift in the production of the fermentation end products is reflected in the reduction of the selected microbial populations.The quantity of VFA produced depends on the amount and composition of the substrate and on the type of microbes present in the large intestine (van Beers-Schreurs et al.,1998).Reduced VFA concentrations indicate that lower amounts of substrate were fermented as a result of a lower microbial activity in the caecum(Htoo et al.,2007).Villous height is generally reduced and crypt depth is increased,which may explain the increased occurrence of diarrhoea and reduced growth after weaning(Pluske et al., 1996).The inclusion of10to50kDa COS in the present study was found to increase the villous height and villous:crypt depth ratio in the duodenum and also in the jejunum com-pared with the control group.Very little data have been published on the effects of COS MW on gut morphology in weaned piglets;thus,the exact mechanism for the increase in villous height and villous:crypt depth ratio is unclear.It may be hypothesised that low MW COS has the potential to promote intestinal morphology through cell proliferation. The COS has been shown to influence colonic cell prolifera-tion,crypt depth and crypt circumference in mice(Torzsas et al.,1996).A study carried out by Liu et al.(2008),on different con-centrations of COS,demonstrated that200mg/kg of COS increased villous height and villous:crypt ratio in the jeju-num and ileum(Liu et al.,2008).The possible explanation for this improved intestinal structure was that COS is com-posed of N-acetyl glucosamine(Kim and Rajapakse,2005), which may bind to certain types of bacteria and possibly interfere with their adhesion to the gut tissue of host animals (Ofek et al.,2003;Liu et al.,2008).This result is in agree-ment with Moura˜o et al.(2006),who reported that an increase in villi length in the ileum of weaned rabbits was correlated to a lower intestinal microflora.A decrease in bacteria load has been shown to increase the proliferation of epithelial cells,which leads to an improved intestinal mor-phology and increased villous height(Moura˜o et al.,2006). In the present study,in pigs fed the lower MW of5to10kDa COS,a strong antimicrobial effect on both Lactobacilli and E.coli populations was observed,with no effect on villous structure,while the higher MW of10to50kDa resulted in a reduction in E.coli numbers in comparison with the control and was optimum for improving villous integrity.There were no effects of COS supplementation in colon tissue on any of the cytokines analysed.This overall lack of an effect on these inflammatory cytokines implies that COS inclusion in the diet had no effects on immune gene expression of the pigs.Mori et al.(1997)also demonstrated that chitin and its derivatives do not stimulate the production of IL-6,IL-1and TNF-a byfibroblasts.In our study,no dif-ferences were observed on growth performance between days0and8post-weaning.In conclusion,MW is an important factor to consider when investigating the biological properties of COS.On the basis of the current study,the lower MW of5to10kDa possessed antibacterial activity while the higher MW of10to50kDaChitooligosaccharide in piglet diets1625。

