Antibacterial activity and specificity of the six human {alpha}-defensins

We developed a kinetic, 96-well turbidimetric procedure that is capable of testing the antimicrobial properties of six human alpha-defensins concurrently on a single microplate. The defensins were prepared by solid-phase peptide synthesis and tested against gram-positive bacteria (Staphylococcus aureus and Bacillus cereus) and gram-negative bacteria (Enterobacter aerogenes and Escherichia coli). Analysis of the growth curves provided virtual lethal doses (vLDs) equivalent to conventional 50% lethal doses (LD(50)s), LD(90)s, LD(99)s, and LD(99.9)s obtained from colony counts. On the basis of their respective vLD(90)s and vLD(99)s, the relative potencies of human myeloid alpha-defensins against S. aureus were HNP2 > HNP1 > HNP3 > HNP4. In contrast, their relative potencies against E. coli and E. aerogenes were HNP4 > HNP2 > HNP1 = HNP3. HD5 was as effective as HNP2 against S. aureus and as effective as HNP4 against the gram-negative bacteria in our panel. HD6 showed little or no activity against any of the bacteria in our panel, including B. cereus, which was highly susceptible to the other five alpha-defensins. The assay described provides a quantitative, precise, and economical way to study the antimicrobial activities of host-defense peptides. Its use has clarified the relative potencies of human alpha-defensins and raised intriguing questions about the in vivo function(s) of HD6.     

Dynamics of {beta}-amyloid reductions in brain, cerebrospinal fluid, and plasma of {beta}-amyloid precursor protein transgenic mice treated with a {gamma}-secretase inhibitor

gamma-Secretase inhibitors are one promising approach to the development of a therapeutic for Alzheimer's disease (AD). gamma-Secretase inhibitors reduce brain beta-amyloid peptide (Abeta), which is believed to be a major contributor in the etiology of AD. Transgenic mice overexpressing the human beta-amyloid precursor protein (APP) are valuable models to examine the dynamics of Abeta changes with gamma-secretase inhibitors in plaque-free and plaque-bearing animals. BMS-299897 2-[(1R)-1-[[(4-chlorophenyl)sulfony](2,5-difluorophenyl)amino]ethyl]-5-fluorobenzenepropanoic acid, a gamma-secretase inhibitor, showed dose- and time dependent reductions of Abeta in brain, cerebrospinal fluid (CSF), and plasma in young transgenic mice, with a significant correlation between brain and CSF Abeta levels. Because CSF and brain interstitial fluid are distinct compartments in composition and location, this correlation could not be assumed. In contrast, aged transgenic mice with large accumulations of Abeta in plaques showed reductions in CSF Abeta in the absence of measurable changes in plaque Abeta in the brain after up to 2 weeks of treatment. Hence, CSF Abeta levels were a valuable measure of gamma-secretase activity in the central nervous system in either the presence or absence of plaques. Transgenic mice were also used to examine potential side effects due to Notch inhibition. BMS-299897 was 15-fold more effective at preventing the cleavage of APP than of Notch in vitro. No changes in the maturation of CD8(+) thymocytes or of intestinal goblet cells were observed in mice treated with BMS-299897, showing that it is possible for gamma-secretase inhibitors to reduce brain Abeta without causing Notch-mediated toxicity.     

Antibacterial activities of rhodamine B-conjugated gelsolin-derived peptides compared to those of the antimicrobial peptides cathelicidin LL37, magainin II, and melittin

