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1.
Augmentation of the Lipopolysaccharide-Neutralizing Activities of Human Cathelicidin CAP18/LL-37-Derived Antimicrobial Peptides by Replacement with Hydrophobic and Cationic Amino Acid Residues
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Isao Nagaoka Satoko Hirota Franois Niyonsaba Michimasa Hirata Yoshiyuki Adachi Hiroshi Tamura Shigenori Tanaka Didier Heumann 《Clinical and Vaccine Immunology : CVI》2002,9(5):972-982
Mammalian myeloid and epithelial cells express various peptide antibiotics (such as defensins and cathelicidins) that contribute to the innate host defense against invading microorganisms. Among these peptides, human cathelicidin CAP18/LL-37 (L1 to S37) possesses not only potent antibacterial activity against gram-positive and gram-negative bacteria but also the ability to bind to gram-negative lipopolysaccharide (LPS) and neutralize its biological activities. In this study, to develop peptide derivatives with improved LPS-neutralizing activities, we utilized an 18-mer peptide (K15 to V32) of LL-37 as a template and evaluated the activities of modified peptides by using the CD14+ murine macrophage cell line RAW 264.7 and the murine endotoxin shock model. By replacement of E16 and K25 with two L residues, the hydrophobicity of the peptide (18-mer LL) was increased, and by further replacement of Q22, D26, and N30 with three K residues, the cationicity of the peptide (18-mer LLKKK) was enhanced. Among peptide derivatives, 18-mer LLKKK displayed the most powerful LPS-neutralizing activity: it was most potent at binding to LPS, inhibiting the interaction between LPS and LPS-binding protein, and attaching to the CD14 molecule, thereby suppressing the binding of LPS to CD14+ cells and attenuating production of tumor necrosis factor alpha (TNF-α) by these cells. Furthermore, in the murine endotoxin shock model, 18-mer LLKKK most effectively suppressed LPS-induced TNF-α production and protected mice from lethal endotoxin shock. Together, these observations indicate that the LPS-neutralizing activities of the amphipathic human CAP18/LL-37-derived 18-mer peptide can be augmented by modifying its hydrophobicity and cationicity, and that 18-mer LLKKK is the most potent of the peptide derivatives, with therapeutic potential for gram-negative bacterial endotoxin shock. 相似文献
2.
Cell Differentiation Is a Key Determinant of Cathelicidin LL-37/Human Cationic Antimicrobial Protein 18 Expression by Human Colon Epithelium 总被引:20,自引:0,他引:20
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Koji Hase Lars Eckmann John D. Leopard Nissi Varki Martin F. Kagnoff 《Infection and immunity》2002,70(2):953-963
Antimicrobial peptides are highly conserved evolutionarily and are thought to play an important role in innate immunity at intestinal mucosal surfaces. To better understand the role of the antimicrobial peptide human cathelicidin LL-37/human cationic antimicrobial protein 18 (hCAP18) in intestinal mucosal defense, we characterized the regulated expression and production of this peptide by human intestinal epithelium. LL-37/hCAP18 is shown to be expressed within epithelial cells located at the surface and upper crypts of normal human colon. Little or no expression was seen within the deeper colon crypts or within epithelial cells of the small intestine. Paralleling its expression in more differentiated epithelial cells in vivo, LL-37/hCAP18 mRNA and protein expression was upregulated in spontaneously differentiating Caco-2 human colon epithelial cells and in HCA-7 human colon epithelial cells treated with the cell differentiation-inducing agent sodium butyrate. LL-37/hCAP18 expression by colon epithelium does not require commensal bacteria, since LL-37/hCAP18 is produced with a similar expression pattern by epithelial cells in human colon xenografts that lack a luminal microflora. LL-37/hCAP18 mRNA was not upregulated in response to tumor necrosis factor alpha, interleukin 1alpha (IL-1alpha), gamma interferon, lipopolysaccharide, or IL-6, nor did the expression patterns and levels of LL-37/hCAP18 in the epithelium of the normal and inflamed colon differ. On the other hand, infection of HCA-7 cells with Salmonella enterica serovar Dublin or enteroinvasive Escherichia coli modestly upregulated LL-37/hCAP18 mRNA expression. We conclude that differentiated human colon epithelium expresses LL-37/hCAP18 as part of its repertoire of innate defense molecules and that the distribution and regulated expression of LL-37/hCAP18 in the colon differs markedly from that of other enteric antimicrobial peptides, such as defensins. 相似文献
3.