Previous Uptake of Apoptotic Neutrophils or Ligation of Integrin Receptors Downmodulates the Ability of Macrophages to Ingest Apoptotic Neutrophils By Lars-Peter Erwig,Sharon Gordon,Garry M.Walsh,and Andrew J.ReesClearance of apoptotic neutrophils(polymorphonuclear leu-kocyte[PMN])by macrophages is thought to play a crucial role in resolution of acute inflammation.There is increasing evidence that ingestion of apoptotic cells modulates macro-phage behavior.We therefore performed experiments to determine whether ingestion of apoptotic PMN modulated the uptake process itself.Rat bone marrow-derived macro-phages(BMDM)ingested apoptotic PMN by a process that was enhanced by tumor necrosis factor(TNF)and attenu-ated by interferon(IFN)-␥,interleukin(IL)-4,and IL-10.It was inhibitable by the tetrapeptide arg-gly-gln-ser(RGDS),there-fore implicating the␣v␤3/CD36/thrombospondin pathway. Interaction of apoptotic PMN with BMDM for30minutes,48 hours before rechallenge reduced uptake of apoptotic PMN by50%compared with previously unchallenged BMDM. Blocking initial uptake with RGDS abrogated the effect of parable and sustained attenuation of up-take was obtained by ligating␣v␤3with the monoclonal antibody(MoAb),F11,after a delay of more than90minutes, whereas MoAbs to CD25and CD45had no effect.Ligation of ␣6␤1and␣1␤2,integrins not previously implicated in the engulfment of apoptotic cells also decreased uptake with similar kinetics to F11.Therefore,apoptotic PMN regulate their own uptake through an integrin-dependent process, which can be reproduced by ligation of other integrins expressed by macrophages.௠1999by The American Society of Hematology.M ACROPHAGES INFLUENCE almost all aspects of immunological and inflammatory responses and play an essential role in linking the innate and acquired immunity.1 Macrophages not only induce injury,but also control key events in the resolution of inflammation and the repair processes that follow it.One of the critical functions in this process is phagocytosis of apoptotic cells via specific recognition mecha-nisms.To date,a number of recognition mechanisms for apoptotic cells have been described:(1)an uncharacterized lectin-dependent interaction2;(2)a complicated charge sensi-tive process involving the CD36/vitronectin receptor(␣v␤3) complex on the macrophage surface interacting with unknown moieties on the apoptotic polymorphonuclear leukocyte(PMN) surface via a thrombospondin bridge3,4;(3)a stereo-specific recognition of phosphatidylserine that is expressed on the surface of the apoptotic cell after loss of membrane asymme-try5,6;(4)macrophage scavenger receptors7;(5)the lipopolysac-charide(LPS)receptor CD148-10and macrosialin or CD68.11,12 The specific removal of apoptotic thymocytes,13eosino-phils,14and neutrophils15by macrophages has been well described.Extensive tissue damage and inflammation both precede and follow neutrophil death by necrosis.The cellular debris is phagocytosed by macrophages,which are activated by the process.In contrast,apoptosis of neutrophils is associated with the swift recognition of intact cells by macrophages followed by their ingestion and degradation.Local inflamma-tion and tissue injury are avoided not only because neutrophils are prevented from releasing their toxic contents,but also because the macrophages usual proinflammatory secretory response to phagocytosis is not activated16and may be biased towards release of the anti-inflammatory cytokine transforming growth factor(TGF)-␤.17These results suggest that uptake of apoptotic neutrophils by macrophages does not merely fail to induce synthesis of proinflammatory cytokines,but actively modulates macrophage function and biases the profile of cytokines they release.This raises the question whether uptake of apoptotic cells‘‘imprints’’a pattern of behavior on macrophages analogous to the effect of exposure to some cytokines.18The specific purpose of the experiments described here was to ascertain whether uptake of apoptotic neutrophils modulates the ability of macrophages to ingest a second challenge with apoptotic PMNs.The results show a substantial reduction in the proportion of macrophages that ingest a second challenge of apoptotic PMNs,but that uptake can still be modulated appropriately by cytokines.The bone marrow-derived macrophages(BMDM)uptake of apop-totic PMN is RGDS-dependent and presumptively occurs by the ␣v␤3/CD36/thrombospondin pathway.After a delay of at least 90minutes,ligation of␣v␤3also downmodulates uptake of apoptotic cells specifically,and ligation of two other integrins,␣6␤1and␣1␤2,have the same effect.This shows that uptake of apoptotic cells is regulated by events that induce signalling through integrin receptors irrespective of whether or not they are directly associated with uptake.This raises the