Induction of immune-related gene expression in Ctenopharyngodon idella kidney cells by secondary metabolites from immunostimulatory Alcaligenes faecalis FY-3

This study was undertaken to isolate active secondary metabolites from immunostimulatory Alcaligenes faecalis FY-3 and evaluate their activities using grass carp Ctenopharyngodon idella kidney (CIK) cells. By applying chromatography techniques and successive recrystallization, three purified metabolites were obtained and identified by spectral data (mass spectrometry and nuclear magnetic resonance) as: (1) phenylacetic acid, (2) p-hydroxyphenylacetylamide and (3) cyclo-(Gly-(L)-Pro). CIK cells were stimulated by different concentrations (1, 10 and 100 μg/ml) of the isolated compounds, and expression of MyD88, IL-1β, TNF-α, type I-IFN and IL-8 genes at different time points (2, 8 and 24 h) post-stimulation was quantified by real-time PCR. The known immunostimulatory agent lipopolysaccharide (LPS) was used as a positive control. To analyse whether these compounds are toxic to the cells, the methyl tetrazolium assay was employed to measure changes in cell viability. The obtained results revealed that transcribing level of MyD88, an important adaptor molecule in toll-like receptor signalling pathway, was augmented remarkably by all the three isolated compounds and LPS as early as 2-h exposure. These compounds also induced gene expression of cytokines such as IL-1β, TNF-α and type I-IFN. Under the experimental conditions, none of the test compounds is toxic to the CIK cells. These findings demonstrate that the immunostimulatory properties of the three metabolites [phenylacetic acid, p-hydroxyphenylacetylamide and cyclo-(Gly-(L)-Pro)] from A. faecalis FY-3 in CIK cells and highlight the potential of using these metabolites as immunostimulants in fish aquaculture.     

Carbon substrate utilization studies of some cultures of Alcaligenes denitrificans, Alcaligenes faecalis, and Alcaligenes odorans isolated from clinical specimens.

One hundred and sixty-two cultures of Alcaligenes species (A. denitrificans, A. faecalis, and A. odorans) of clinical origin were characterized by routine diagnostic and carbon substrate utilization techniques. The microorganisms were tested for their ability to utilize a total of 188 substrates. Substrate utilization was assayed by (i) growth stimulation and (ii) substrate alkalinization. The A. denitrificans and A. odorans cultures had unique substrate utilization profiles for each species. The A. faecalis isolates were redefined by colonial morphology into two biotypes: (i) biotype I, morphologically and biochemically similar to the A. denitrificans cultures and (ii) biotype II, morphologically similar to the A. odorans cultures.     

PER-1 extended-spectrum beta-lactamase production in an Alcaligenes faecalis clinical isolate resistant to expanded-spectrum cephalosporins and monobactams from a hospital in Northern Italy

An Alicaligenes faecalis (FL-424/98) resistant to expanded-spectrum cephalosporins and aztreonam was isolated from the urine of an inpatient at the Intensive Care Unit of the Varese Hospital (Northern Italy) after antimicrobial chemotherapy with cefazolin, vancomycin, and amikacin. Clavulanic acid restored the activity of expanded-spectrum cephalosporins, suggesting the production of an extended-spectrum beta-lactamase (ESbetaL). A crude extract of FL-424/98 showed the presence of two beta-lactamase activities focusing at pH 5.3 and 7.6, respectively. The ESbetaL activity, purified by means of three chromatographic steps, was found to correspond to the pI 5.3 enzyme. Determination of kinetic parameters confirmed that the enzyme efficiently hydrolyzed expanded-spectrum cephalosporins and aztreonam. A colony-blot hybridization revealed the presence of blaPER-related sequences in FL-424/98, and sequencing confirmed the identity of this determinant with blaPER-1, previously detected in Pseudomonas aeruginosa, Acinetobacter, and Salmonella clinical isolates from Turkey. Finding of blaPER-1 in a species that can be part of the resident human microbiota raises the possibility that it could be an efficient shuttle for spreading of this resistance gene among other opportunistic pathogens that are normally members of the resident microbiota. Kinetic parameters determined for the PER-1 enzyme with some cephalosporin substrates were somewhat different from those previously reported.     

