The use of the antimicrobial peptide piscidin (PCD)-1 as a novel anti-nociceptive agent

The antimicrobial peptide piscidin (PCD)-1 has been reported to have antibacterial and immunomodulatory functions. Here, we investigated the anti-neuropathic properties of PCD-1, in order to determine its potential as a compound to alleviate pain. Treatment with PCD-1 suppressed the inflammatory proteins COX-2 and iNOS in murine macrophage (RAW264.7) and microglial (BV2) cell lines stimulated by lipopolysaccharide (LPS). For studies of the effect of PCD-1 in vivo, mononeuropathy in rats was induced by chronic constriction injury (CCI), and the resulting anti-nociceptive behaviors were compared between CCI controls and CCI rats given intrathecal injections of PCD-1. Much like gabapentin, PCD-1 exerts anti-nociceptive effects against thermal hyperalgesia, with a median effective dose (ED50) of 9.5 μg in CCI rats. In CCI rats, PCD-1 exerted effects against mechanical and cold allodynia, thermal hyperalgesia, and weight-bearing deficits. Furthermore, CCI-mediated activation of microglia and astrocytes in the dorsal horn of the lumbar spinal cord were decreased by PCD-1. In addition, PCD-1 suppressed up-regulation of interleukin-1β (IL-1β) and phosphorylated mammalian target of rapamycin (phospho-mTOR) in CCI rats. Finally, CCI-induced down-regulation of transforming growth factor-β1 (TGF-β1) in rats was attenuated by injection of PCD-1. Taken together, the present findings demonstrate that the marine antimicrobial peptide PCD-1 has anti-nociceptive effects, and thus may have potential for development as an alternative pain-alleviating agent.     

Modulation of immune responses by the antimicrobial peptide, epinecidin (Epi)-1, and establishment of an Epi-1-based inactivated vaccine

Current efforts to improve the effectiveness of vaccines include incorporating antimicrobial peptides mixed with a virus. The antimicrobial peptide, epinecidin (Epi)-1, was reported to have an antiviral function, and an Epi-1-based inactivated vaccine was postulated as a model and discussed. In this report, we demonstrated modulation of immune responses by Epi-1 and an Epi-1-based Japanese encephalitis virus (JEV)-inactivated vaccine against JEV infection in mice. Under in vitro conditions, Epi-1 prevented JEV infection-mediated loss of cell viability in BHK-21 cells. When Epi-1 and JEV were co-injected into mice and mice were re-challenged with JEV after 14 days, all mice survived. In addition, Epi-1 modulated the expressions of immune-responsive genes like interleukin (IL)-6, IL-10, MCP-1, tumor necrosis factor-α, interferon-γ and IL-12, and elevated the levels of anti-JEV-neutralizing antibodies in the serum. The presence of Epi-1 suppressed the multiplication of JEV in brain sections at 4 days after an injection. Mice immunized with the developed vaccine showed complete survival against JEV infection, and it was superior to the traditional formalin-based JEV-inactivated vaccine. This study demonstrates the use of Epi-1 to develop an inactivated vaccine can provide guidelines for the future design of Epi-1-virus formulations for various in vivo applications.     

Psoriasin (S100A7) is a principal antimicrobial peptide of the human tongue

The human tongue is particularly resistant to bacterial infections although the mouth is continuously exposed to a complex and abundant ensemble of microbes, such as the common intestinal bacterium Escherichia coli. We show that lingual epithelia produce and release, as a primary E. coli-killing compound, the S100 protein psoriasin. No significant reduction in psoriasin release could be achieved through repeated rinsing of the epithelial surface of the tongue. Psoriasin is produced in the upper layers of the lingual epithelia but is lacking in the most superficial and basal cells. It displays a gradient pattern of expression with decreasing expression from the anterior one-third to the posterior portion of the tongue. Thus, psoriasin may be the key to the resistance of the human tongue toward E. coli.     