question whether uptake of apoptotic cells via␣v␤3reciprocally influ-ences functions of integrins responsible for cell adhesion and facilitate the emigration of macrophages from an inflamed focus as described by Bellingan et al.19MATERIALS AND METHODSReagents.Recombinant human tumor necrosis factor(rhTNF)-␣, rhTGF-␤,and recombinant rat interferon(IFN)-␥were obtained from Boehringer(Ingelheim,Germany),Sigma Chemical Co(Dorset,UK), and Bradsure Biologicals Ltd(Loughborough,UK),respectively. Recombinant rat interleukin(IL)-4was produced in-house as described previously20using a Chinese hamster ovary(CHO)cell line generously donated by Dr Neil Barclay(MRC Cellular Immunology Unit,Oxford, UK).The rat monoclonal antibody(MoAb),F11,against the integrin␤3From the Department of Medicine and Therapeutics,University ofAberdeen,Aberdeen,UK.Submitted May8,1998;accepted October13,1998.Supported by Grant No.ER254/1-1from the Deutsche Forschungsge-meimschaft(to L.-P.E.),Grant No.044988/2/95/2from the WellcomeTrust(to G.M.W.),and the National Kidney Research Fund.Address reprint requests to Lars-Peter Erwig,MD,University ofAberdeen,Department of Medicine and Therapeutics,Institute ofMedical Sciences,Foresterhill,Aberdeen AB252ZD,UK;e-mail:L.P.Erwig@.The publication costs of this article were defrayed in part by pagecharge payment.This article must therefore be hereby marked‘‘adver-tisement’’in accordance with18U.S.C.section1734solely to indicatethis fact.௠1999by The American Society of Hematology.0006-4971/99/9304-0010$3.00/01406Blood,Vol93,No4(February15),1999:pp1406-1412chain21was a gift from Prof Michael Horton(Bone and Mineral Centre, University College London Medical School,London,UK).The mouse antirat integrin antibody␣6␤1,CD18,CD116,anti-CD45,anti-CD25, anti-ED3,and mouse antihuman CD21were obtained from Serotec (Oxford,UK).The rabbit antihuman erythrocyte membrane antibody was obtained from DAKO(Glostrup,Denmark).The tetrapeptides arg-gly-asp-ser(RGDS),arg-gly-glu-ser(RGES),and phospho-L-serine were obtained from Sigma Chemical Co.Isolation and culture of BMDM.Rat BMDM were obtained using a technique previously described in detail.22Briefly,bone marrow cells wereflushed aseptically from the dissected femurs of male Spraque Dawley rats with a jet of complete medium directed through a25-gauge needle to form a single cell suspension.The cells were cultured in Dulbecco’s modified Eagle’s medium(DMEM)containing2mmol/L glutamine,100U/mL penicillin,and100U/mL streptomycin,10% heat-inactivated fetal calf serum,and10%L929conditioned medium as a source of macrophage-colony stimulating factor(M-CSF).After7 days in culture,BMDM were dispensed into24-well culture plates (Corning,Corning,NY)at a concentration of5ϫ105cells/well and rested in medium without added M-CSF for24hours before use in experiments.Inhibition of uptake of apoptotic neutrophils.BMDM were incu-bated with a series of inhibitors at concentrations of1mmol/L for15 minutes at4°C and were washed immediately before interaction with apoptotic neutrophils.Phospho-L-serine was used as a stereo-specific inhibitor of the macrophage phosphatidylserine receptor,using condi-tions described by Fadok et al.5The tetrapeptide arg-gly-asp-ser (RGDS)was used as described,4and the noninhibitory peptide arg-gly-glu-ser(RGES)added as a control.Assay for uptake of apoptotic neutrophils.BMDM were transferred to24-well plates at a density of5ϫ105cells/well and rested for24 hours before the medium was changed and the cells incubated with various cytokines.Uptake of apoptotic neutrophils was assessed after 48hours using a microscopically quantified phagocytic assay,which has previously been described and illustrated in detail.4,23Apoptotic neutrophils were prepared from PMN isolated from fresh heparinized normal human blood by dextran sedimentation and Percoll centrifuga-tion.They were aged in teflon bags for approximately24hours in RPMI 1640supplemented with antibiotics and10%fetal calf serum.More than98%of these cells excluded trypan blue while apoptosis was verified by oil immersion light microscopy of May-Giemsa–stained cytospin preparations as previously described.23The apoptotic cells were washed once and resuspended in RPMI at a concentration of2.5ϫ106/mL.A total of1mL of cells was added to each well and allowed to interact with the macrophages for30minutes at37°C in a5%CO2 atmosphere.The wells were washed in saline at4°C to remove noningested PMN,fixed with2%gluteraldehyde in0.9%saline,and stained for myeloperoxidase to identify ingested PMN.The proportion of macrophages that had ingested neutrophils was then counted by inverted light microscopy.To determine the effect of previous ingestion of apoptotic neutrophils on macrophages,rat BMDM were transferred to24-well plates at a density of5ϫ105cells/well and rested