Curdlan, a (1----3)-beta-D-glucan from Alcaligenes faecalis var. myxogenes IFO13140, activates the alternative complement pathway by heat treatment

From the results of consumption experiments of guinea pig complement, Curdlan, a (1----3)-beta-D-glucan obtained from Alcaligenes faecalis var. myxogenes IFO13140, has been found to lack the ability to activate complement when unheated or preheated at either 40 degrees C or 50 degrees C. However, Curdlan heated at or above 60 degrees C increased complement consumption. This activation, dependent on the temperature of the Curdlan, was via the alternative complement pathway as assessed by cleavage of factor B into Ba and Bb fragments. These results suggest a substantial change must occur in Curdlan with heat treatment for alternative pathway activation.     

Fluorescence in situ hybridization for rapid identification of Achromobacter xylosoxidans and Alcaligenes faecalis recovered from cystic fibrosis patients

Achromobacter xylosoxidans is frequently isolated from the respiratory secretions of cystic fibrosis (CF) patients, but identification with biochemical tests is unreliable. We describe fluorescence in situ hybridization assays for the rapid identification of Achromobacter xylosoxidans and Alcaligenes faecalis. Both assays showed high sensitivities and high specificities with a collection of 155 nonfermenters from CF patients.     

Biological activities of C3 beta c, a novel neutrophil chemoattractant derived from the beta-chain of rat complement C3.

Biological activities of C3 beta c, which is a C-terminal fragment of the beta-chain of rat complement C3, have been studied by in vivo and in vitro experiments. C3 beta c was purified as a novel neutrophil chemoattractant from the exudate of the chronic phase of rat carrageenin-induced inflammation. The purified C3 beta c induced neutrophil chemotaxis in vivo when C3 beta c was injected into the preformed air-pouch on the back of rats. C3 beta c transiently increased the intracellular free Ca2+ concentration of neutrophils and enhanced the adhesion of neutrophils to fibrinogen in vitro, suggesting that C3 beta c has the ability to express an adhesion molecule of rat neutrophils. In addition, C3 beta c at low concentrations (10(-10)-10(-11) M) stimulated rat macrophages to produce cytokine-induced neutrophil chemoattractant-2, a member of the interleukin-8 family. Furthermore, C3 beta c enhanced vascular permeability in vivo, which is suppressed by cyproheptadine, suggesting that C3 beta c may have the characteristics of an anaphylatoxin. Our results suggest that C3 beta c contributes to oedema formation and neutrophil accumulation at inflammatory sites in rats.     

Liver-protective activities of aucubin derived from traditional oriental medicine

The iridoid glycosides including aucubin (AU), catalpol (CA), swertimarin (SW), and gardenoside (GA) are frequently found as natural constituents of many traditional oriental medicinal plants including Chinese herbs. Among these iridoid glycosides, AU was systematically studied for its potent liver-protective activities using experimental systems of hepatic damage. AU showed high liver-protective activity against carbon tetrachloride-induced hepatic damage in mice. Also AU showed significant protective activity against alpha-amanitin-induced hepatic damage in mice, and it prevented a depression of liver RNA biosynthesis caused by alpha-amanitin administration. Potent antidotal effects on mushroom poisoning in beagle dogs ingested with aqueous extract of Amanita virosa was observed; beagle dogs completely survived, even when AU administration was withheld for half an hour after mushroom poisoning. In addition, AU was found to suppress hepatitis B viral DNA replication in vitro. Conversion of AU to its aglycone form appeared to be a prerequisite step for an exhibition of such antiviral activity.     

Molecular cloning and characterization of two types of CD8alpha from ginbuna crucian carp, Carassius auratus langsdorfii

We cloned and sequenced full-length cDNAs for two types of CD8alpha from the S3n strain of ginbuna crucian carp (Carassius auratus langsdorfii) and quantified the expression of CD8alpha genes after sensitization by scale grafting, employing a model system of clonal triploid ginbuna and tetraploid ginbuna-goldfish hybrids. RT-PCR yielded four different fragments of CD8alpha homologue from the S3n strain of ginbuna and these sequences were classified into two groups. The two types of ginbuna CD8alpha (gbCD8alpha) were also found in other strains of triploid ginbuna and goldfish, which are a subspecies of ginbuna. The gbCD8alpha chains consisted of a signal peptide, Ig superfamily (IgSf) V-like domain, hinge, transmembrane domain, and cytoplasmic domain similar to other known CD8alpha. Phylogenetic analysis indicated that both types of gbCD8alpha are closely related to CD8alpha from other vertebrates. Expression of both types of gbCD8alpha mRNA was detected in the gill, thymus, head kidney, posterior kidney, spleen, intestine and peripheral blood leucocytes. In addition, quantitative real-time PCR analysis demonstrated that copy numbers of both gbCD8alpha gene products in kidney cells increased significantly following grafting with allogeneic but not isogeneic scales, and that regulation of expression correlated with that of TCRbeta. Expression of both gbCD8alpha genes after second scale allografting was elevated compared to that after the first set of grafting. These results suggest that expression analysis of these two gbCD8alpha sequences provides a useful tool to address the involvement of cytotoxic T-lymphocytes during the cell-meditated immune response in fish.     