Antimicrobial activity of truncated alpha-defensin (human neutrophil peptide (HNP)-1) analogues without disulphide bridges

Antimicrobial peptides play an important role in host defence, particularly in the oral cavity where there is constant challenge by microorganisms. The alpha-defensin antimicrobial peptides comprise 30-50% of the total protein in the azurophilic granules of human neutrophils, the most abundant of which is human neutrophil peptide 1 (HNP-1). Despite its antimicrobial activity, a limiting factor in the potential therapeutic use of HNP-1 is its chemical synthesis with the correct disulphide topology. In the present study, we synthesised a range of truncated defensin analogues lacking disulphide bridges. All the analogues were modelled on the C-terminal region of HNP-1 and their antimicrobial activity was tested against a range of microorganisms, including oral pathogens. Although there was variability in the antimicrobial activity of the truncated analogues synthesised, a truncated peptide named 2Abz(23)S(29) displayed a broad spectrum of antibacterial activity, effectively killing all the bacterial strains tested. The finding that truncated peptides, modelled on the C-terminal beta-hairpin region of HNP-1 but lacking disulphide bridges, display antimicrobial activity could aid their potential use in therapeutic interventions.     

Antibacterial activity of lactoferrin and a pepsin-derived lactoferrin peptide fragment.

Although the antimicrobial activity of lactoferrin has been well described, its mechanism of action has been poorly characterized. Recent work has indicated that in addition to binding iron, human lactoferrin damages the outer membrane of gram-negative bacteria. In this study, we determined whether bovine lactoferrin and a pepsin-derived bovine lactoferrin peptide (lactoferricin) fragment have similar activities. We found that both 20 microM bovine lactoferrin and 20 microM lactoferricin release intrinsically labeled [3H]lipopolysaccharide ([3H]LPS) from three bacterial strains, Escherichia coli CL99 1-2, Salmonella typhimurium SL696, and Salmonella montevideo SL5222. Under most conditions, more LPS is released by the peptide fragment than by whole bovine lactoferrin. In the presence of either lactoferrin or lactoferricin there is increased killing of E. coli CL99 1-2 by lysozyme. Like human lactoferrin, bovine lactoferrin and lactoferricin have the ability to bind to free intrinsically labeled [3H]LPS molecules. In addition to these effects, whereas bovine lactoferrin was at most bacteriostatic, lactoferricin demonstrated consistent bactericidal activity against gram-negative bacteria. This bactericidal effect is modulated by the cations Ca2+, Mg2+, and Fe3+ but is independent of the osmolarity of the medium. Transmission electron microscopy of bacterial cells exposed to lactoferricin show the immediate development of electron-dense "membrane blisters." These experiments offer evidence that bovine lactoferrin and lactoferricin damage the outer membrane of gram-negative bacteria. Moreover, the peptide fragment lactoferricin has direct bactericidal activity. As lactoferrin is exposed to proteolytic factors in vivo which could cleave the lactoferricin fragment, the effects of this peptide are of both mechanistic and physiologic relevance.     

Safety and tolerability of omalizumab

Background Omalizumab (Xolair^®) is a recombinant humanized monoclonal anti-IgE antibody with proven efficacy in patients with moderate-to-severe and severe persistent allergic (IgE-mediated) asthma.
Objective To review clinical study data to assess the safety profile of omalizumab.
Methods We analysed the safety of omalizumab using data from completed clinical studies (up to 1 year) involving more than 7500 patients with asthma, rhinitis or related conditions and up to 4 years in one study of patients with severe allergic asthma, as well as post-marketing safety data. Analysis focuses on the risk of immune-system effects, hypersensitivity reactions, malignant neoplasia, parasitic infections and thrombocytopenia.
Results Omalizumab exhibited a good safety and tolerability profile that was maintained up to 4 years in one study. The incidence of anaphylaxis was 0.14% in omalizumab-treated patients and 0.07% in control patients. No omalizumab-treated patient developed measurable anti-omalizumab antibodies. Post-marketing, based on estimated exposure of 57 300 patients (June 2003–December 2006), the frequency of anaphylaxis attributed to omalizumab use was estimated to be at least 0.2% of patients. Current clinical trial data do not support an increased risk of malignant neoplasia or thrombocytopenia with omalizumab.
Conclusion Data indicate that the proven efficacy of add-on omalizumab in patients with moderate-to-severe or severe allergic asthma is accompanied by a favourable safety and tolerability profile.     