The growing number of antibiotic-resistant bacteria necessitates the search for new antimicrobial agents and the principles by which they work. We report that cell membrane-permeant rhodamine B (RhB)-conjugated peptides based on the phosphatidylinositol-4,5-bisphosphate binding site of gelsolin can kill the gram-negative organisms Escherichia coli and Pseudomonas aeruginosa and the gram-positive organism Streptococcus pneumoniae. RhB linkage to the QRLFQVKGRR sequence in gelsolin was essential for the antibacterial function, since the unconjugated peptide had no effect on the bacteria tested. Because RhB-QRLFQVKGRR (also termed PBP10), its scrambled sequence (RhB-FRVKLKQGQR), and PBP10 synthesized from D-isomer amino acids show similar antibacterial properties, the physical and chemical properties of these derivatives appear to be more important than specific peptide folding for their antibacterial functions. The similar activities of PBP10 and all-D-amino-acid PBP10 also indicate that a specific interaction between RhB derivatives and bacterial proteins is unlikely to be involved in the bacterial killing function of PBP10. By using a phospholipid monolayer system, we found a positive correlation between the antibacterial function of PBP10, as well as some naturally occurring antibacterial peptides, and the intrinsic surface pressure activity at the hydrophobic-hydrophilic interface. Surprisingly, we observed little or no dependence of the insertion of these peptides into lipid monolayers on the phospholipid composition. These studies show that an effective antimicrobial agent can be produced from a peptide sequence with specificity to a phospholipid not found in bacteria, and comparisons with other antimicrobial agents suggest that the surface activities of these peptides are more important than specific binding to bacterial proteins or lipids for their antimicrobial functions.     

Shared reactivity of V{delta}2(neg) {gamma}{delta} T cells against cytomegalovirus-infected cells and tumor intestinal epithelial cells

Long-lasting expansion of Vdelta2(neg) gammadelta T cells is a hallmark of cytomegalovirus (CMV) infection in kidney transplant recipients. The ligands of these cells and their role remain elusive. To better understand their immune function, we generated gammadelta T cell clones from several transplanted patients. Numerous patient Vdelta1(+), Vdelta3(+), and Vdelta5(+) gammadelta T cell clones expressing diverse Vgamma chains, but not control Vgamma9Vdelta2(+) T clones, displayed strong reactivity against CMV-infected cells, as shown by their production of tumor necrosis factor-alpha. Vdelta2(neg) gammadelta T lymphocytes could also kill CMV-infected targets and limit CMV propagation in vitro. Their anti-CMV reactivity was specific for this virus among herpesviridae and required T cell receptor engagement, but did not involve major histocompatibility complex class I molecules or NKG2D. Vdelta2(neg) gammadelta T lymphocytes expressed receptors essential for intestinal homing and were strongly activated by intestinal tumor, but not normal, epithelial cell lines. High frequencies of CMV- and tumor-specific Vdelta2(neg) gammadelta T lymphocytes were found among patients' gammadelta T cells. In conclusion, Vdelta2(neg) gammadelta T cells may play a role in protecting against CMV and tumors, probably through mucosal surveillance of cellular stress, and represent a population that is largely functionally distinct from Vgamma9Vdelta2(+) T cells.     

Bortezomib sensitizes malignant human glioma cells to TRAIL, mediated by inhibition of the NF-{kappa}B signaling pathway

Previous studies have shown that the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has significant apoptosis-inducing activity in some glioma cell lines, although many lines are either moderately or completely resistant, which has limited the therapeutic applicability of this agent. Because our recent studies showed that inhibition of proteasomal function may be independently active as an apoptosis-inducing stimulus in these tumors, we investigated the sensitivity of a panel of glioma cell lines (U87, T98G, U373, A172, LN18, LN229, LNZ308, and LNZ428) to TRAIL alone and in combination with the proteasome inhibitor bortezomib. Analysis of these cell lines revealed marked differences in their sensitivity to these treatments, with two (LNZ308 and U373) of the eight cell lines revealing no significant induction of cell death in response to TRAIL alone. No correlation was found between sensitivity of cells to TRAIL and expression of TRAIL receptors DR4, DR5, and decoy receptor DcR1, caspase 8, apoptosis inhibitory proteins XIAP, survivin, Mcl-1, Bcl-2, Bcl-Xl, and cFLIP. However, TRAIL-resistant cell lines exhibited a high level of basal NF-κB activity. Bortezomib was capable of potentiating TRAIL-induced apoptosis in TRAIL-resistant cells in a caspase-dependent fashion. Bortezomib abolished p65/NF-κB DNA-binding activity, supporting the hypothesis that inhibition of the NF-κB pathway is critical for the enhancement of TRAIL sensitization in glioma cells. Moreover, knockdown of p65/NF-κB by shRNA also enhanced TRAIL-induced apoptosis, indicating that p65/NF-κB may be important in mediating TRAIL sensitivity and the effect of bortezomib in promoting TRAIL sensitization and apoptosis induction.     