Guoming Li Jing Wu Renqi Li Dong Yuan Yunxia Fan Jianjun Yang Muhuo Ji Sihai Zhu 《Inflammation》2016,39(1):54-64
Oxidative stress causes mitochondrial impairment, the failure of energy production, and consequent organ dysfunctions. The aim of the present study was to investigate the potential therapeutic effects of mitochondrial antioxidant SS-31 on sepsis-induced organ dysfunctions and to explore the possible mechanism. Sepsis was induced by cecal ligation and puncture. Immediately and at 5 h after the operation, SS-31 (5 mg/kg) or vehicle was administered intraperitoneally. The levels of organ dysfunctions, malondialdehyde, superoxide dismutase, proinflammatory cytokines, pulmonary wet-to-dry weight ratio, myeloperoxidase activity, histological scores, nuclear factor kappa B p65, inducible nitric oxide synthase, reactive oxygen species, adenosine triphosphate, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells were assessed at the indicated time points. The 7-day survival rate was estimated by the Kaplan-Meier method. In the present study, SS-31 treatment significantly improved sepsis-induced organ dysfunctions as evidenced by decreased histological scores, increased arterial partial oxygen tension, and deceased serum alanine aminotransferase, urea nitrogen, and creatinine levels, which was accompanied by decreased levels of malondialdehyde, tumor necrosis factor-alpha, pulmonary myeloperoxidase activity, nuclear factor kappa B p65, inducible nitric oxide synthase, reactive oxygen species, and TUNEL-positive cells. In conclusion, our data suggested that the protective effects of SS-31 on sepsis-induced organ dysfunctions were associated with the inhibition of proinflammatory cytokines, oxidative stress, and apoptosis. 相似文献
4.
A 20-Kilodalton N-Terminal Fragment of the D15 Protein Contains a Protective Epitope(s) against Haemophilus influenzae Type a and Type b
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Yan-ping Yang Wayne R. Thomas Pele Chong Sheena M. Loosmore Michel H. Klein 《Infection and immunity》1998,66(7):3349-3354
A conserved 80-kDa minor outer membrane protein, D15, of Haemophilus influenzae has been shown to be a protective antigen in laboratory animals against H. influenzae type a (Hia) or type b (Hib) infection. To localize the protective B-cell epitope(s) within the D15 protein and to further explore the possibility of using synthetic peptides as vaccine antigens, a 20-kDa N-terminal fragment of D15 protein (truncated D15 [tD15]) was expressed as a fusion protein with glutathione S-transferase in Escherichia coli. The tD15 moiety was cleaved from glutathione S-transferase by using thrombin and purified to homogeneity. The purified soluble tD15 appeared to contain immunodominant protective epitope(s) against Hia and Hib, since rabbit antisera directed against tD15 were capable of protecting infant rats from Hia or Hib bacteremia. The ease of purification of soluble tD15, therefore, makes it a better candidate antigen than the full-length recombinant D15 which is produced as inclusion bodies in E. coli. Furthermore, both the purified tD15 fragment and a mixture of tD15-derived peptides spanning amino acid residues 93 to 209 of the mature D15 protein were capable of inhibiting the protection against Hib conferred on infant rats by rabbit anti-tD15 antiserum, indicating that the protective epitopes of D15 may not be conformational. However, the administration of pooled rabbit immune sera raised against the same panel of peptides failed to protect infant rats from Hib infection. 相似文献
5.
Immunoreactivity of Five Monoclonal Antibodies against the 37-Kilodalton Common Cell Wall Protein (PsaA) of Streptococcus pneumoniae
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Jennifer Crook Jean A. Tharpe Scott E. Johnson Derrick B. Williams Annie R. Stinson Richard R. Facklam Edwin W. Ades George M. Carlone Jacquelyn S. Sampson 《Clinical and Vaccine Immunology : CVI》1998,5(2):205-210
Five monoclonal antibodies (MAbs) were produced against the Streptococcus pneumoniae pneumococcal surface adhesin A (PsaA) 37-kDa common cell wall protein. These antibodies were used in a dot immunoblot and Western blot study of clinical isolates of S. pneumoniae to detect the presence of the protein. By both assays, the MAbs reacted with clinical isolates representing the 23 type-specific serotypes present in the licensed pneumococcal polysaccharide vaccine. Western blot analysis confirmed the presence of a protein migrating in the gel with a molecular mass of 37 kDa. An extension of the study by using dot immunoblot analysis that included an analysis of the 90 serotypes of S. pneumoniae showed that all five MAbs reacted with 89 of the 90 serotypes tested. MAb 1B6, the exception, did not react with S. pneumoniae serotype 16F. Dot immunoblot analysis of the MAbs with Enterococcus faecalis and viridans streptococci showed varied reactivity patterns, depending on the species. The MAbs against the 37-kDa antigen did not react with Escherichia coli, respiratory pathogens, or nonpathogens representing 22 genera and 29 species of bacteria. All five MAbs also reacted with five multidrug-resistant strains of S. pneumoniae. In summary, these MAbs may be useful for detection of pneumococcal antigen and may lead to the development of diagnostic assays for pneumococcal disease. 相似文献
6.