Antibody CR1-2B11 recognizes a non-polymorphic epitope of human CR1 (CD35)

Measurement of erythrocyte [red blood cells (RBC)] complement receptor type 1 (CR1, CD35) has the potential to serve as a sensitive assessment of complement activation and immune complex clearance. All previously reported monoclonal antibodies (MoAb) to the extracellular region of CR1 recognize epitopes within the long homologous repeats (LHR) of CR1 and the epitopes for the most frequently used MoAbs are repeated at least twice per CR1 molecule. Furthermore, CR1 exhibits structural polymorphism characterized by a variable number of LHR per molecule. Thus, accurate enumeration of cell surface CR1 using currently available MoAb would require that the results be corrected for the number of antibody epitopes per CR1 molecule encoded by each individual's alleles. To obtain a MoAb to a non-polymorphic epitope on human CR1, hybridomas were generated from mice immunized with recombinant soluble CR1 (sCR1) and MoAb were screened for those that recognized the full-length extracellular domain but failed to bind to all four recombinant LHR fragments. A single antibody, CR1-2B11, was identified and was found to recognize an epitope located wholly within SCR29-30 of CR1, NH2-terminal to an elastase cleavage site. Like other CR1 MoAb, the CR1-2B11 epitope expression decreased on old erythrocytes compared to younger cells and CR1-2B11 did not identify a CR1 'stump' on RBC. Importantly, CR1-2B11 immunofluorescence did not change with storage or handling of RBC, unlike the apparent decrease in immunofluorescence observed with other MoAb. CR1-2B11 should be useful for the accurate enumeration of RBC CR1.     

Functional characterization of the brain-to-blood efflux clearance of human amyloid-beta peptide (1-40) across the rat blood-brain barrier

The present study sought to characterize the brain-to-blood efflux transport of human amyloid-beta peptide (hAbeta)(1-40) across the blood-brain barrier (BBB) in rats. We determined the apparent brain-to-blood [(125)I]hAbeta(1-40) efflux clearance in rats and found it to be 11.0 microL/(ming brain). There were no significant gender differences in the apparent brain-to-blood [(125)I]hAbeta(1-40) efflux clearance. The brain-to-blood [(125)I]hAbeta(1-40) efflux transport was significantly inhibited by unlabeled hAbeta(1-40) and hAbeta(1-42) by 79.1% and 36.4%, respectively, but was not inhibited by hAbeta(1-43) and hAbeta(40-1), and was significantly facilitated by hAbeta(17-40) by 16.0%, which is one of the major proteolytic fragments of hAbeta(1-40) generated by the action of Abeta degradation enzymes, such as endothelin-converting enzyme. Pre-administration of human receptor-associated protein, a low-density lipoprotein receptor-related protein (LRP) antagonist, reduced the elimination of [(125)I]hAbeta(1-40) by 20.3%, while quinidine or verapamil, P-glycoprotein (P-gp) inhibitors, did not significantly affect the elimination. Western blot analysis suggested that LRP-1 is expressed in rat brain capillary endothelial cells. In conclusion, the partial contribution of LRP-1 and the minor contribution of P-gp suggest that the hAbeta(1-40) elimination from rat brain is mediated by as yet unidentified molecules.     

An enzyme-linked immunosorbent assay (ELISA) for quantification of human collectin 11 (CL-11, CL-K1)

Collectin 11 (CL-11), also referred to as collectin kidney 1 (CL-K1), is a pattern recognition molecule that belongs to the collectin group of proteins involved in innate immunity. It interacts with glycoconjugates on pathogen surfaces and has been found in complex with mannose-binding lectin-associated serine protease 1 (MASP-1) and/or MASP-3 in circulation. Mutation in the CL-11 gene was recently associated with the developmental syndrome 3MC. In the present study, we established and thoroughly validated a sandwich enzyme-linked immunosorbent assay (ELISA) based on two different monoclonal antibodies. The assay is highly sensitive, specific and shows excellent quantitative characteristics such as reproducibility, dilution linearity and recovery (97.7-104%). The working range is 0.15-34 ng/ml. The CL-11 concentration in two CL-11-deficient individuals affected by the 3MC syndrome was determined to be below 2.1 ng/ml. We measured the mean serum CL-11 concentration to 284 ng/ml in 100 Danish blood donors, with a 95% confidence interval of 269-299 ng/ml. There was no significant difference in the CL-11 concentration measured in matched serum and plasma samples. Storage of samples and repeated freezing and thawing to a certain extent did not influence the ELISA. This ELISA offers a convenient and reliable method for studying CL-11 levels in relation to a variety of human diseases and syndromes.     

