Evaluation of the pulmonary and systemic immunogenicity of Fluidosomes, a fluid liposomal-tobramycin formulation for the treatment of chronic infections in lungs.

In previous studies, we have developed a fluid bactericidal liposomal formulation containing tobramycin, called Fluidosomes, which has been shown to be highly bactericidal both in in vitro and in in vivo studies against Pseudomonas aeruginosa and other related and unrelated bacteria. One foreseeable application of these Fluidosomes is the treatment of chronic pulmonary infections in cystic fibrosis patients colonized with P. aeruginosa and other related bacteria. Considering the capacity of some liposomal preparations to play an adjuvant role in vaccines, the non-immunogenicity of Fluidosomes has to be demonstrated. The systemic and local immunogenicity of Fluidosomes were assessed by effectuating repeated intraperitoneal (i.p.) and intratracheal (i.t. ) immunizations in BALB/c mouse. No significant mucosal and serum immune responses against Fluidosomes and/or tobramycin were detected as compared with preimmune sera. These data suggest that Fluidosomes could be administered repeatedly without adverse immune responses to control chronic pulmonary infections in cystic fibrosis.     

Mechanism of Antibacterial Activity of Liposomal Linolenic Acid against Helicobacter pylori

Helicobacter pylori infects approximately half of the world population and is a major cause of gastritis, peptic ulcer, and gastric cancer. Moreover, this bacterium has quickly developed resistance to all major antibiotics. Recently, we developed a novel liposomal linolenic acid (LipoLLA) formulation, which showed potent bactericidal activity against several clinical isolated antibiotic-resistant strains of H. pylori including both the spiral and coccoid form. In addition, LipoLLA had superior in vivo efficacy compared to the standard triple therapy. Our data showed that LipoLLA associated with H. pylori cell membrane. Therefore, in this study, we investigated the possible antibacterial mechanism of LipoLLA against H. pylori. The antibacterial activity of LipoLLA (C18:3) was compared to that of liposomal stearic acid (LipoSA, C18:0) and oleic acid (LipoOA, C18:1). LipoLLA showed the most potent bactericidal effect and completely killed H. pylori within 5 min. The permeability of the outer membrane of H. pylori increased when treated with LipoOA and LipoLLA. Moreover, by detecting released adenosine triphosphate (ATP) from bacteria, we found that bacterial plasma membrane of H. pylori treated with LipoLLA exhibited significantly higher permeability than those treated with LipoOA, resulting in bacteria cell death. Furthermore, LipoLLA caused structural changes in the bacterial membrane within 5 min affecting membrane integrity and leading to leakage of cytoplasmic contents, observed by both transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Our findings showing rapid bactericidal effect of LipoLLA suggest it is a very promising new, effective anti-H. pylori agent.     

矾冰纳米乳对临床常见病原菌体外抗菌活性的研究

研究纳米化提高白矾与冰片复合物体外抗菌活性的效果。分别采用琼脂扩散法、体外杀菌试验及试管稀释法,测定白矾与冰片O/W型复合纳米乳对临床常见病原菌的体外抑菌、杀菌效果及最低抑菌浓度(MIC),实验中以等浓度矾冰液作为对照。结果显示,矾冰纳米乳对金黄色葡萄球菌、表皮葡萄球菌、大肠埃希菌、铜绿假单胞菌、白假丝酵母菌的抑制及杀灭活性均明显强于矾冰液(P0.05)。矾冰纳米乳对金黄色葡萄球菌、铜绿假单胞菌、大肠埃希菌临床菌株MIC90值分别为1.02、2.04和2.04 mg/mL,均明显低于矾冰液的MIC90值(P0.05)。上述实验结果提示,矾冰纳米乳与矾冰液均有广谱体外抑菌及杀菌活性,白矾及冰片复合物纳米化可提高抗菌效果。     

Biological Action of Liposomes on Main Pathogenetic Mechanisms of Inflammation

Abstract

Small neutral liposomes from phosphatidylholine and cholesterol (7:5 molar ratio) were used to test their antiexudative and antialterative action on rats with acute foot oedema and chemical necrosis of soft hip tissues. The action of liposomes on erythrocytes and liver cells homogenates depending on the peroxidation level of liposomal lipids was studied in vitro. Bactericidal action of liposomes was tested on main types of conditionally pathogenic microorganisms. The interaction of liposomes with blood serum components was studied in vitro. Showed that liposomes reduced the degree of tissue hyperhydratation and shortened time of healing chemically induced necrosis. Rate of liposomal lipids oxidation had a great influence on erythrocytes resistance and lipids oxidation in liver cells homogenates. Showed that liposomes had an evident bactericidal action and could adsorbe toxic components (middle mass peptides) of blood serum. The results showed that liposomes could be used as independent drug to influence on main pathogenetic mechanisms of local and general inflammatory disease.     