Livia Cristina Liporagi Lopes Allan J. Guimar?es Mariana Duarte de Cerqueira Beatriz L. Gómez Joshua D. Nosanchuk 《Clinical and Vaccine Immunology : CVI》2010,17(7):1155-1158
Monoclonal antibodies to Histoplasma capsulatum can modify pathogenesis. We now show that monoclonal antibody H1C to a 70-kDa antigen increases intracellular fungal growth and reduces macrophage nitric oxide release but has no effect on fungal burden or survival in murine infection. This further demonstrates the complexities of host-pathogen interactions.Histoplasma capsulatum is a dimorphic fungal pathogen that is the causative agent of histoplasmosis. After Candida species, H. capsulatum is the most common causative agent of systemic mycoses in North America and either the first- or second-most-common cause in Central and South America (4, 7). We previously identified an IgG1 isotype monoclonal antibody (MAb), H1C, that reacts with a 70-kDa H. capsulatum cell wall antigen (6). The MAb is highly specific for H. capsulatum, as it did not react with yeast cell antigens from related microbes. An inhibition enzyme-linked immunosorbent assay (ELISA) system using H1C is also highly specific for the detection of antigenemia and antigenuria in patients with histoplasmosis (6). Furthermore, the inhibition ELISA may be useful for monitoring treatment responses in patients with histoplasmosis (5).We have shown that MAbs to the H. capsulatum cell surface proteins histone 2B (H2B) and heat shock protein 60 (Hsp60) can alter the intracellular fate of the fungus, modify the inflammatory response to infection, and prolong the survival of lethally infected mice (8, 9). In particular, we demonstrated the marked protective efficacy of IgG1 and IgG2a MAbs to Hsp60. In the present study, we assessed the capacity of H1C to modify host-pathogen interactions.H1C was purified from cell culture supernatants using protein G-agarose beads (Pierce Biotechnology) according to the manufacturer''s instructions. Aliquots of H1C were screened to ensure the absence of endotoxin with the Limulus amebocyte lysate assay (BioWhittaker Inc.). An irrelevant, isotype-matched mouse IgG1 MAb (SouthernBiotech) was used as a control for all experiments. H. capsulatum G217B was used as the reference strain for all the studies (gift from G. Deepe, University of Cincinnati). H. capsulatum yeast cells were grown in Ham''s F-12 medium at 37°C with rotary shaking and collected as described previously (9). Enumeration of the yeast cells was accomplished with a hemacytometer.Immunofluorescence and immunoblotting were performed as described previously (8), except that H1C labeled with Alexa 488 was used in lieu of MAbs to Hsp60. Fluorescence analysis revealed that H1C diffusely labeled the fungal cell surface in a punctuate manner (Fig. (Fig.11 A), and H1C reacted with a 70-kDa protein, as shown by immunoblotting (Fig. (Fig.1B).1B). A whole-cell H. capsulatum yeast ELISA was also used to examine the binding of H1C to H. capsulatum, as described previously (9). Significant binding occurred at concentrations >1 μg/ml H1C (Fig. (Fig.1C).1C). Hence, H1C readily interacted with the 70-kDa protein at the H. capsulatum cell surface.Open in a separate windowFIG. 1.MAb H1C binds to a 70-kDa protein on the cell surface of H. capsulatum. (A) Immunofluorescence analysis demonstrates that H1C diffusely reacts with the fungal cell surface. (B) H1C reacts with a 70-kDa protein in cell wall extracts from H. capsulatum. (C) Yeast cell ELISA demonstrates the reactivity of H1C with intact fungal cells. Experiments were repeated at least three times, with consistent results.Phagocytosis and killing assays were accomplished as described by confocal microscopy and fluorescence-activated cell sorting (9) using J774.16 macrophagelike cells. H1C increased the association of H. capsulatum with the J774.16 cells (Fig. (Fig.22 A). Although increases in yeast cell attachment occurred at 25 and 50 μg/ml of H1C (Fig. (Fig.2B),2B), phagocytosis of H. capsulatum increased at all the concentrations tested (Fig. (Fig.2C).2C). Further, H. capsulatum opsonized with H1C had significantly increased intracellular survival after 24 h compared to controls (Fig. (Fig.2D).2D). GraphPad Prism version 5.00 for Windows (GraphPad Software) was used for statistical analyses. Kruskal-Wallis nonparametrical tests were used for one-way analysis of variance to compare differences between groups, and individual comparisons of groups were performed with the Bonferroni posttest.Open in a separate windowFIG. 