Susceptibilities of Mycoplasma fermentans and Mycoplasma hyorhinis to membrane-active peptides and enrofloxacin in human tissue cell cultures

Mycoplasmas, which are bacteria that are devoid of a cell wall and which belong to the class Mollicutes, are pathogenic for humans and animals and are frequent contaminants of tissue cell cultures. Although contamination of cultures with mycoplasma can easily be monitored with fluorescent dyes that stain DNA and/or with molecular probes, protection and decontamination of cultures remain serious challenges. In the present work, we investigated the susceptibilities of Mycoplasma fermentans and Mycoplasma hyorhinis to the membrane-active peptides alamethicin, dermaseptin B2, gramicidin S, and surfactin by growth inhibition and lethality assays. In the absence of serum, the four peptides killed mycoplasmas at minimal bactericidal concentrations that ranged from 12.5 to 100 microM, but in all cases the activities were decreased by the presence of serum. As a result, under standard culture conditions (10% serum) only alamethicin and gramicidin S were able to inhibit mycoplasma growth (MICs, 50 microM), while dermaseptin B2 and surfactin were ineffective. Furthermore, 8 days of treatment of HeLa cell cultures experimentally contaminated with either mycoplasma species with 70 microM enrofloxacin cured the cultures of infection, whereas treatment with alamethicin and gramicidin S alone was not reliable because the concentrations and treatment times required were toxic to the cells. However, combination of alamethicin or gramicidin S with 70 microM enrofloxacin allowed mycoplasma eradication after 30 min or 24 h of treatment, depending on the mycoplasma and peptide considered. HeLa cell cultures experimentally infected with mycoplasmas should prove to be a useful model for study of the antimycoplasma activities of antibiotics and membrane-active peptides under conditions close to those found in vivo.     

Design, synthesis, and analysis of a polyethelene glycol-modified (PEGylated) small molecule inhibitor of integrin {alpha}4{beta}1 with improved pharmaceutical properties

Integrin alpha4beta1 plays an important role in inflammatory processes by regulating the migration of leukocytes into inflamed tissues. Previously, we identified BIO5192 [2(S)-{[1-(3,5-dichloro-benzenesulfonyl)-pyrrolidine-2(S)-carbonyl]-amino}-4-[4-methyl-2(S)-(methyl-{2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl}-amino)-pentanoylamino]-butyric acid], a highly selective and potent (K(D) of 9 pM) small molecule inhibitor of alpha4beta1. Although BIO5192 is efficacious in various animal models of inflammatory disease, high doses and daily treatment of the compound are needed to achieve a therapeutic effect because of its relatively short serum half-life. To address this issue, polyethylene glycol modification (PEGylation) was used as an approach to improve systemic exposure. BIO5192 was PEGylated by a targeted approach in which derivatizable amino groups were incorporated into the molecule. Two sites were identified that could be modified, and from these, five PEGylated compounds were synthesized and characterized. One compound, 2a-PEG (K(D) of 19 pM), was selected for in vivo studies. The pharmacokinetic and pharmacodynamic properties of 2a-PEG were dramatically improved relative to the unmodified compound. The PEGylated compound was efficacious in a rat model of experimental autoimmune encephalomyelitis at a 30-fold lower molar dose than the parent compound and required only a once-a-week dosing regimen compared with a daily treatment for BIO5192. Compound 2a-PEG was highly selective for alpha4beta1. These studies demonstrate the feasibility of PEGylation of alpha4beta1-targeted small molecules with retention of activity in vitro and in vivo. 2a-PEG, and related compounds, will be valuable reagents for assessing alpha4beta1 biology and may provide a new therapeutic approach to treatment of human inflammatory diseases.     