Design of synthetic bactericidal peptides derived from the bactericidal domain P(18-39) of aprotinin.

A bactericidal domain, P(18-39), of the proteinase inhibitor aprotinin, possesses the structural feature of two antiparallel beta-sheets connected by a short turn. In order to understand the structural requirements for antibacterial activity, two peptides, each having the sequence corresponding to a single beta-sheet structure of P(18-39), were synthesized and their antibacterial properties investigated. One peptide, P(18-28), with the sequence IIRYFYNAKAG, was active against almost all the bacterial strains investigated. However, the bactericidal activity of P(18-28) was reduced compared to the parent molecule, P(18-39). The other peptide, P(29-39), with the sequence LCQTFVYGGCR, was only weakly bactericidal against Pseudomonas aeruginosa. A peptide, P(18-26), devoid of the C-terminus dipeptide Ala-Gly of P(18-28), retained the bactericidal activity of P(18-28) against most of the bacterial strains investigated. Only Klebsiella pneumoniae, P. aeruginosa and Staphylococcus aureus were resistant to P(18-26). Replacement of lysine 26 by arginine in P(18-26) (IIRYFYNAR) improved the bactericidal activity. The retropeptide, RANYFYRII, retained the antibacterial activity of IIRYFYNAR toward Gram-negative bacteria, but it was less active against Gram-positive bacteria. The random peptide, IANRIYRYF, was as bactericidal as IIRYFYNAR. Moreover, the random peptide possessed, in contrast to IIRYFYNAR, a strong antifungal activity against Candida albicans. Elimination of the N-hydrophobic terminal Ile-Ile from P(18-26) (RYFYNAK) strongly reduced the bactericidal potency of the peptide. Attaching the hydrophobic peptide, FFVAP, to the C-terminal of P(18-26) (IIRYFYNAKFFVAP) increased the bactericidal potency of the peptides considerably. We concluded that the order of the amino acids in the sequence of the peptides is not, per se, a critical feature for bactericidal activity. Hydrophobic interaction between peptide and bacterial membrane is probably the most important feature involved in the bactericidal mechanism of the antibiotic peptides.     

Antibiotic resistance plasmids and bactericidal effect of chlorhexidine on Enterobacteriaceae

The effect of plasmid RP4 on the bactericidal effect of chlorhexidine on five Enterobacteriaceae has been tested. Escherichia coli, Serratia marcescens and Proteus mirabilis strains harbouring RP4 were more susceptible than R^- strains to this antiseptic. The role of 17 other plasmids harboured in the same bacterial host ( E. coli ) on the bactericidal effect of chlorhexidine has also been examined. Six plasmids (R751, R702, R144, RP4, pME206, S-a), of which four belonged to incompatibility group P, produced an increased bactericidal effect of chlorhexidine.     

Genetic study of the functional organization of the colicin E1 molecule.

Colicin E1 fragments obtained by genetic manipulations of the ColE1 plasmid were tested for bactericidal activity, binding to bacterial cells, and reactions with a series of anticolicin monoclonal antibodies. Two of the fragments were also tested for ability to form channels in liposomal vesicles. The results are in agreement with studies from chemically and enzymatically derived colicin fragments, assigning the receptor binding activity to the central part of the molecule and the killing activity to a region near the carboxyl terminus.     

Resistance of Pseudomonas pseudomallei to Normal Human Serum Bactericidal Action

The effect of human normal serum (HNS) on Pseudomonas pseudomallei was determined. It is apparent from our data that the organism is resistant to the normal serum bactericidal mechanism. Ancillary experiments to confirm this serum-resistant property of P. pseudomallei were done by examining the effects of growth phase conditions of the bacteria (i.e., logarithmic and stationary phases) and different buffered systems used as diluent in our bactericidal assay. Results obtained showed similar degree of resistance to serum bactericidal killing by 5 strains of the organisms tested. The possible survival advantage of serum-resistant property to P. pseudomallei as bacterial pathogens known to invade the blood stream is discussed.     