2.MAb H1C alters H. capsulatum-macrophage interactions. (A) H1C alters H. capsulatum association with J774.16 macrophage-like phagocytosis. H1C opsonization affects H. capsulatum attachment (B) and phagocytosis (C). (D) H1C-opsonized H. capsulatum has significantly improved intracellular survival. (E) Opsonization of H. capsulatum with H1C reduces nitric oxide production. (F) Opsonization of H. capsulatum with H1C increases macrophage death. Macrophage toxicity data are at 2 h of coculture, and similar results were present at 4 h. For each graph, three independent experiments were performed, with each condition tested in triplicate, except for the nitric oxide experiments, which were performed twice with triplicates. Graphs were generated with pooled data from the experiments. The bars are the mean values, with error bars representing the standard deviations. *, P < 0.05, which indicates a comparison between the condition and H. capsulatum in the absence of antibody. “Hc” represents yeast without antibody in the presence of macrophages, “irrelevant” indicates an IgG1 isotype control antibody with yeast cells and macrophages, and the concentrations listed represent the specific amounts of H1C MAb present with H. capsulatum yeast and macrophages.Since intracellular growth increased in the presence of H1C, we examined the production of nitric oxide and superoxide by J774.16 cells cocultured with H. capsulatum exposed to H1C, irrelevant antibody, or phosphate-buffered saline (PBS), as described previously (10), and also assessed J774.16 viability. Nitric oxide formation was assessed with a commercial Griess reagent kit (Promega), and superoxide dismutase activity was quantified with a kit (Cayman Chemical). H1C-opsonized H. capsulatum induced significantly less nitric oxide release than controls at concentrations ≥25 μg/ml after 2 h of coculture (Fig. (Fig.2E).2E). In contrast, there were no differences in superoxide radicals (data not shown). J774.16 viability after exposure to H. capsulatum was measured using an MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay (Sigma-Aldrich). H1C concentrations ≥25 μg/ml in the presence of H. capsulatum yeast cells resulted in a dose-dependent toxicity on the J774.16 cells at 2 h of coculture (Fig. (Fig.2D).2D). H1C alone in the presence of the J774.16 cells had no effect on the macrophages (data not shown). Given the damage to the macrophages caused by the opsonized H. capsulatum yeast cells, it is possible that the reduction in nitric oxide was a direct effect of the reduction in viable macrophages.C57BL/6 mice (6 to 8 weeks old, female; NCI) were used for survival studies, which were approved by the Animal Institute Committee of the Albert Einstein College of Medicine. C57BL/6 mice were injected intraperitoneally with 250 μg of H1C, an isotype-matched control MAb, or PBS. Two hours later, the mice were intranasally challenged with either 5 × 106 CFU (sublethal dose) or 1.25 × 107 CFU (lethal dose for survival studies) of H. capsulatum yeast. The mice were closely monitored. CFU studies revealed that there was a slight trend toward an increase in numbers of CFU at day 3 after infection in animals given H1C, but the differences at days 3 and 7 in the fungal burdens between H1C-treated animals and controls were not significant (Fig. (Fig.33 A). Lethally challenged mice receiving H1C died at day 10 ± 1, whereas control mice died between days 10 and 12, which results are not statistically different by Kaplan-Meier analysis (P > 0.05). We also tested the efficacy of 100 and 500 μg of H1C in the lethal-challenge model, but these doses similarly failed to alter survival (data not shown).Open in a separate windowFIG. 3.Pretreatment with intraperitoneal injections of MAb H1C. A dose of 100 μg does not alter the pulmonary fungal burden in sublethally infected mice (A) or prolong survival in mice receiving lethal challenge (B). CFU studies were preformed with three mice per condition for each day assessed. Survival studies were performed using groups of 10 mice, and the experiment was repeated, with similar results.MAbs have been shown to protect against several fungal diseases, including candidiasis, cryptococcosis, aspergillosis, and pneumocystosis (2, 3). We have previously demonstrated that IgM, IgG2a, and IgG1 MAbs to H. capsulatum H2B or Hsp60 modify histoplasmosis (8, 9), but an IgG2b to Hsp60 is disease enhancing (8, 9). Hence, this report presents the second nonprotective MAb to H. capsulatum. Isotype has been shown to influence the outcome of cryptococcosis with human and murine MAbs to the major polysaccharide of Cryptococcus neoformans; in addition, a recent study showed that human IgG1-treated mice had accelerated death (1). Our findings do not rule out the possibility that a protective antibody to the 70-kDa protein could be generated, either one with a different isotype or one corresponding to a different epitope on the protein. Notably, although we found no differences in fungal burden or survival in our murine infection model, our results nevertheless suggest a possible role for the 70-kDa molecule in the virulence of the fungus, since H1C-opsonized H. capsulatum had increased intracellular survival, modified nitric oxide production, and increased macrophage damage. Our data further demonstrate the complexities involved in antibody-pathogen interactions.(The data in this paper are from a thesis to be submitted by A. J. Guimarães in partial fulfillment of the requirements for a Ph.D. degree from the Sue Golding Graduate Division of Medical Science, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY.) 相似文献
7.