Occurrence and molecular analysis of extended-spectrum {beta}-lactamase-producing Proteus mirabilis in Hong Kong, 1999-2002

OBJECTIVES: A study was conducted to evaluate the occurrence and characterization of extended-spectrum beta-lactamases (ESBLs) among blood isolates of Proteus mirabilis collected over a 4 year period in Hong Kong. METHODS: Production of ESBLs among 99 consecutive and non-duplicate isolates was evaluated by the double-disc synergy test. The ESBLs were characterized by isoelectric focusing and PCR sequencing using specific primers. The epidemiological relationship of the isolates was studied by the Dienes test and PFGE. RESULTS: ESBLs were identified in 13 isolates, from none in 1999-2000 and up to 18.5% (5/27) in 2001 and 25.8% (8/31) in 2002. The ESBL-producing isolates were more resistant to ceftriaxone than to ceftazidime, and were more likely than non-ESBL-producers to have resistance to ciprofloxacin (76.9% versus 14%) and gentamicin (38.5% versus 9.3%). The ESBL content included CTX-M-13 (n=8), CTX-M-14 (n=3), SHV-5 (n=2), TEM-11 (n=1), and an unidentified ESBL with a pI of 7.5. The Dienes test revealed that the genetic background in the 99 isolates was highly heterogeneous, with 54 distinct types among 92 isolates and seven were non-typeable. Among the 13 ESBL-producing isolates, five different backgrounds, including one cluster (Dienes-pulsotype A) with nine isolates, were identified by both Dienes test and PFGE, thus suggesting both clonal and multi-clonal spread of the CTX-M enzymes. CONCLUSIONS: Our findings indicate the emergence of CTX-M enzymes among P. mirabilis in Hong Kong. More ESBL screening of this species is required to improve their recognition.     

Plasma Noradrenaline and Adrenaline Concentrations and Dopamine-{beta}-hydroxylase Activity in Patients With Shock Due to Septicaemia, Trauma and Haemorrhage

Plasma adrenaline and noradrenaline concentrations and dopamine-ß-hydroxylaseactivities were measured in patients with septicaemic, traumaticor haemorrhagic shock. Irrespective of the type of shock plasmaadrenaline and noradrenaline concentrations were increased abovethe normal range. This is in keeping with the clinical featuresof increased sympathetic nervous system and adrenal medullaryactivity present in these patients. Plasma dopamine-ß-hydroxylaseactivities were within the normal limits in all forms of shockindicating the poor relationship of this measurement to sympatheticnervous system activity. In patients who died plasma noradrenalineconcentrations remained persistently elevated above normal whilein those who survived there was a rapid decline towards thenormal range.     

TGF-{beta}-dependent CD103 expression by CD8(+) T cells promotes selective destruction of the host intestinal epithelium during graft-versus-host disease

Destruction of the host intestinal epithelium by donor effector T cell populations is a hallmark of graft-versus-host disease (GVHD), but the underlying mechanisms remain obscure. We demonstrate that CD8(+) T cells expressing CD103, an integrin conferring specificity for the epithelial ligand E-cadherin, play a critical role in this process. A TCR transgenic GVHD model was used to demonstrate that CD103 is selectively expressed by host-specific CD8(+) T cell effector populations (CD8 effectors) that accumulate in the host intestinal epithelium during GVHD. Although host-specific CD8 effectors infiltrated a wide range of host compartments, only those infiltrating the intestinal epithelium expressed CD103. Host-specific CD8 effectors expressing a TGF-beta dominant negative type II receptor were defective in CD103 expression on entry into the intestinal epithelium, which indicates local TGF-beta activity as a critical regulating factor. Host-specific CD8 effectors deficient in CD103 expression successfully migrated into the host intestinal epithelium but were retained at this site much less efficiently than wild-type host-specific CD8 effectors. The relevance of these events to GVHD pathogenesis is supported by the finding that CD103-deficient CD8(+) T cells were strikingly defective in transferring intestinal GVHD pathology and mortality. Collectively, these data document a pivotal role for TGF-beta-dependent CD103 expression in dictating the gut tropism, and hence the destructive potential, of CD8(+) T cells during GVHD pathogenesis.     