for24hours before the medium was changed and cells were incubated with either medium alone,RGDS peptide followed by apoptotic neutrophils,or apoptotic neutrophils alone.After30minutes incubation,the wells were washed in saline at 4°C to remove noningested PMN,and the macrophages were rested for 48hours in control medium or medium containing various cytokines. They were then reincubated for30minutes with a second challenge of apoptotic neutrophils and the proportion of macrophages that took up PMN assessed.The assay for uptake of opsonized erythrocytes was performed exactly as previously described.24Ligation of the integrin receptors.MoAbs to␣v␤3,␣6␤1,␣1␤2, CD25,and CD45were used to assess the effect of ligation of macrophage cell surface receptors on uptake of apoptotic neutrophils.Macrophages were incubated at various MoAb concentrations ranging from0.01to10µg/mL,for30minutes,at4°C in saline,or were incubated with various concentrations of mouse antihuman CD21as an irrelevant isotype-matched control.The cells were then washed before incubation in medium for various times before the start of the standard interaction assay with apoptotic PMN.Quantitation of nitric oxide(NO)generation.Generation of NO was measured by assaying culture supernatants for nitrite,a stable reaction product of NO.Aliquots of200µL of each cell-free culture supernatant were incubated with50µL of Griess reagent(0.5% sulphanilamide,0.05%N-(1-naphtyl)ethylendiamine dihydrochloride in2.5%phosphoric acid)in96-flat–bottomed tissue culture plates for 10minutes at room temperature.The optical densities of the assay samples were then measured at540nm using a solution of phenol red free DMEM.In most experiments,nitrite was measured48hours after exposure to cytokines.RESULTSCytokines regulate uptake of apoptotic neutrophils by BMDM. The initial experiment was designed to confirm our previous observations that pro and antiinflammatory cytokines influence uptake of apoptotic human neutrophils by uncommitted rat BMDM.20TNF caused a36%increase in the proportion of BMDM that took up apoptotic neutrophils compared with controls,whereas IL-4,IL-10,and IFN-␥inhibited uptake by 56%,22%,and42%,respectively,and TGF-␤had no effect (Table1).Incubation with cytokines modulated not only the number of macrophages taking up apoptotic cells,but also the average number of neutrophils per macrophage,ie,IL-4caused a56%decrease in the number of macrophages taking up apoptotic neutrophils and a40%reduction in the number of neutrophils per macrophage.Thesefindings differ from those reported for human monocyte-derived macrophages.In these cells,incubation with proinflammatory cytokines(IFN and TNF)increased their ability to ingest neutrophils,whereas antiinflammatory cytokines(IL-4,IL-6,and IL-10)had no effect.25These differences could reflect the source and species of the macrophages used or the conditions in which they were matured.Recently,Bonder et al26have shown that human 7-day–cultured monocytes did not express the functionally active IL-2receptor␥-chain,a component of the IL-4receptor, whereas macrophages did,which may explain the different effect of IL-4on uptake of apoptotic neutrophils by monocyte-derived macrophages and BMDM.BMDM use an integrin-dependent mechanism to recognize apoptotic PMN.Human monocyte-like cell lines and murine peritoneal macrophages use the phosphatidylserine receptor Table1.Effect of Cytokines on the Number of Macrophages ThatTake up Apoptotic NeutrophilsCytokine(Concentration)Uptake of Apoptotic PMNs(%)Control31Ϯ2.8IFNϩTNF(20U/mL,10ng/mL)18Ϯ1.9*IFN(20U/mL)21.6Ϯ2†TNF(10ng/mL)42.6Ϯ2.8*TGF-␤(7.5ng/mL)28.2Ϯ2.5IL-4(5µL/mL)13Ϯ2.5*IL-10(100ng/mL)22.8Ϯ2.7Nϭ10.*PϽ.01relative to unstimulated controls.†PϽ.05relative to unstimulated controls.MODULATION OF NEUTROPHIL UPTAKE BY MACROPHAGES1407(PSR)for recognition of apoptotic cells.27Human monocyte-derived macrophages and murine BMDM have been reported to use the␣v␤3/CD36/thrombospondin pathway.3In our studies,1 mmol/L RDGS specifically inhibited uptake of apoptotic PMN by unstimulated rat BMDM and by macrophages incubated for 48hours with IFN-␥,TNF,IL-4,or TGF-␤.Neither the control peptide RGES,nor phospho-L-serine,which inhibits PS-mediated recognition of apoptotic cells,5had any effect on uptake by cytokine-stimulated or unstimulated macrophages (Table2).Thus,both uncommitted or cytokine-stimulated rat BMDM use an integrin-dependent recognition mechanism, presumptively the CD36/␣v␤3/thrombospondin system rather than a PSR-dependent mechanism.This conclusion is strength-ened by the demonstration of␣v␤3on the surface of BMDM by immunofluorescence using the MoAb,F11,directed against the ␤3subunit of the receptor(data not shown).To verify the recognition mechanism,it would be necessary to block either CD36or␣v␤3on the macrophage