Characterization of a rabbit cationic protein (CAP18) with lipopolysaccharide-inhibitory activity. 总被引:3,自引:4,他引:3
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M Hirata Y Shimomura M Yoshida J G Morgan I Palings D Wilson M H Yen S C Wright J W Larrick 《Infection and immunity》1994,62(4):1421-1426
Cationic antibacterial proteins (CAP) were purified from rabbit granulocytes, and the effects of CAP on lipopolysaccharide (LPS)-induced tissue factor generation by murine peritoneal macrophages and human blood monocytes were studied. CAP were purified from rabbit peritoneal leukocytes by using as an assay the agglutination of erythrocytes coated with Re-LPS. Two proteins with CAP activity, CAP18 (18 kDa) and CAP7 (7 kDa), were isolated by acid extraction, ethanol precipitation, affinity chromatography, gel filtration, and reverse-phase high-pressure liquid chromatography. On the basis of protein sequencing, CAP7 was identified as the C-terminal fragment of CAP18, designated CAP18(106-142). Various forms of LPS (S-LPS, Re-LPS, and lipid A) activate murine macrophages and human blood monocytes to generate tissue factor (tissue thromboplastin). Incubation of LPS for 18 h with partially purified CAP (heparin-Sepharose fraction) inhibited the capacity of LPS to induce tissue factor; however, purified CAP18 inhibited about 75% of the activity of S-LPS after 1 h of incubation. CAP more effectively inhibited S-LPS than Re-LPS or lipid A. Synthetic CAP18(106-142) inhibited LPS-induced tissue factor generation by murine macrophages. CAP18(106-142) has greater LPS-binding and LPS-neutralizing activities than CAP18. We hypothesize that CAP18 and the derivative peptide, CAP18(106-142), bind to LPS and alter the capacity of LPS to initiate disseminated intravascular coagulation. In this regard, CAP may have therapeutic potential for sepsis and endotoxin shock. 相似文献
8.
The Lipopolysaccharide of Bordetella bronchiseptica Acts as a Protective Shield against Antimicrobial Peptides
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Resistance profiles of the two Bordetella species B. bronchiseptica and B. pertussis against various antimicrobial peptides were determined in liquid survival and agar diffusion assays. B. bronchiseptica exhibited significantly higher resistance against all tested peptides than B. pertussis. The most powerful agents acting on B. bronchiseptica were, in the order of their killing efficiencies, cecropin P > cecropin B > magainin-II-amide > protamine > melittin. Interestingly, for B. bronchiseptica, the resistance level was significantly affected by phase variation, as a bvgS deletion derivative showed an increased sensitivity to these peptides. Tn5-induced protamine-sensitive B. bronchiseptica mutants, which were found to be very susceptible to most of the cationic peptides, were isolated. In two of these mutants, the genetic loci inactivated by transposon insertion were identified as containing genes highly homologous to the wlbA and wlbL genes of B. pertussis that are involved in the biosynthesis of lipopolysaccharide (LPS). In agreement with this finding, the two peptide-sensitive mutants revealed structural changes in the LPS, resulting in the loss of the O-specific side chains and the prevalence of the LPS core structure. This demonstrates that LPS plays a major role in the resistance of B. bronchiseptica against the action of antimicrobial peptides and suggests that B. pertussis is much more susceptible to these peptides due to the lack of the highly charged O-specific sugar side chains. 相似文献
9.
CD4+ T-Cell- and Gamma Interferon-Dependent Protection against Murine Malaria by Immunization with Linear Synthetic Peptides from a Plasmodium yoelii 17-Kilodalton Hepatocyte Erythrocyte Protein
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Yupin Charoenvit Victoria Fallarme Majam Giampietro Corradin John B. Sacci Jr. Ruobing Wang Denise L. Doolan Trevor R. Jones Esteban Abot Manuel E. Patarroyo Fanny Guzman Stephen L. Hoffman 《Infection and immunity》1999,67(11):5604-5614
Most work on protective immunity against the pre-erythrocytic stages of malaria has focused on induction of antibodies that prevent sporozoite invasion of hepatocytes, and CD8(+) T-cell responses that eliminate infected hepatocytes. We recently reported that immunization of A/J mice with an 18-amino-acid synthetic linear peptide from Plasmodium yoelii sporozoite surface protein 2 (SSP2) in TiterMax adjuvant induces sterile protection that is dependent on CD4(+) T cells and gamma interferon (IFN-gamma). We now report that immunization of inbred A/J mice and outbred CD1 mice with each of two linear synthetic peptides from the 17-kDa P. yoelii hepatocyte erythrocyte protein (HEP17) in the same adjuvant also induces protection against sporozoite challenge that is dependent on CD4(+) T cells and IFN-gamma. The SSP2 peptide and the two HEP17 peptides are recognized by B cells as well as T cells, and the protection induced by these peptides appears to be directed against the infected hepatocytes. In contrast to the peptide-induced protection, immunization of eight different strains of mice with radiation-attenuated sporozoites induces protection that is absolutely dependent on CD8(+) T cells. Data represented here demonstrate that CD4(+) T-cell-dependent protection can be induced by immunization with linear synthetic peptides. These studies therefore provide the foundation for an approach to pre-erythrocytic-stage malaria vaccine development, based on the induction of protective CD4(+) T-cell responses, which will complement efforts to induce protective antibody and CD8(+) T-cell responses. 相似文献
10.