Activity of three {beta}-lactams (ertapenem, meropenem and ampicillin) against intraphagocytic Listeria monocytogenes and Staphylococcus aureus

OBJECTIVES: Assessment of the activity of three beta-lactams [ertapenem (a carbapenem with a prolonged half-life), meropenem and ampicillin] against intraphagocytic Listeria monocytogenes and Staphylococcus aureus. METHODS: Quantitative measurements of cfu changes in broth and in THP-1 macrophages (post-phagocytosis) over time (5 and 24 h) at concentrations spanning from sub-MICs to C(max) (maximal concentration typically observed in patients' serum upon administration of conventional doses); morphological studies using an electron microscope; evaluation of drug stability (HPLC), protein binding (equilibrium dialysis) and measurement of drug cellular accumulation (microbiological assay). RESULTS: Ertapenem was unable to control L. monocytogenes growth in THP-1 macrophages at all concentrations and times tested, even under conditions where ampicillin and meropenem were bactericidal. This behaviour could not be ascribed to drug instability, protein binding or lack of cell accumulation in comparison with ampicillin or meropenem. Ertapenem, ampicillin and meropenem were equally effective at reducing the post-phagocytosis inoculum of S. aureus ( approximately 1 log cfu), and caused conspicuous changes in the morphology of intracellular bacteria consistent with their lysis. These effects were obtained, however, only at large multiples (100-fold or more) of the MIC maintained over 24 h. Because of the high intrinsic antimicrobial potency of the beta-lactams studied, these concentrations were below the C(max). CONCLUSIONS: Ertapenem will probably be ineffective against intraphagocytic forms of L. monocytogenes for reasons that remain to be discovered. Conversely, ertapenem could be an alternative to ampicillin and meropenem against intraphagocytic S. aureus since its longer half-life may allow high concentrations to be maintained for more prolonged times.     

Characterization of In100, a new integron carrying a metallo-{beta}-lactamase and a carbenicillinase, from Pseudomonas aeruginosa

In100, a new integron carrying a carbapenemase gene (bla(VIM-2)) associated with a carbenicillinase (blaP1b) and aminoglycoside resistance genes (aacA4 and aadA2), was detected in a Pseudomonas aeruginosa clinical isolate. The particular gene cassette organization of In100 seems to reflect the evolution of antibiotic usage in therapeutics.     

Thymic stromal lymphopoietin is released by human epithelial cells in response to microbes, trauma, or inflammation and potently activates mast cells

Compelling evidence suggests that the epithelial cell-derived cytokine thymic stromal lymphopoietin (TSLP) may initiate asthma or atopic dermatitis through a dendritic cell-mediated T helper (Th)2 response. Here, we describe how TSLP might initiate and aggravate allergic inflammation in the absence of T lymphocytes and immunoglobulin E antibodies via the innate immune system. We show that TSLP, synergistically with interleukin 1 and tumor necrosis factor, stimulates the production of high levels of Th2 cytokines by human mast cells (MCs). We next report that TSLP is released by primary epithelial cells in response to certain microbial products, physical injury, or inflammatory cytokines. Direct epithelial cell-mediated, TSLP-dependent activation of MCs may play a central role in "intrinsic" forms of atopic diseases and explain the aggravating role of infection and scratching in these diseases.     

Susceptibilities of human cytomegalovirus clinical isolates to BAY38-4766, BAY43-9695, and ganciclovir

BAY38-4766 and BAY43-9695 are nonnucleosidic compounds with activities against human cytomegalovirus (HCMV). Two phenotypic assays were used to determine the drug susceptibilities of 36 HCMV clinical isolates to the BAY compounds and ganciclovir. Using either assay, both BAY compounds at a concentration of approximately 1 microM inhibited the replication of all 36 HCMV clinical isolates, including 11 ganciclovir-resistant clinical isolates, by 50%.     