Hemagglutinating and hemolytic activities of Enterococcus faecalis strains isolated from different human clinical sources

A total of 95 Enterococcus faecalis strains isolated from different human clinical sources were investigated for hemagglutinating activities and hemolysin (Hly) production in the presence of erythrocytes from a wide range of species. MRHA (mannose-resistant hemagglutination) activity was found in all clinical strains tested in this study. MRHA of E. faecalis strains isolated from different sources was most frequently observed with human (both group O and A) and guinea pig erythrocytes. None of the strains agglutinated horse erythrocytes in the presence of 1% alpha-D-mannose. It should be emphasized that our data indicate the absence of a relationship between sources and MRHA. In contrast, all 95 strains investigated in this report were negative for MSHA (mannose-sensitive hemagglutination) activity. Regarding hemolysin production, it was seen that E. faecalis, and particularly urinary strains, preferably lysed horse erythrocytes. On the other hand, none of the 95 clinical strains tested in this study showed hemolytic activity against bovine and sheep erythrocytes. In general, these results show that E. faecalis strains isolated from different clinical sources possessed a diversity of hemagglutinins and a limited repertoire of hemolysin activities.     

Immunostimulatory and immunomodulatory peptides derived from the alpha1 domain of HLA-B27 in experimental autoimmune diseases in Lewis rats

Peptides derived from amino acid sequence 60-80 of HLA-B27 (B27PA, aa 60-72 and B2702PA, aa 60-80) mimic cytokeratin and are able to induce in vitro proliferation of human peripheral blood lymphocytes as well as arthritis in Lewis rats. Here we show that the pathogenic epitope recognized by autoaggressive rat T cells is located at the N-terminus of the sequence, between aa 60 and 72. A C-terminally elongated 25mer peptide (B2702.60-84) showed increased pathogenicity, indicating either a second arthritogenic epitope or an immunomodulatory region within this peptide. B2702.60-84 has been described to inhibit murine and human CD8 + cytotoxic T cells (CTL) and was even successfully used for the treatment of allograft rejection. In addition to pathogenicity we have investigated the immunomodulatory effect of peptide B2702.60-84 in our rat model of experimental autoimmune uveitis (EAU), induced with retinal S-Antigen peptide PDSAg. We found that disease exacerbated following coimmunization of PDSAg with B2702.60-84. In vitro, the B27-peptide enhanced the proliferation of CD4+ T cell lines specific for retinal autoantigen peptides during coincubation of B2702.60-84 with the respective antigen. Oral tolerance induction, an effective mechanism to prevent uveitis in Lewis rats, is abrogated by cofeeding peptide B2702.60-84 with the tolerogen PDSAg. In rat EAU, naturally occurring regulatory T cells and orally induced gamma deltaTCR+ suppressor cells are CD8+ which might be impeded by peptide B2702.60-84. As a consequence of their abrogated suppressive capacity disease was exacerbated. We propose a similar role of HLA-B27 in man: disturbing the mechanisms down-regulating self-responses might lead to autoimmune diseases. This could explain the high association of HLA-B27 with a variety of autoimmune diseases targeting different organs or tissues.     

Chemokine CCR3 ligands-binding peptides derived from a random phage-epitope library