J. A. Schrumpf M. A. J. A. van Sterkenburg R. M. Verhoosel S. Zuyderduyn P. S. Hiemstra 《Infection and immunity》2012,80(12):4485-4494
Vitamin D is an important regulator of the expression of antimicrobial peptides, and vitamin D deficiency is associated with respiratory infections. Regulating expression of antimicrobial peptides, such as the human cathelicidin antimicrobial peptide 18 (hCAP18)/LL-37, by vitamin D in bronchial epithelial cells requires local conversion of 25(OH)-vitamin D3 (25D3) into its bioactive metabolite, 1,25(OH)2-vitamin D3 (1,25D3), by CYP27B1. Low circulating vitamin D levels in childhood asthma are associated with more-severe exacerbations, which are often associated with infections. Atopic asthma is accompanied by Th2-driven inflammation mediated by cytokines such as interleukin 4 (IL-4) and IL-13, and the effect of these cytokines on vitamin D metabolism and hCAP18/LL-37 expression is unknown. Therefore, we investigated this with well-differentiated bronchial epithelial cells. To this end, cells were treated with IL-13 with and without 25D3, and expression of hCAP18/LL-37, CYP27B1, the 1,25D3-inactivating enzyme CYP24A1, and vitamin D receptor was assessed by quantitative PCR. We show that IL-13 enhances the ability of 25D3 to increase expression of hCAP18/LL-37 and CYP24A1. In addition, exposure to IL-13 resulted in increased CYP27B1 expression, whereas vitamin D receptor (VDR) expression was not significantly affected. The enhancing effect of IL-13 on 25D3-mediated expression of hCAP18/LL-37 was further confirmed using SDS-PAGE Western blotting and immunofluorescence staining. In conclusion, we demonstrate that IL-13 induces vitamin D-dependent hCAP18/LL-37 expression, most likely by increasing CYP27B1. These data suggest that Th2 cytokines regulate the vitamin D metabolic pathway in bronchial epithelial cells. 相似文献
11.
Inhibition of Natural Killer Cell Cytotoxicity by a Monoclonal Antibody Directed against Adhesion-Mediating Protein gp 90 (CD18) 总被引:1,自引:0,他引:1
I. AXBERG U. RAMSTEDT M. PATARROYO P. BEATTY H. WIGZELL 《Scandinavian journal of immunology》1987,26(5):547-554
Contact is required between the effector natural killer (NK) or cytotoxic killer T cell and its corresponding target in order for efficient lysis to occur. Several surface molecules of different types are involved in this process. Here we could show that Fab fragments from a murine monoclonal antibody reacting with gp 90, the human leucocyte common antigen CD18, are extremely efficient in blocking human NK of killer T cells, regardless of whether the target has or does not have the antigen. In contrast, no impact of the antibody was observed when the effector cells were of murine origin, again regardless of whether the target cell did or did not display the leucocyte common antigen. The inhibition could be shown to occur at the level of blockage of target-conjugate formation. This means that the functional display of effector/target gp 90 on the effector but not the target cell is necessary for efficient lysis to occur both in NK and killer T cell systems. 相似文献
12.
13.
Long-wang Chen Wei Chen Zhi-qiang Hu Jia-lan Bian Lan Ying Guang-liang Hong Qiao-meng Qiu Guang-ju Zhao Zhong-qiu Lu 《Inflammation》2016,39(2):575-582
Acute kidney injury (AKI) is a serious complication of sepsis, which has a high mortality rate. Growth arrest-specific protein 6 (Gas6), the protein product of the growth arrest specific gene 6, has been shown to have an anti-apoptotic effect as well as pro-survival capability. Here, we investigated the effects of Gas6 on sepsis-associated AKI in mice subjected to cecal ligation and puncture (CLP). We found that the administration of rmGas6 significantly reduced serum urea nitrogen and creatinine and improved the survival of septic mice. Furthermore, the renal pathological damage induced by CLP was attenuated by rmGas6 treatment. Finally, rmGas6 reduced the renal tissue apoptotic index and the expression of Bax, while it upregulated the expression of Bcl-2. The data suggest that rmGas6 might be used as a potential therapeutic agent for sepsis-induced AKI. 相似文献
14.