Visualization of binding and transcytosis of botulinum toxin by human intestinal epithelial cells

Botulinum toxin is an unusually potent oral poison, which means that the toxin must have an efficient mechanism for escaping the lumen of the gut to reach the general circulation. Previous work involving iodination of toxin and analysis of its movement demonstrated a specific process of transepithelial transport. In the present study, botulinum toxin labeled with Alexa Fluor 488 was used to visualize the discrete steps of binding, internalization, transcytosis, and release. The data revealed that binding sites for the toxin were distributed across the apical surface of epithelial cells, and there was no evidence of significant clustering. The amount of toxin bound to receptors at saturation was too large to be accommodated in a single wave of endocytosis. Toxin that entered epithelial cells did not remain in the vicinity of the endocytosing membrane, which is in striking contrast to events in neuronal cells. Instead, the toxin began to spread across the length of cells, eventually being released on the basolateral surface. Migration of toxin through epithelial cells required redistribution to the cell periphery. This migration pattern could be attributed to the large and centrally located nucleus, which physically displaced transport vesicles. Transcytosed toxin began to reach the contralateral surface within ca. 5 min, and transcytosis was essentially complete within 20 to 30 min.     

LL-37, the neutrophil granule- and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and T cells

We have previously shown that antimicrobial peptides like defensins have the capacity to mobilize leukocytes in host defense. LL-37 is the cleaved antimicrobial 37-residue, COOH-terminal peptide of hCAP18 (human cationic antimicrobial protein with a molecular size of 18 kD), the only identified member in humans of a family of proteins called cathelicidins. LL-37/hCAP18 is produced by neutrophils and various epithelial cells. Here we report that LL-37 is chemotactic for, and can induce Ca(2+) mobilization in, human monocytes and formyl peptide receptor-like 1 (FPRL1)-transfected human embryonic kidney 293 cells. LL-37-induced Ca(2+) mobilization in monocytes can also be cross-desensitized by an FPRL1-specific agonist. Furthermore, LL-37 is also chemotactic for human neutrophils and T lymphocytes that are known to express FPRL1. Our results suggest that, in addition to its microbicidal activity, LL-37 may contribute to innate and adaptive immunity by recruiting neutrophils, monocytes, and T cells to sites of microbial invasion by interacting with FPRL1.     

Susceptibilities of 428 gram-positive and -negative anaerobic bacteria to Bay y3118 compared with their susceptibilities to ciprofloxacin, clindamycin, metronidazole, piperacillin, piperacillin-tazobactam, and cefoxitin.

The susceptibilities of 428 gram-negative and gram-positive anaerobes (including selected cefoxitin-resistant strains) to Bay y3118 (a new fluoroquinolone), ciprofloxacin, clindamycin, metronidazole, cefoxitin, piperacillin, and piperacillin-tazobactam were tested. Organisms comprised 115 Bacteroides fragilis group, 116 non-B. fragilis Bacteroides, Prevotella, and Porphyromonas spp., 40 fusobacteria, 58 peptostreptococci, 48 gram-positive non-spore-forming rods, and 51 clostridia. beta-Lactamase production was demonstrated in 87% of the gram-negative rods but in none of the gram-positive organisms. Overall, Bay y3118 was the most active agent, with all organisms inhibited at an MIC of < or = 2.0 micrograms/ml (MICs for 50% [MIC50] and 90% [MIC90] of strains tested, 0.125 and 0.5 microgram/ml, respectively). By contrast, ciprofloxacin was much less active, with only 42% of strains susceptible at a breakpoint of 2.0 micrograms/ml (MIC50, 4.0 micrograms/ml; MIC90, 16.0 micrograms/ml). Metronidazole was active against all gram-negative rods, but 7% of peptostreptococci, 83% of gram-positive non-spore-forming rods, and 4% of non-Clostridium perfringens, non-Clostridium difficile clostridia were resistant to this agent (MICs, > 16.0 micrograms/ml). Clindamycin was active against 94% of Bacteroides, Prevotella, and Porphyromonas spp., 91% of peptostreptococci, and 100% of gram-positive non-spore-forming rods, but was active against only 70% of fusobacteria and 53% of clostridia. Cefoxitin was active against > or = 90% of all groups except the B. fragilis group and non-Propionibacterium acnes gram-positive non-spore-forming rods (both 85%) and C. difficile (20%). Significant enhancement of piperacillin by tazobactam was seen in all beta-lactamase-positive strains (99% susceptible; MIC90, 8.0 micrograms/ml), and all beta-lactamase-negative strains were susceptible to piperacillin (MIC90, 8.0 micrograms/ml). Clinical studies are required to delineate the role of Bay y3118 in the treatment of anaerobic infections.