Eosinophils are major effectors cells implicated in a number of chronic inflammatory diseases in humans, particularly bronchial asthma and allergic rhinitis. The human chemokine receptor C-C receptor 3 (hCCR3) provides a mechanism for the recruitment of eosinophils into tissue and thus has recently become an attractive biological target for therapeutic intervention. In order to develop peptides antagonists of hCCR3-hCCL11 (human eotaxin) interactions, a random bacteriophage hexapeptide library was used to map structural features of hCCR3 by determining the epitopes of neutralizing anti-hCCR3 mAb 7B11. This mAb t is selective for hCCR3 and exhibit potent antagonist activity in receptor binding and functional assays. After three rounds of biopanning, four mAb7B11-binding peptides were identified from a 6-mer linear peptide library. The phage bearing the peptides showed specific binding to immobilized mAb 7B11 with over 94% of phages bound being competitively inhibited by free synthetic peptides. In FACScan analysis all selected phage peptides were able to strongly inhibit the binding of mAb 7B11 to hCCR3-transfected preB-300-19 murine cells. Furthermore, synthetic peptides of the corresponding phage epitopes were effective in blocking the antibody–hCCR3 interactions and to inhibit the binding of hCCL11 to hCCR3 transfectants. Chemically synthesized peptides CKGERF, FERKGK, SSMKVK and RHVSSQ, effectively competed for ¹²⁵I-hCCL11 binding to hCCR3 with IC₅₀ ranging from 3.5 to 9.7 μM. Calcium release and chemotaxis of hCCR3 transfectants or human eosinophils were inhibited by all peptides in a dose-dependent manner. Furthermore, they showed inhibitory effects on chemotaxis of human eosinophils induced by hCCL11, hCCL5, hCCL7, hCCL8, and hCCL24. Specificities of all selected peptides were assessed with hCXCR1, hCXCR2, hCXCR3, and hCCR5 receptors. Peptides CKGERF and FERKGK showed inhibitory effects on eosinophil chemotaxis in a murine model of mCCL11-induced peritoneal eosinophilia. The development of peptides inhibiting the interactions between hCCR3 and its chemokine ligands will facilitate the development of small peptides antagonists with the hope of ameliorating chronic inflammatory diseases in humans.     

Trypanoplasma borreli cysteine proteinase activities support a conservation of function with respect to digestion of host proteins in common carp

Trypanoplasma borreli is an extracellular parasite that is transmitted by a leech vector and is naturally found in the blood of cyprinid fish. High parasitemia and associated severe anemia together with splenomegaly are typical of infection of common carp, Cyprinus carpio L. Papain-like cysteine proteinases expressed by trypanosome parasites contribute to the pathogenicity of trypanosomes, and are considered an important target for the development of new trypanocidal drugs. T. borreli is a member of the Parabodonida, sharing a common ancestor with the other Kinetoplastida. We demonstrate the presence of a cysteine proteinase expressed by T. borreli. Alignment of the sequence with other kinetoplastid cysteine proteinase sequences supports the phylogenetic hypotheses based on analyses of ribosomal RNA genes. We expressed the T. borreli cysteine proteinase in Escherichia coli, refolded the purified protein into a biologically active proteinase and showed it has cathepsin L-like activity. Addition of the (non)active proteinase to in vitro-derived carp head kidney-derived macrophages did not significantly modulate macrophage activity. Immunization of carp with the recombinant proteinase did induce a very high increase in proteinase-specific antibodies but only slightly lowered parasitemia. Digestion of host hemoglobin and immunoglobulin by the cysteine proteinase likely contribute to the pathogenicity of T. borreli. The possibility that digestion by the cysteine proteinase of host transferrin could contribute to an innate activation profile of macrophages in vivo is discussed. Our findings suggest a conservation of function with respect to cysteine proteinase activity in the Parabodonida in support of the hypotheses on the phylogeny of the Kinetoplastida.     

Isolation of Enterococcus faecalis from a case of nonpuerperal breast abscess

Nonpuerperal breast abscess (NPBA) has different etiology as compared to the mastitis occurring in post partum women. The condition presents either as acute suppurative infection or chronic type. Organisms usually implicated are Staphylococcus aureus, coagulase negative staphylococci, and anaerobes. Mostly the infection is polymicrobial in nature. Herein, we report the isolation of Enterococcus faecalis from a case of acute suppurative NPBA.     

Human interleukin-3-like activity, basophil and eosinophil growth promoting activities and colony stimulating factor derived from several cell lines

Human IL-3-like activity, colony stimulating factor (CSF) and basophil/eosinophil growth promoting activity (Ba/Eo GPA) in serum-free conditioned media (CM) derived from various cell lines of human origin were examined. Squamous cell carcinoma (Colo-16), osteogenic sarcoma (R97KL4) and human placental (HP) cells produced 10-20% IL-3 activity present in supernatants from a mouse myelomonocytic cell line (WEHI-3BCM) when assayed using a murine IL-3 dependent cell line (32Dcl/H4). The human T-cell leukemic cell line (Mo) and several neuroblastoma cell lines did not produce IL-3-like activity, nor did purified human erythroid potentiating activity (EPA) from Mo contain IL-3. CSF and Ba/Eo GPA were detected in CMs from Mo, HP, Colo-16 but not from R97KL4. No IL-2 activity was detected in any of these CMs. These observations point to the existence of diverse sources of human IL-3-like activity and to the probable distinctiveness of human IL-3, basophil or eosinophil GPA, and EPA. Analogies drawn between human and murine hemopoietic activities need to be made with caution.     