Bactericidal antibody response against P6, protein D, and OMP26 of nontypeable Haemophilus influenzae after acute otitis media in otitis-prone children

The bactericidal antibody response to three nontypeable Haemophilus influenzae (NTHi) outer membrane proteins (D, P6, and OMP26) was studied in 24 otitis-prone children (aged 7-28?months) after an acute otitis media (AOM) caused by NTHi. The study was carried out to understand the contribution of antigen-specific bactericidal antibody responses in the class of children who are most vulnerable to recurrent otitis media infections. Levels of protein D (P?=?0.005) and P6 (P?=?0.026) but not OMP26 antibodies were higher in bactericidal sera compared with nonbactericidal sera. For five (24%) and 16 (76%) of 21 bactericidal sera tested, removal of anti-protein D and P6 antibody, respectively, resulted in a two- to fourfold drop in bactericidal antibody. Antibodies to OMP26 did not make any contribution to the overall bactericidal activity in any serum samples. Eleven of 21 sera (52%) had bactericidal activity against a heterologous NTHi (86-028 NP) strain but the titers were significantly lower (P?相似文献   

The immediate bactericidal effect of lactic acid on meat-borne pathogens

P. VAN NETTEN, J.H.J. HUIS IN'T VELD AND D.A.A. MOSSEL. 1994. The kinetics of the bactericidal effect of lactic acid decontamination (LAD) on meat-borne pathogens ( Salmonella spp., Campylobacter jejuni and Listeria monocytogenes ) was studied in an in-vitro model. The bactericidal effect was greatest on organisms in the lactic acid film that replaced the natural fluid on the meat surface during LAD. A stepwise increase in pH from 2.6 to 3.5 and 4.0 progressively reduced the bactericidal effect of decontamination. For treatment with 2% lactic acid for 30–90 s at 21C, the immediate death of Salmonella spp. decreased from about 0.5–2 log₁₀ cfu at pH 2.6 to an insignificant level at pH 4.0. The immediate death for Camp. jejuni decreased from 2.6 to >5.3 at pH 2.6 to 0.3–1.0 at pH 4.0. The decrease in bactericidal effect with increasing pH could, however, be countered by an increase in the temperature from 21C to 37C. It is suggested that 2% LAD at 37C for 30–90 s is suitable for elimination of salmonellas on meat but not for L. monocytogenes. Decontamination with 1% lactic acid at pH 3.0 and 21C for at least 30 s was effective for Camp. jejuni. Mesophilic Enterobacteriaceae were reliable indicators of the LAD-induced bactericidal effect on Salmonella spp. and Camp. jejuni.     

Enhanced killing of Pseudomonas aeruginosa by human polymorphonuclear leukocytes in presence of subinhibitory concentrations of carbenicillin and ticarcillin

The effect of carbenicillin and ticarcillin on the killing of Pseudomonas aeruginosa was studied with an in vitro system using peripheral blood polymorphonuclear (PMN) leukocytes collected from human donors. No corticosteroid was given to the donor prior to leukocytes collection by a continuous flow cell separator. The assay was carried out with or without serum. P. aeruginosa yield after a 4 hour-incubation was estimated by colony counting. In Hanks' balanced salt solution, P. aeruginosa strains 74 and 78 were resistant to human PMN leukocytes. The presence of subinhibitory concentrations of carbenicillin or ticarcillin (1/10th the minimal inhibitory concentration (MIC) for P. aeruginosa 74, 1/4th the MIC for P. aeruginosa 78) enhanced the bactericidal activity of human leukocytes. Difference between the numbers of bacteria recovered with PMN cells and without cells increased with concentration of carbenicillin or ticarcillin. The synergistic effect was not observed when serum (heated fetal calf serum or heated pooled human serum) was used. The mode of action of carbenicillin and ticarcillin on bactericidal activity of phagocytic cells was not elucidated, but we suggest the effect is due not to action on the phagocytic cells themselves but on the microorganisms.     