Development and Evaluation of an Epstein-Barr Virus (EBV) Immunoglobulin M Enzyme-Linked Immunosorbent Assay Based on the 18-Kilodalton Matrix Protein for Diagnosis of Primary EBV Infection 总被引:1,自引:3,他引:1
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K. H. Chan R. X. Luo H. L. Chen M. H. Ng W. H. Seto J. S. M. Peiris 《Journal of clinical microbiology》1998,36(11):3359-3361
A new immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) based on the recombinant Epstein-Barr virus (EBV) matrix protein was developed. Compared to indirect immunofluorescence for the detection of IgM antibody to the EBV capsid antigen on clinical specimens, the sensitivity and specificity of the new IgM ELISA were 96 and 96%, respectively. 相似文献
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用纯化的36kD蛋白免疫Balb/c小鼠。取免疫脾细胞与小鼠骨髓瘤细胞SP2/0以PEG进行细胞融合,融合率为86%。采用ELISA筛选出杂交瘤细胞阳性孔。经过4~7次有限稀释克隆化,共获得7株能稳定分泌36kD相应MCAb的杂交瘤细胞。将杂交瘤细胞分别注射小鼠腹腔,测定腹水McAb的效价为1:5×10~3~1:1×10~5,抗体亚型均为IgG1。用荧光显微镜观察,结果发现EC2作用的人精子头颈部出现明显荧光反应,提示共同抗原存在于人精子头颈部。这些结果有助于进一步探讨细菌感染引起男性不育的机制以及研制细菌避孕疫苗等工作。 相似文献
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Human and Murine Immune Responses to a Novel Leishmania major Recombinant Protein Encoded by Members of a Multicopy Gene Family 总被引:1,自引:0,他引:1
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John R. Webb Antonio Campos-Neto Pamela J. Ovendale Tricia I. Martin Erika J. Stromberg Roberto Badaro Steven G. Reed 《Infection and immunity》1998,66(7):3279-3289
Vaccination of BALB/c mice with Leishmania major promastigote culture filtrate proteins plus Corynebacterium parvum confers resistance to infection with L. major. To define immunogenic components of this protein mixture, we used sera from vaccinated mice to screen an L. major amastigote cDNA expression library. One of the immunoreactive clones thus obtained encoded a novel protein of L. major with a molecular mass of 22.1 kDa. The predicted amino acid sequence of this clone exhibited significant homology to eukaryotic thiol-specific-antioxidant (TSA) proteins. Therefore, we have designated this protein L. major TSA protein. Southern blot hybridization analyses indicate that there are multiple copies of the TSA gene in all species of Leishmania analyzed. Northern blot analyses demonstrated that the TSA gene is constitutively expressed in L. major promastigotes and amastigotes. Recombinant TSA protein containing an amino-terminal six-histidine tag was expressed in Escherichia coli with the pET17b system and was purified to homogeneity by affinity chromatography. Immunization of BALB/c mice with recombinant TSA protein resulted in the development of strong cellular immune responses and conferred protective immune responses against infection with L. major when the protein was combined with interleukin 12. In addition, recombinant TSA protein elicited in vitro proliferative responses from peripheral blood mononuclear cells of human leishmaniasis patients and significant TSA protein-specific antibody titers were detected in sera of both cutaneous-leishmaniasis and visceral-leishmaniasis patients. Together, these data suggest that the TSA protein may be useful as a component of a subunit vaccine against leishmaniasis. 相似文献
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Saeyoung Park Archana R. Parameswar Alexei V. Demchenko Moon H. Nahm 《Infection and immunity》2009,77(8):3374-3379
Streptococcus pneumoniae, a major human pathogen, expresses at least 91 serologically distinct carbohydrate capsules. Since pneumococcal vaccines are designed to elicit antibodies against many different capsular polysaccharides (PSs), it is important to identify the epitopes involved in eliciting anti-capsular PS antibodies. We investigated the epitopes recognized by Dob1, which is a hybridoma-secreting human immunoglobulin G2 antibody to the PS of serotype 6B (Y. Sun et al., Infect. Immun. 67:1172-1179, 1999). We found that Dob1 bound synthetic capsular carbohydrates Gal(1→3)α-d-Glcp(1→3)α-l-Rhap(1→3)Rib-ol and α-d-Glcp(1→3)α-l-Rhap(1→3)Rib-ol but did not bind α-l-Rhap(1→3)Rib-ol. The critical epitope α-d-Glcp(1→3)α-l-Rhap is found in the capsular PSs of serotypes 6A, 6B, 6C, and 19A but not in the 19F PS. Consistent with this observation, Dob1 bound to the PSs of serotypes 6A, 6B, 6C, and 19A but did not bind the 19F PS and 23 additional unrelated pneumococcal capsular PSs. Also, Dob1 could opsonize pneumococci expressing serotypes 6A, 6B, 6C, and 19A but did not opsonize 19F pneumococci. In addition, ca. 7% of immune sera (12 of 175 sera) had significant amounts of Dob1-like antibodies, i.e., reacted with 6B and 19A PSs, but not with 19F PS. Humans can produce antibodies to the Dob1 epitope and the antibodies to that epitope cross-react with the four serotypes 6A, 6B, 6C, and 19A that belong to different serogroups. This epitope may be useful for producing a totally synthetic, simple chemical structure that is capable of generating protective antibodies to multiple pneumococcal serogroups.Streptococcus pneumoniae is a major human pathogen that is responsible for a large percentage of the cases of pneumonia, bacteremia, meningitis, and otitis media in young children and old adults (5). Most pathogenic pneumococci express a carbohydrate capsule, which is recognized as their most important virulence factor. Because of their importance, pneumococcal capsules have been the subject of extensive chemical and serological studies. These studies have found that pneumococci, as a species, produce at least 91 different pneumococcal serotypes (22). In some cases, capsular polysaccharides (PSs) from two serotypes are sufficiently similar in structure that antibodies to one capsule type can cross-react with the similar capsule type (14). For instance, serotype 6B PS, which differs from 6A PS in only one chemical linkage (Table (Table1),1), can elicit antibodies that cross-react with 6A PS (31). Such serologically related serotypes are grouped together to form a single serogroup (8, 15). Also, for such cross-reacting antibodies to be cross-protective, they should opsonize pneumococci expressing cross-reactive serotypes as well.