surface.To our knowledge, the one antirat antibody available for this purpose is the mouse MoAb F11against the␤3subunit of the vitronectin receptor, which is a poor blocking antibody under our experimental conditions.BMDM incubated with F11for45minutes and then seeded in vitronectin-coated plates adhered as efficiently as control macrophages.There was no difference in the number ofnonadherent cells(less than1%of the seeded cells in both groups)when aliquots of the supernatants of control and F11-treated macrophages were examined2,4,12,and24hours after seeding(data not shown).Previous uptake of apoptotic PMNs reduces the ability of BMDM to ingest apoptotic PMN.To determine the effect of uptake of apoptotic neutrophils on macrophage function,uncom-mitted rat BMDM were challenged for30minutes with apoptotic neutrophils in medium alone or in the presence of RGD peptide to prevent uptake.They were then rested for48 hours in medium before being reexposed to freshly prepared apoptotic neutrophils.Macrophages that had previously in-gested apoptotic PMN had a markedly reduced ability to engulf apoptotic neutrophils compared with control macrophages (Fig1),whereas their ability to take up opsonized erythrocytes was unchanged(data not shown).The difference cannot be attributed to a nonspecific effect of the neutrophils because macrophages challenged with PMN in the presence of RGDS-peptide retain their subsequent ability to take up apoptotic neutrophils(Fig1).The degree of inhibition was comparable to that observed when BMDM are exposed to IFN-␥,IL-4,or IL-10,which we have previously shown cannot be reversed by treatment with TNF.28By contrast,uptake of apoptotic cells by BMDM did not abrogate the modulatory effects of TNF or other cytokines on uptake of apoptotic cells when added to the medium after the initial challenge.However,in each,their capacity to take up apoptotic PMNs was reduced by50%.Furthermore,prior uptake of PMNs did not affect the ability of IFN-␥to prime macrophages for generation of NO(Table3).In this set of experiments,there was no significant difference in IFN/TNF-induced NO generation between uncommitted BMDM,macro-phages that had ingested apoptotic neutrophils,and macro-phages that have been incubated with RGDS peptide followed by apoptotic neutrophils.Thus,uptake of apoptotic cells specifically inhibits BMDM ability to engulf apoptotic cells without interfering with their ability to respond to a range of pro and antiinflammatory cytokines.Ligation of the␣v␤3receptor and other integrins reduce uptake of apoptotic PMNs.Overloading the macrophage phagocytic capacity provides the most obvious explanation as to why uptake of apoptotic cells prevented further uptake.Table2.Effect of Inhibitors on the Number of Macrophages Takingup Apoptotic PMN(%)ControlRGDS(1mmol/L)RGES(1mmol/L)Phospho-LSerine(1mmol/L)Control32Ϯ2.49.2*Ϯ130.6Ϯ228.2Ϯ2.2 IFN-TNF(20U/mL,10ng/mL)18.0Ϯ2.37.9*Ϯ1.517.9Ϯ4.816.3Ϯ3.2 TNF(10ng/mL)42.6Ϯ2.415*Ϯ1.839.7Ϯ3.143Ϯ3 TGF(7.5ng/mL)28Ϯ4.214.1†Ϯ2.127.5Ϯ3.324.8Ϯ3IL-4(5µL/mL)12.6Ϯ2.2 5.7†Ϯ1.913.5Ϯ112.1Ϯ1.3This table shows the effect of inhibitors on recognition of apoptotic PMNs by control and cytokine-stimulated BMDM.*PϽ.01relative to controls.†PϽ.05relative tocontrols.Fig1.Figure1shows the percentage uptake of apoptotic neutro-phils by BMDM.The macrophages were incubated48hours before the interaction assay with apoptotic neutrophils,RGDS followed by apoptotic neutrophils or medium.They were then washed and cultured in medium containing cytokines or medium alone before washing and a30-minute interaction with apoptotic PMN;mean؎standard error(SE),n؍10;*P F.01.Table3.Effect of Uptake of Apoptotic PMN or Ligation of Integrins on IFN/TNF-Induced NO Generation(Arbitrary Units)PMNRGDS(1mmol/L)ϩPMN MediumF11(1µg/mL)CD18(1µg/mL) IFN-TNF(20U/mL,10ng/mL)21.2Ϯ1.223.4Ϯ0.922.7Ϯ1.119.4Ϯ3.422.5Ϯ1.9 Control 2.1Ϯ1.2 2.9Ϯ0.5 2.5Ϯ0.7 2.09Ϯ0.9 1.8Ϯ1.11408ERWIG ET ALHowever,this seems unlikely for three reasons.First,uptake of opsonized erythrocytes did not downmodulate the ability of macrophages to ingest apoptotic neutrophils 48hours later (Table 4).Second,incubation of BMDM with neutrophils from different donors known to induce high or low uptake showed that irrespective of whether 20%or 40%of macrophages took up apoptotic cells and regardless of substantial differences in the number of PMN ingested per macrophage,the degree of downmodulation was about 50%(data not shown).Finally,the 48hours between the PMN challenges should be sufficient to allow the macrophages to recover.The fact that inhibition was prevented by incubation in the presence of RGDS suggested that the mechanism might involve the ␣v ␤3/CD36/thrombospon-din pathway.4This was addressed by incubation of BMDM for 30minutes with various concentrations of the MoAb,F11,against the ␤3subunit of the ␣v ␤3receptor.21F11blocks the calcium response after peptide binding to the vitronectin receptor in rat osteoclast.29It binds to ␣v ␤3on the surface of macrophages,but did not alter their adhesion to vitronectin,nor did it block uptake of apoptotic PMN by BMDM.Despite this,ligation of ␣v ␤3with F1112to 36hours before the interactionassay caused substantial reduction of PMN uptake (Fig 2),comparable to that induced by apoptotic PMN themselves.These data indicate that ligation of ␣v ␤3integrin decreases uptake of apoptotic neutrophils after a delay of at least 90minutes,whereas an isotype-matched control mouse antihuman CD21MoAb,which recognized neither PMNs nor macro-phages,had no effect.To examine the specificity of the effects of F11,the experiments were repeated,first using MoAb against ␣6␤1,another integrin receptor expressed by macrophages,but not known to be involved in recognition of apoptotic neutro-phils,and secondly using a MoAb against CD45,another molecule on the macrophage plasma membrane.Strikingly the MoAb against ␣6␤1also downregulated uptake of PMNs with identical kinetics to antibodies against ␣v ␤3,whereas the MoAb against CD45had no effect (Fig 3).Thus,ligation of integrins,but not of other receptors on the surface of macrophages,specifically downregu-lates uptake of apoptotic cells after a delay of more than 90minutes,but did not affect the uptake of other particles such as opsonized red blood cells (data not shown).To confirm these observations,we performed another set of experiments examining the role of receptor ligation on uptake of apoptotic PMNs by macrophages after ligation of the receptors for 45minutes 12hours before the interaction assay.Ligation of the integrin receptors CD11b (percentage uptake 14.4Ϯ2.1,P Ͻ.01)and CD18(13.4Ϯ3.4,P Ͻ.01)significantly decreased uptake of apoptotic cells by macro-phages,whereas incubation with MoAbs against the IL-2receptor CD25(28.7Ϯ3.5)present on the macrophage surface,or ED3(29Ϯ4),only expressed by activated or tissueTable 4.Effect of Uptake of Opsonized Erythrocytes on the Number of Macrophages That Take up Apoptotic Neutrophils 48Hours LaterInitial ChallengeUptake of Apoptotic PMNs (%)Control25.8Ϯ7.3Opsonized erythrocytes 27.2Ϯ4.8N ϭ5.Fig 2.Figure 2shows the effect of ligation of ␣v ␤3(1␮g/mL)and an isotype-matched control CD21(10␮g/mL)on uptake of apoptotic neutrophils by BMDM.The macrophages were incubated for 30minutes at various times before the start of the interaction assay.Mean (percentage of uptake)؎SE,n ؍8.*P F.01.Fig 3.Figure 3shows the effect of ligation of ␣6␤1and CD45(10␮g/mL)on uptake of apoptotic neutrophils by BMDM.The macro-phages were incubated for 30minutes at various times before the start of the interaction assay.Mean (percentage of uptake)؎SE,n ؍8.*P F .01.MODULATION OF NEUTROPHIL UPTAKE BY MACROPHAGES 1409macrophages,were not different from controls(29.8Ϯ3.4). Thus,ligation of three different integrin receptors specifically downmodulated macrophage ingestion of apoptotic neutrophils, whereas ligation of two other receptors on the macrophage surface did not.It seems likely that at least some of the modulating effects of apoptotic PMNs themselves can be attributed to this mechanism.In addition,there was no signifi-cant difference in IFN/TNF-induced NO generation between uncommitted BMDM and macrophages,which have been incubated with antibodies that ligate their integrin receptors (Table3).Thus,similar to uptake of apoptotic cells,ligation of integrin receptors specifically inhibits BMDM ability to engulf apoptotic cells without interfering with their ability to respond to a range of pro and antiinflammatory cytokines.DISCUSSIONThe specific uptake of apoptotic neutrophils by macrophages is one of the critical steps in the resolution of inflammation.30It provides a way to remove neutrophils before granulocyte lysis and release of the neutrophils’cytotoxic contents16and does not activate the macrophages usual proinflammatory response to phagocytosis.Indeed,Fadok et al17have recently provided evidence that uptake of apoptotic cells induces macrophages to synthesize the antiinflammatory cytokine,TGF-␤.The impor-tance of the process is illustrated by the observation that insufficient or impaired capacity for phagocytic clearance leads to disintegration of the cells undergoing apoptosis and worsen-ing of tissue damage.31We hypothesized that alterations in the process responsible for removal of apoptotic neutrophils might contribute to these observations.In some situations,induction of a single episode of acute inflammation resolves quickly, whereas a second episode results in progressive tissue damage. One explanation for this might be that the difference was caused by a reduced capacity to remove apoptotic neutrophils.32This prompted us to