Structure and function of collectin liver 1 (CL-L1) and collectin 11 (CL-11, CL-K1)

The collectins are a group of innate immune proteins structurally characterized by their content of a carbohydrate recognition domain and a collagen-like region. Collectin liver 1 (CL-L1) and collectin 11 (CL-11, alias collectin kidney 1, CL-K1) are the more recently described members of this group. Their genomic organization and protein structure reveal many similarities. However, CL-11 is a serum protein, whereas CL-L1 appears to be restricted to the cytosol of cells such as hepatocytes. Specificity analyses of the CRDs reveal some differences in their preferences for saccharides: CL-11 binds most avidly to l-fucose and d-mannose, whereas CL-L1 shows preference for d-mannose, d-fucose, N-acetylglucosamine, and surprisingly also d-galactose. CL-11 binds to various microorganisms including Escherichia coli, Candida albicans and Influenza A virus. Polymorphisms in the CL-11 gene (COLEC11) leading to deficiencies have recently been identified as causative for 3MC syndrome. The 3MC syndrome is associated with a wide spectrum of developmental features including facial dysmorphism, cognitive impairment, hearing loss and vesicorenal anomalies. Similar polymorphic associations were reported for the mannan-binding lectin-associated serine protease 3 (MASP-3), and falls into line with the observation that CL-11 is found in circulating complexes with MASP-1/3. These findings suggest dual or overlapping functions of CL-11 in innate immunity and in fetal development.     

Management in Senegal of the 1st efficacy and tolerability studies of ivermectin (MK 933) in human onchocerciasis

In 1981, 32 adult Senegalese males infected with Onchocerca volvulus but without ocular involvement were treated by groups of 8 with single oral dosages of 5, 10, 30 or 50 micrograms/kg of ivermectin . As soon as the second day after treatment, we observed a marked decrease of the dermal microfilaremic charge after a posology of 30 micrograms/kg. However the decrease is most important in the group subjected to 50 micrograms/kg and 75% of them had no more microfilariae in the dermis 28 days following treatment.     

High-performance liquid chromatographic determination of [11C]1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide in mouse plasma and tissue and in human plasma

The high-performance liquid chromatographic determination of 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide ([11C]PK 11195) is described. The method was successfully applied for plasma and tissue analysis after i.v. injection of [11C]PK 11195 in mice and for plasma analysis after administration of [11C]PK 11195 to humans. Separation is effected on a RP-C18 column, using a mixture of acetonitrile-water-triethylamine (65:35:0.5, v/v). Quantitative measurements of radioactivity are performed on a one-channel gamma-ray spectrometer equipped with a 2 x 2 in. NaI(Tl) detector. For humans rapid metabolisation of [11C]PK 11195 was observed. At 5, 20 and 35 min post injection 5%, 22% and 32%, respectively, of the plasma activity consisted of at least two more polar metabolites. Despite the extensive metabolisation rate in mice (up to 42% at 10 min post injection of [11C]PK 11195), no 11C-labelled metabolites could be detected in the extracts of brain and heart.     

Glucagon-like peptide 1 (GLP-1) and eating

New information regarding gastrointestinal mechanisms that participate in the control of food intake has extended our understanding of appetite control. Although each new signaling pathway discovered in the gut is a potential target for drug development in the treatment of obesity, the growing number of such signaling molecules indicates that a highly complex process controls food intake. The present summary focuses on the role of glucagon-like peptide 1 (GLP-1) in this regulatory process. The different biological effects of GLP-1 (glucose-lowering properties, inhibition of appetite and food intake) provide a powerful impetus for development of GLP-1-based new drugs.     

Determinants of a genotoxic effect of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in human diploid fibroblasts

Summary The induction of micronuclei was studied in human diploid fibroblasts incubated in the presence of the tobacco-specific nitrosamine NNK. We used four fibroblast strains having a high capacity of 0⁶-alkylguanine DNA alkyltransferase (13.0–23.3 pmol 0⁶-methylguanine repaired per 8 × 10⁶ cells) and four strains that showed no detectable repair capacity. Incubation with NNK doubled the frequency of micronuclei in repair-deficient cells but failed to evoke any effect in the proficient cell strains. Control experiments were performed with the direct methylating agent MNNG and in the presence of inhibitors of either metabolic activation or alkyltransferase. The results showed that the genotoxicity of NNK is dependent on the relationship between its metabolic activation and the constitutive DNA repair. This supports earlier findings that low constitutive levels of 0⁶-alkylguanine DNA alkyltransferase may increase susceptibility to lung cancer after exposure to DNA methylating agents.Abbreviations NNK 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone - MNNG N-methyl-N-nitro-N-nitrosoguanidin - MN micronuclei - O⁶-AT O⁶-alkylguanine-DNA-alkyl-transferase This work is part of a thesis for a medical doctorate of CP     