Open in a separate windowSince antibodies to the carbohydrate capsules of pneumococci can protect the host, currently available pneumococcal vaccines are designed to elicit antibodies to the capsule. Old adults are routinely immunized with the 23-valent pneumococcal PS vaccine (PPV23) (25), and young children are immunized with a PS-protein conjugate vaccine. A widely used conjugate vaccine is the 7-valent pneumococcal conjugate vaccine (PCV7). Although the “conjugate vaccine” has been found to be highly effective, chemical conjugation is technically difficult. In addition, the conjugate vaccines are expensive to produce and usually contain only a limited number of serotypes (e.g., 7 to 13 serotypes) (24, 33). To allow more serotypes to be included in conjugate vaccines, extensive efforts are under way to increase the efficiency of conjugating PS to proteins.Another approach to improving conjugate vaccines is to identify simple chemical structures that can elicit antibodies to the capsule (11, 13, 27). Various groups of researchers have chemically synthesized parts of capsular PS and tested those “parts” for their ability to elicit antibodies in animals (27). However, animal studies provide unreliable guidance since the immunogenicity of the chemical constructs is species dependent. This limitation may be overcome if various chemical constructs can first be tested for their ability to bind to human immune sera. When we explored this idea using the human monoclonal antibody Dob1, which was previously reported to be specific for pneumococcal serogroup 6 PSs (30), we discovered a novel epitope shared between 19A PS and serogroup 6 PSs. 相似文献
TABLE 1.
Structure of pneumococcal PSs and synthetic carbohydrates used in this studyCategory | Serotype | Structure |
---|---|---|
Pneumococcal PSs | 2 | {4)-β-d-Glcp-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-β-l-Rhap-(1→}n |
2 | ||
↑ | ||
1 | ||
α-d-GlcpA-(1→6)-α-d-Glcp | ||
6A | {2)-α-d-Galp-(1→3)-α-d-Glcp-(1→3)-α-l-Rhap-(1→3)-d-ribitol-5-P-(O→}n | |
6B | {2)-α-d-Galp-(1→3)-α-d-Glcp-(1→3)-α-l-Rhap-(1→4)-d-ribitol-5-P-(O→}n | |
6C | {2)-Glcp-(1→3)-Glcp-(1→3)-Rhap-(1→3)-ribitol-5-P-(O→}n | |
14 | {4)-β-d-Glcp-(1→6)-β-d-GlcpNAc-(1→3)-β-d-Galp-(1→}n | |
4 | ||
↑ | ||
1 | ||
β-d-Galp | ||
19A | {4)-β-d-ManpNAc-(1→4)-α-d-Glcp-(1→3)-α-l-Rhap-1-P-(O→}n | |
19F | {4)-β-d-ManpNAc-(1→4)-α-d-Glcp-(1→2)-α-l-Rhap-1-P-(O→}n | |
Synthetic carbohydrates | (6A Di)-BSA | α-l-Rhap-(1→3)-d-ribitol |
(6A Tri)-BSA | α-d-Glcp-(1→3)-α-l-Rhap-(1→3)-d-ribitol | |
(6A Tetra)-BSA | α-d-Galp-(1→3)-α-d-Glcp-(1→3)-α-l-Rhap-(1→3)-d-ribitol | |
(6B Di)-BSA | α-l-Rhap-(1→4)-d-ribitol | |
(6B Tri)-BSA | α-d-Glcp-(1→3)-α-l-Rhap-(1→4)-d-ribitol | |
(6B Tetra)-BSA | α-d-Galp-(1→3)-α-d-Glcp-(1→3)-α-l-Rhap-(1→4)-d-ribitol |