Antigen-specific and non-specific helper activities derived from supernatants of human influenza virus-specific T-cell lines.

A T-cell line (H3) was established by culturing human peripheral blood mononuclear cells with influenza virus A/X31 and maintained in long term culture with Interleukin-2 (TCGF). Supernatants were prepared by culturing these cells overnight in the absence of Interleukin-2 but with A/X31 and irradiated autologous E rosette negative cells as a source of antigen presenting cells, and harvesting by centrifugation. The supernatants were shown to replace T cells in helping E- (B) cells to produce antibody specific to A/X31 which was measured by enzyme immunoassay (EIA). Although maximal help was obtained with autologous or semi allogeneic B cells (in the latter case bearing HLA-DR 3 loci) there was still significant antibody production with allogeneic combinations. The supernatants were subsequently fractionated into specific and non-specific helper activities by gel filtration, giving an approximate mol. wt of 50-70,000 and 10-30,000 for each respectively. The specific HF was shown to be genetically restricted in its action upon B cells and also to generate antibody to A/X31 only. The lower molecular weight material acted on any responding B cell regardless of HLA-DR type and produced antibody non-specifically in culture with E- cells even in the absence of antigen. The apparent lack of restriction was therefore due to the masking effect of non-specific and non-restricted HF(s) on the genetically restricted specific HF produced by this line.     

Binding sites of interleukin-3-mimetic monoclonal autoantibodies derived from a MRL/lpr mouse

MRL/lpr mouse-derived interleukin-3 (IL-3)-mimetic monoclonal antibodies were examined for their binding sites. One of these five antibodies (B10, F8, F9, F12, H11), F9 interacted with the IL-3 receptor, as if it were an anti-idiotypic antibody; the IL-3-mimetic activity of F9 was blocked by a neutralizing rat monoclonal anti-IL-3 antibody. IL-3 mRNA was not detected in hybridoma F9, as analyzed by the S1 protection assay, Thus, the activity neutralized by the rat antibody is of the F9 antibody itself but not the IL-3 type. Such blocking was not observed with the IL-3-mimetic activity of the other MRL/lpr-derived monoclonal antibodies. On the other hand, the binding of all these monoclonal antibodies to IL-3-dependent cells was inhibited by each other and vice versa, as analyzed by two-color flow cytometry. This indicates that the binding sites of the five monoclonal antibodies are located so close to each other that the binding of one would interfere with the binding of any one of the others (since the binding experiment was done on ice, it is unlikely that the inhibition is due to down-modulation of the receptors). Taken together the results obtained by the enzyme digestion study, we discussed that all five IL-3-mimetic monoclonal antibodies are directed to the IL-3 receptor, but only F9 binds to the portion directly responsible for the binding of IL-3 and the other antibodies (B10, F8, F12, H11) bind to different portions, respectively, which are adjacent or overlapping to the binding site of F9.     

粪肠球菌噬菌体 IME-EF3 的分离及其治疗优势的发掘简

目的利用医院污水分离到临床耐药粪肠球菌的噬菌体IME-EF3,分析其生物学特性,发掘其治疗特性,为噬菌体制剂个体化治疗耐药粪肠球菌感染及利用噬菌体消除耐药粪肠球菌污染提供应用基础。方法利用医院污水,经污水富集法分离噬菌体,以粪肠球菌临床耐药分离株为宿主菌。经双层平板法确定噬菌斑形态及大小,纯化后测定其宿主谱范围、最佳感染复数（MOI）、一步生长曲线,并检测温度、紫外线照射对IME-EF3活性的影响。取适量噬菌体液,利用蛋白酶K俗DS法提取其基因组,对其进行酶切处理．确定其遗传物质的组成及形态。用酶切法进行了随机克隆,测序结果进行在线Blastx,并构建系统进化树分析与已知噬菌体的进化关系。结果成功分离到一株噬菌体斑．边缘清晰,斑体透明的裂解性粪肠球菌噬菌体IME-EF3热稳定性较好．对紫外线敏感。一步生长曲线显示IME-EF3的潜伏期为10～20min,呈爆发性增长,裂解量为75。其遗传物质为双链DNA,通过Blastx结果初步确定IME-EF3为尾病毒目,长尾噬菌体科。通过系统进化树可看出IME—EF3与其他噬菌体的亲缘关系较远。结论从医院废水成功分离到裂解性噬菌体IME-EF3,其爆发力强、潜伏期短、高裂解性、较好的热稳定性、与已知噬菌体远缘性等一系列的优良特点,为其噬菌体个体化治疗及与药物的联合使用提供了依据。