analyze the effect of ingestion of apoptotic cells on the ability of macrophages to take up a second pulse of apoptotic cells48hours later.The results show that uptake of the second pulse48hours after thefirst is consistently reduced by50%,which could have a substantial effect on tissue repair. The characteristics of the mechanisms responsible for im-paired uptake demonstrate that it involves specific interaction between the neutrophils and macrophages:(1)neutrophil up-take by BMDM was inhibited by RGDS,which interrupts integrin-dependent recognition;(2)it was not influenced by uptake of opsonized erythrocytes and was independent of the magnitude of thefirst‘‘neutrophil meal’’and thus unlikely to be due simply to macrophage‘‘indigestion’’;(3)the effect was sustained for at least48hours;(4)it did not interfere with cytokine-induced modulation of uptake of apoptotic cells;and (5)uptake of apoptotic neutrophils does not influence IFN-␥/ TNF-induced generation of NO by macrophages.Taken to-gether,these characteristics suggest that modulation is caused by the specific interactions between macrophage receptors and ligands on the PMN.A number of different macrophage receptor-mediated path-ways have been described to be involved in uptake of apoptotic neutrophils:(1)an uncharacterized lectin-dependent interac-tion2;(2)a complicated charge sensitive process involving the CD36/vitronectin receptor(␣v␤3)complex on the macrophage surface interacting with unknown moieties on the apoptotic PMN surface via a thrombospondin bridge3,4;(3)a stereo-specific recognition of phosphatidylserine that is expressed on the surface of the apoptotic cell after loss of membrane asymmetry5,6;(4)macrophage scavenger receptors7;(5)the LPS receptor,CD148-10and macrosialin or CD68.11,12Inhibition by RGDS,but not PS,suggests that uptake by rat BMDM in our experiments is mediated by the␣v␤3/CD36/thrombospondin recognition pathway,which has been extensively characterized by Savill et al.3,4The importance of␣v␤3was identified in blocking experiments using MoAbs.Our experiments were conducted using the MoAb,F11,an antibody to the␤3chain, which blocks some␣v␤3-dependent functions,but not the ability of BMDM to bind to vitronectin under our experimental conditions.Despite this,it caused a sustained downmodulation of the macrophages’ability to take up apoptotic cells after a delay of more than90minutes,comparable in degree to that seen after ingestion of apoptotic cells.This effect was not observed with isotype-matched control antibodies or with antibodies to CD25or CD45on the macrophage surface. Strikingly,however,antibodies to three other integrin receptors,␣6␤1,CD11b,and CD18,not known to be associated with uptake of apoptotic neutrophils,had the same effect.Thus, ligation of␤1,␤2,and␤3integrins all cause sustained specific downmodulation of uptake of apoptotic cells,but no effect on uptake of opsonized erythrocytes,and presumptively␤1and␤2 integrins downmodulate the function of␣v␤3in neutrophil uptake.There are precedents for cross-inhibition between integrin receptors,including interactions involving␣v␤3.Blystone et al33have previously shown that ligation of␣v␤3blocks high-affinity phagocytic function,but not adhesive function of thefibronectin receptor␣5␤1,possibly by influencing serine/ threonine kinase activity of the cytoplasmic portion␤1chain. Similarly␣4␤1ligation inhibits␣5␤1-dependent expression of metalloproteinases.34Diaz-Gonzalez et al35and Fenczik et al36 have analyzed the cross-talk between different integrins in detail and introduced the term transdominant inhibition to describe this phenomenon.They showed that ligation of␣ll b␤3 suppresses adhesive properties of␣5␤1and␣2␤1.35,36They demonstrated that the phenomenon was dependent on the assumption of the high-affinity state of␣ll b␤3and was attribut-able to conformational changes in the cytoplasmatic portion of the␤1chain.37It is not yet clear at what level changes in integrins might modulate uptake of apoptotic cells,partly because of uncertainties about the signalling pathways in-volved.The intracellular signalling pathways that control uptake of apoptotic cells have not been studied systematically.However, Rossi et al38have recently reported that activation of cyclic adenosine monophosphate(cAMP)signalling pathways by inflammatory mediators downmodulates macrophage ingestion of apoptotic cells and that alteration in cAMP concentrations might be responsible for the observation that ligation of CD44 specifically enhances phagocytosis of apoptotic neutrophils.39 Our results suggest that uptake is also regulated by cross-talk between integrins and emphasize the multiple levels of control for macrophage removal of apoptotic cells.The ability of macrophages to ingest apoptotic cells can be dynamically1410ERWIG ET AL。

