Host innate inflammatory factors and staphylococcal protein A influence the duration of human Staphylococcus aureus nasal carriage.

Human Staphylococcus aureus (SA) nasal carriage provides a reservoir for the dissemination of infectious strains; however, factors regulating the establishment and persistence of nasal colonization are mostly unknown. We measured carriage duration and nasal fluid inflammatory markers after nasally inoculating healthy participants with their previously isolated SA strains. Out of 15 studies, 10 resulted in rapid clearance (9±6 days) that corresponded with upregulated chemokines, growth factors, and predominantly Th1-type cytokines, but not interleukin (IL)-17. Nasal SA persistence corresponded with elevated baseline levels of macrophage inflammatory protein-1ß, IL-1ß, and IL-6, no induction of inflammatory factors after inoculation, and decreased IL-1 receptor antagonist/IL-1ß ratio. SA-expressed staphylococcal protein A (SpA) levels correlated positively with carriage duration. Competitive inoculation studies revealed that isogenic SpA knockout (ΔSpA) strains were cleared faster than wild type only in participants with upregulated inflammatory markers after inoculation. The remaining participants did not mount an inflammatory response and did not clear either strain. ΔSpA strains demonstrated lower growth rates in carrier nasal fluids and lower survival rates when incubated with neutrophils. Collectively, the presented studies identify innate immune effectors that cooperatively modulate nasal carriage duration, and confirm SpA as a bacterial codeterminant of SA nasal carriage.

Colonoscopy in rural communities: a systematic review of the frequency and quality.

INTRODUCTION: In this systematic review, the authors review studies of rural colonoscopy to determine specialty types providing rural colonoscopy and the quality of these procedures. METHODS: A systematic MEDLINE search was conducted for articles pertaining to rural colonoscopy. Inclusion criteria were rural location, report of quality outcomes, or report of endoscopy workforce in rural areas. Two investigators independently reviewed and abstracted included articles. The following information was obtained from each study: author identification, citation, study design, source of funding, study duration and follow-up, study population, sample size, study setting, population characteristics, outcomes and results. Standard abstraction forms were used to summarize and assess the quality of evidence. RESULTS: From 121 articles in the MEDLINE search, 11 met inclusion criteria. One additional article found from a reference list was included. Eleven articles from three countries reported on 8703 colonoscopies performed by 25 rural generalists. Reach-the-cecum rates (RCR) ranged from 36% to 96.5% with more recent studies showing higher RCRs. Adenoma detection rates ranged from 16.6% to 46%. The rate of complications was low in all studies. One study of the rural endoscopist workforce reported that general surgeons performed most rural colonoscopies in Canada. CONCLUSIONS: Rural generalist physicians can safely and effectively perform colonoscopies. More research is needed on the rural endoscopist workforce.

Endogenous c-Myc is essential for p53-induced apoptosis in response to DNA damage in vivo.

Recent studies have suggested that C-MYC may be an excellent therapeutic cancer target and a number of new agents targeting C-MYC are in preclinical development. Given most therapeutic regimes would combine C-MYC inhibition with genotoxic damage, it is important to assess the importance of C-MYC function for DNA damage signalling in vivo. In this study, we have conditionally deleted the c-Myc gene in the adult murine intestine and investigated the apoptotic response of intestinal enterocytes to DNA damage. Remarkably, c-Myc deletion completely abrogated the immediate wave of apoptosis following both ionizing irradiation and cisplatin treatment, recapitulating the phenotype of p53 deficiency in the intestine. Consistent with this, c-Myc-deficient intestinal enterocytes did not upregulate p53. Mechanistically, this was linked to an upregulation of the E3 Ubiquitin ligase Mdm2, which targets p53 for degradation in c-Myc-deficient intestinal enterocytes. Further, low level overexpression of c-Myc, which does not impact on basal levels of apoptosis, elicited sustained apoptosis in response to DNA damage, suggesting c-Myc activity acts as a crucial cell survival rheostat following DNA damage. We also identify the importance of MYC during DNA damage-induced apoptosis in several other tissues, including the thymus and spleen, using systemic deletion of c-Myc throughout the adult mouse. Together, we have elucidated for the first time in vivo an essential role for endogenous c-Myc in signalling DNA damage-induced apoptosis through the control of the p53 tumour suppressor protein.