Prostaglandin and thromboxane in liposomes: suppression of the primary immune response to liposomal antigens

Liposomes containing lipid A as adjuvant and also containing prostaglandin E2 or thromboxane B2 were examined for the ability to influence induction of humoral immunity against liposomal protein or lipid antigens in rabbits. The protein antigen consisted of cholera toxin that was bound to ganglioside GM1 on the surface of the liposomes. High titers of anti-cholera toxin antibodies were produced and IgM and IgG responses were detected. When the immunizing liposomes contained either prostaglandin E2 or thromboxane B2 as part of the lipid bilayer, the primary immune response, involving both IgM and IgG antibodies, was greatly reduced. The secondary immune response observed after a boosting immunization was not suppressed by liposomal eicosanoids. A similar inhibitory effect on the primary response was observed when liposomal lipid antigens were examined instead of cholera toxin. An inhibitory effect of liposomal prostaglandin E2 on the phagocytic uptake of opsonized liposomes by cultured macrophages was also observed, suggesting that liposomal eicosanoids can have direct local effects on macrophages that might influence the immune response to liposomal antigens.     

The effect of ethylenediaminetetra-acetate on Pseudomonas alcaligenes and the composition of the bacterial cell wall

1. EDTA in borate buffer has a marked bactericidal effect on Pseudomonas alcaligenes, which is more sensitive than Pseudomonas aeruginosa. The bactericidal effect is accompanied by solubilization of lipopolysaccharide and release of intracellular solutes. These effects are more pronounced at pH9.2 than 7.1. 2. Cell walls of P. alcaligenes were prepared and from them were obtained the readily extracted lipids and the fractions given by treatment with aqueous phenol. 3. The cell walls and the above components were analysed and results are compared with those for P. aeruginosa. 4. Lipopolysaccharide obtained by treatment of cell walls with aqueous phenol is contaminated with glycosaminopeptide to a variable extent. 5. The lipopolysaccharide contains less neutral sugar but more phosphorus than the lipopolysaccharide of P. aeruginosa; fucosamine is not a component of the lipopolysaccharide of P. alcaligenes.     

甘草提取物对四种牙周常见致病菌的抑菌作用

目的 体外评价甘草提取物对牙龈卟啉单胞菌、中间普氏菌、具核梭杆菌和伴放线放线杆菌四种牙周常见致病菌的抑制效果。方法 以牙龈卟啉单胞菌、中间普氏菌、具核梭杆菌和伴放线放线杆菌四种牙周常见致病菌作为供试菌,采用液体稀释法,考察甘草提取物对这四种细菌的最小抑菌浓度（MIC）和最小杀菌浓度（MBC）;并采用不同浓度的甘草提取物溶液,绘制甘草提取物对四种牙周致病菌的时间‒杀菌曲线。结果 甘草提取物对牙龈卟啉单胞菌、中间普氏菌、具核梭杆菌和伴放线放线杆菌的MIC值分别为1.50、1.50、0.75和1.50 mg/mL,MBC值分别为6、3、3和3 mg/mL。当甘草提取物达到对四种细菌的MBC值时,对于牙龈卟啉单胞菌、中间普氏菌、伴放线放线杆菌可在2 h后可达到杀菌效果,对于具核梭杆菌可在4 h后达到杀菌效果。结论 甘草提取物对以上四种牙周常见致病菌具有良好的抑菌及杀菌作用。     

Suppression of penicillin-induced lysis of Staphylococcus aureus by cibacron blue 3G-A

The effect of cibacron blue 3G-A (CB) on bacteriolysis induced by penicillin G was investigated using Staphylococcus aureus FDA 209P. Penicillin-induced lysis was completely inhibited by 30 microM CB. However, the bactericidal effect of penicillin G was not influenced by CB. These results indicate that a bacteriolytic process is not essential for penicillin to kill S. aureus.     

Production of Photons in the Bactericidal Effect of Transient Electric Arcs in Aqueous Systems

The radiation in the visible and ultraviolet regions from submerged, transient electrical arcs was measured with a K(3)Fe(C(2)O(4))(3) chemical actinometer and was compared to the bactericidal effect obtained with the same electrical arrangements. Photon production and bactericidal effect were obtained at lower voltages with a smaller electrode separation than with a wider one. At higher voltages, both increased with wider electrode separations. The voltages at maximal photon production efficiency coincided with those of maximal bactericidal efficiency. However, the same photon radiation produced by different electrical arrangements did not always yield the same bactericidal effect in the small discharge vessel usually employed. In a larger discharge vessel, the bactericidal effect was closely correlated with the photon production. The efficiency of photon production by transient arcs was smaller than that of germicidal mercury lamps, particularly with respect to wavelengths of great bactericidal activity. The mechanisms of inactivation and their use for practical disinfection purposes are discussed.     