小鼠肝Kupffer细胞分离方法探讨【摘要】目的探讨分离BALB/c小鼠肝Kupffer细胞(KC)的方法。

方法采用在体酶灌注和离体酶消化、不连续密度梯度离心、选择性贴壁三步法分离KC，并比较链霉蛋白酶、Ⅳ型胶原酶及联用链霉蛋白酶和Ⅳ型胶原酶等3种不同酶消化分离方法所得KC得率及纯度。

结果 3种不同酶消化分离方法细胞得率分别为(6.32±0.5)×106 g-1，(3.66±0.4)×106g-1，(10.3±0.7)×106 g-1；细胞纯度分别为(93.2±1.7)％，(90.7±1.5)％，(94.5±1.9)％。

结论联合链霉蛋白酶和Ⅳ型胶原酶在体灌注和离体消化是分离小鼠KC的较好方法。

【关键词】肝；枯否细胞；离心法，梯密度；小鼠，近交BABLc ；细胞分离肝Kupffer细胞（Kupffer cell，KC）为定居在肝窦内的巨噬细胞，约占全身单核巨噬细胞总数的80％～90％。

肝KC能吞噬、杀灭病原微生物，清除体内的内毒素，并具有抗原递呈、分泌细胞因子等免疫调节作用，同时影响肝细胞、贮脂细胞及内皮细胞的生物学功能[1]。

近期发现KC能诱导同种异体T淋巴细胞凋亡，在调节肝移植免疫耐受中发挥重要作用[2]。

如何获得较多数量和较高纯度的KC是研究其在机体中作用机制的首要条件。

而传统的分离方法往往因数量和纯度不足而影响实验结果。

本试验采用在体酶灌注和离体酶消化、不连续密度梯度、选择性贴壁三步法分离KC，探讨分离小鼠肝KC的较好方法。

1 材料与方法1.1 材料1.1.1实验动物 BABL/c小鼠，雄性，10～12周龄，清洁级，由四川大学华西医学中心试验动物中心提供（许可证号：046）。

试验前禁食12 h，自由饮水，随机分为3组。

1.1.2 主要试剂和仪器Ⅳ型胶原蛋白酶、DNAaseⅠ、Percoll及HEPES （美国Sigma公司）；兔抗人溶菌酶（lysozyme，美国DAKO公司）；链霉蛋白酶E（瑞士Roche公司）；RPMI��1640培养基（美国Gibco公司）。

催化基础知识普及、探讨帖之五：催化期刊及投稿催化基础知识普及、探讨帖之五：催化期刊及投稿催化知识普及、探讨系列帖第 5 帖——催化期刊及投稿此帖主题相信大家平时了解的比较多，恐怕也是大家最为关心的问题之一。

小木虫论文投稿专版关于此方面的介绍比较多也比较详细，且我们催化专版也有几个帖子专门进行了探讨和讨论，而我对这方面了解比较少（主要是没发过什么文章，哈哈），此帖内容主要是对网络上的一些投稿知识进行汇总（加入了少的可怜的自己对催化期刊的认识及投稿经验）。

目的还是办此系列帖的主旨：介绍催化相关基础知识、抛砖引玉、相互学习、分享经验及教训。

催化是一门跨学科、跨专业的科学，按理论上讲化学类，甚至物理等类的期刊都可以收录催化相关的文章，因此本贴并不打算介绍诸如《科学》《自然》《德国应用化学》、、、JACS 等等这些高等次的通用型期刊，此帖只局限于催化专业期刊。

简而言之：只介绍含有“催化”两字的相关期刊。

具体介绍各个催化期刊之前，有必要对现今几大出版社或数据库简要介绍一下（一般催化期刊都是这四个出版社或数据库名下的）：（1）Elsevier Science 出版社Elsevier 出版的期刊是世界公认的高品位学术期刊，且大多数为核心期刊，被世界上许多著名的二次文献数据库所收录。

SDOS 目前收录1700 多种数字化期刊，该数据库涵盖了食品、数学、物理、化学、生命科学、商业及经济管理、计算机科学、工程技术、能源科学、环境科学、材料科学和社会科学等众多学科。

该数据库不仅涵盖了以上各个学科的研究成果，还提供了简便易用的智能检索程序。

通过Science Direct Onsite（SDOS）中国集团的数据库支持，用户可以使用Elsevier Science 为其特别定制的科学、技术方面的学术期刊并共享资源。

目前 (截止到 2005 年 11 月 16 日)该数据库已有期刊种数1,734,期刊期数145,078 ，文章篇数2，576，316，最早年份为1995 年。

连敏，高艺玮，年新，等. 黑果枸杞花色苷的提取、纯化及降解动力学研究[J]. 食品工业科技，2024，45（6）：24−31. doi:10.13386/j.issn1002-0306.2023080105LIAN Min, GAO Yiwei, NIAN Xin, et al. Study on Extraction, Purification and Degradation Kinetics of Anthocyanins from Lycium ruthenicum [J]. Science and Technology of Food Industry, 2024, 45(6): 24−31. (in Chinese with English abstract). doi:10.13386/j.issn1002-0306.2023080105· 特邀主编专栏—枸杞、红枣、沙棘等食药同源健康食品研究与开发（客座主编：方海田、田金虎、龚桂萍） ·黑果枸杞花色苷的提取、纯化及降解动力学研究连　敏，高艺玮，年　新，王梦泽*（宁夏大学食品科学与工程学院，宁夏银川 750021）摘　要：以花色苷提取量为主要考察指标，通过单因素和正交试验优化冻干黑果枸杞花色苷提取工艺，并在此条件下研究花色苷纯化工艺及其降解动力学，探讨不同温度、pH 下花色苷提取量的变化。

结果表明，提取最佳工艺条件为：料液比1:25（g :mL ）、乙醇浓度60%、pH4、提取时间2 h ，此条件下花色苷提取量达36.507±0.325 mg/g 。

研究显示AB-8大孔树脂纯化黑果枸杞花色苷效果最好，对花色苷吸附量和解吸量的影响效果最佳，其最佳条件为：上样液浓度200 mg/100 g ，解吸乙醇浓度80%，上样流速2 mL/min ，洗脱流速2 mL/min ，上样体积为5 BV ，纯化率为90.02%。