Gene for glutathione S-transferase-1 (GST1) is on human chromosome 11

The glutathione S-transferases (GST) are a group of related enzymes that can detoxify potentially carcinogenic electrophiles by conjugating them with reduced glutathione (GSH). The chromosomal location of one of the enzyme forms, GST1,reported recently to be polymorphic, was determined utilizing man-mouse somatic cell hybrids segregating human chromosomes. The expression of GST1by hybrid clones was compared with that of 34 enzyme markers representing 23 chromosomes, and karyotypes of selected cell hybrids were analyzed. The evidence indicated that GST1is assigned to chromosome 11 in humans. Utilizing an X/11 translocation segregating in hybrids, GST1was localized to the p13 qter region of chromosome 11.     

Adrenomedullin and proadrenomudullin N-terminal 20 peptide (PAMP) are present in human colonic epithelia and exert an antimicrobial effect

The hypotensive and vasorelaxing peptides adrenomedullin (AM) and its gene-related peptide, proadrenomedullin N-terminal 20 peptide (PAMP), were found to be distributed on the surface of the colonic mucosa. AM and PAMP showed dose-dependent antimicrobial activity against E. coli. The results suggest that the novel vasoactive peptides AM and PAMP play an important role in mucosal defence.     

人抗菌肽hCAP18全长cDNA克隆及原核表达

目前发现，人体主要的抗菌肽有两大类；防御素（defensins）和cathelicidins。cathelicidins 家族成员中人类只有一个，即human cathelicidin antimicrobial peptide 简称hCAP18或LL-37。     

Killing of Candida albicans by lactoferricin B,a potent antimicrobial peptide derived from the N-terminal region of bovine lactoferrin

Candida albicans was found to be highly susceptible to inhibition and inactivation by lactoferricin B, a peptide produced by enzymatic cleavage of bovine lactoferrin. Effective concentrations of the peptide varied within the range of 18 to 150 g/ml depending on the strain and the culture medium used. Its effect was lethal, causing a rapid loss of colony-forming capability. ¹⁴C-labeled lactoferricin B bound to C. albicans and the rate of binding appeared to be consistent with the rate of killing induced by the peptide. The extent of binding was diminished in the presence of Mg²⁺ or Ca²⁺ ions which acted to reduce its anticandidal effectiveness. Binding occurred optimally at pH 6.0 and killing was maximal near the same pH. Such evidence suggests the lethal effect of lactoferricin B results from its direct interaction with the cell surface. Cells exposed to lactoferricin B exhibited profound ultrastructural damage which appeared to reflect its induction of an autolytic response. These findings suggest that active peptides of lactoferrin could potentially contribute to the host defense against C. albicans.     

Parathyroid hormone-(1-34) peptide activates cyclic adenosine 3',5'-monophosphate in the human placenta.

Dually perfused human term placental lobules were exposed to forskolin, bovine parathyroid hormone (bPTH(1-34)) and human parathyroid hormone related-peptides, hPTHrP(1-34), hPTHrP(67-86)NH2 or PTHrP(107-138) for 15 min in the presence of 3-iso-butyl-1-methyl-xanthine (IBMX); control lobules were exposed to IBMX alone. Homogenates of these tissues were then assayed for cyclic adenosine 3',5'-monophosphate (cyclic AMP) and results normalized per mg of protein. Exposure to forskolin or bPTH(1-34) on both sides, and exposure to bPTH(1-34) at a concentration of 30 nM on the maternal side of the placenta or 120 nM on the fetal side of the placenta, significantly enhanced tissue cyclic AMP production compared with tissue exposed to IBMX alone. Exposure to hPTHrP(1-34), hPTHrP(67-86)NH2 and hPTHrP(107-138) at a concentration of 30 nM on both sides of the placenta had no significant effect upon tissue cyclic AMP production.