Evaluation of liposomal forms of rifampicin and prodigiozan in the treatment of experimental chronic tonsillitis

Therapeutic effect of liposomal dosages of rifampicin and prodigiozan was studied on rabbits with simulated chronic tonsillitis in comparison to that of commercial ones of the drugs. The treatment schemes included daily intra-tonsillar++ injections of the dosage forms for 5 days. A high efficacy of their liposomal dosage forms in treatment of experimental chronic tonsillitis was confirmed microbiologically and immunologically. Approval of the liposomal dosage forms used in the therapy of patients with chronic tonsillitis requires clinical trials.     

Potentiation of Inhibitory Activity of Colistin on Pseudomonas aeruginosa by Sulphamethoxazole and Sulphamethizole

Potentiation of colistin by sulphamethoxazole and sulphamethizole was demonstrated with 19 out of 20 strains of Pseudomonas aeruginosa. This enhancement was bactericidal as well as bacteriostatic. Synergy between trimethoprim and sulphamethoxazole was also demonstrated with four strains of Ps. aeruginosa, but even when the two drugs were combined high concentrations of trimethoprim were still required to produce a bactericidal effect. Combinations of sulphamethoxazole and gentamicin appeared to be synergistic when the bacteriostatic effect was measured, but the combined bactericidal effect was indifference. The bactericidal and bacteriostatic effect of combinations of carbenicillin with sulphamethoxazole was also indifference.     

IgM but not IgG monoclonal anti-Nocardia brasiliensis antibodies confer protection against experimental actinomycetoma in BALB/c mice

Nocardia brasiliensis is a facultative intracellular microorganism that produces a human chronic infection known as actinomycetoma. Human and mouse anti- N. brasiliensis antibody response identify P24, P26 and P61 immunodominant antigens. In this work, we generated immunoglobulin M (IgM) and IgG monoclonal antibodies (mAbs) specific to immunodominant P61 antigen. The monoclonal IgM (NbM1) and IgG2a (NbG1) antibodies were assessed for their in vitro bactericidal activity, in vivo protective effect and ability to block catalase activity. These mAbs specifically recognized P61, but they did not inhibit its enzyme activity. The in vitro bactericidal effect of NbG1 was higher than the killing ability of the IgM mAb. In vivo experiments with a murine model of experimental infection with N. brasiliensis injected into rear footpads was used to test the effect of NbM1 and NbG1. The negative untreated group developed a chronic actinomycetoma within 4 weeks. IgM mAbs conferred protection to BALB/c mice infected with N. brasiliensis . IgG mAb lacked this protective effect. IgM mAb showed a dose–response correlation between antibody concentration and lesion size. These results demonstrate that humoral immune response mediated by antigen-specific IgM antibody protects against an intracellular bacterial infection.     

HPC脂质体的制备及抗HL-60细胞增殖作用的初探

采用十六烷基磷酸胆碱（ＨＰＣ）作为脂质体膜材,配以胆固醇和双十六烷基磷酸盐,反相蒸发法制备出ＨＰＣ脂质体,连续５周每周测定一次它对ＣＦ（ｃａｒｂｏｘｙｆｌｕｏｒｅｓｃｅｉｎ,羧基荧光素）的包封率,可知制备的脂质体在前２周内相当稳定,５周后包封率仅减少２４％,可满足实际应用的需要．冰冻蚀刻法测定脂质体的平均直径在５００ｎｍ左右,该直径的脂质体较适于和细胞发生相互作用且稳定性比小单层脂质体好．四氮唑（ｄｉｍｅｔｈｙｌｔｈｉａｚｏｌｄｉｐｈｅｈｙｌｔｅｔｒａｚｏｌｉｕｍｂｒｏ－ｍｉｄｅ,ＭＴＴ）分析可知,在脂质体浓度达１５μｍｏｌ／Ｌ,对ＨＬ－６０细胞的增殖具有抑制作用．在相同的脂浓度下,ＨＰＣ脂质体抑制ＨＬ－６０细胞生长比游离ＨＰＣ有较强的抑制细胞增殖作用,当ＨＰＣ浓度低于５μｍｏｌ／Ｌ时,细胞生长不受抑制．当ＨＰＣ浓度在１０μｍｏｌ／Ｌ时,ＨＰＣ脂质体表现出对细胞生长的抑制作用,而游离ＨＰＣ在此浓度下的抑制作用较低