Formulation development of retrocyclin 1 analog RC-101 as an anti-HIV vaginal microbicide product.

RC-101 is a synthetic microbicide analog of retrocyclin, which has shown in vitro activity against X4 and R5 HIV-1. In an effort to develop a safe and effective RC-101 vaginal microbicide product, we assessed safety in ex vivo macaque and human models and efficacy using in vitro and ex vivo models. A polyvinyl-alcohol vaginal film containing RC-101 (100 µg/film) was developed. Formulation assessment was conducted by evaluating disintegration, drug content, mechanical properties, and stability. Efficacy was evaluated by in vitro peripheral blood mononuclear cells (PBMC) assay and ex vivo human ectocervical tissue explant model. Ex vivo safety studies were conducted by exposing RC-101 to an excised monkey reproductive tract and excised human ectocervical tissue. RC-101 100 µg films were shown to be safe to human and monkey tissue and effective against HIV-1 in vitro and ex vivo in human ectocervical tissue. The 90% inhibitory concentration (IC90) for RC-101 films at 2,000 µg (IC90=57.5 µM) using an ex vivo model was 10-fold higher than the IC90 observed using an in vitro model (IC90=5.0 µM). RC-101 films were stable for 1 month at 25°C, with in vitro bioactivity maintained for up to 6 months. RC-101 was developed in a quick-dissolve film formulation that was shown to be safe in an ex vivo model and effective in in vitro and ex vivo models. RC-101 film formulations were shown to maintain bioactivity for a period of 6 months. Findings from the present study contribute to the development of a safe and effective topical microbicide product.

Innate host defense of human vaginal and cervical mucosae.

Host defense responses of the human female genital tract mucosa to pathogenic microbes and viruses are mediated in part by the release of antimicrobial substances into the overlying mucosal fluid. While host defense has long been considered a prominent function of vaginal and cervical mucosae, evidence that cationic antimicrobial peptides and proteins have fundamental roles in the innate host defense of this tissue has only recently become available. This chapter explores elements of the physical and chemical defense barriers of the cervicovaginal mucosa, which protect against infections of the lower genital tract. Cationic antimicrobial and antiviral polypeptide components of cervicovaginal fluid are discussed in detail, with special emphasis placed on the defensin family of peptides as well as polypeptides that are active against viruses such as HIV-1. The reader should be cognizant that each polypeptide by itself does not provide complete protection of the genital tract. On the contrary, the abundance and multiplicity of antimicrobial peptides and proteins suggest protection of the cervicovaginal mucosa may be best realized from the aggregate effector molecules.

A theta-defensin composed exclusively of D-amino acids is active against HIV-1.

The ability of certain theta-defensins, including retrocyclin-1, to protect human cells from infection by HIV-1 marks them as potentially useful molecules. Theta-defensins composed of L-amino acids are likely to be unstable in environments that contain host and microbial proteases. This study compared the properties of two enantiomeric theta-defensins, retrocyclin-1, and RC-112. Although these peptides have identical sequences, RC-112 is composed exclusively of D-amino acids, whereas retrocyclin-1 contains only L-amino acids. We compared the ability of these peptides to protect JC53-BL human cells from infection by 30 primary HIV-1 isolates. JC53-BL cells are modified HeLa cells that express surface CD4, CXCR4, and CCR5. They also contain reporter cassettes that are driven by the HIV-1 LTR, and express beta-galactosidase and luciferase. The HIV-1 isolates varied in co-receptor specificity and included subtypes A, B, C, D, CRF01-AE, and G. RC-112 was several fold more potent than retrocyclin-1 across the entire HIV-1 panel. Although RC-112 bound immobilized gp120 and CD4 with lower affinity than did retrocyclin-1, surface plasmon resonance experiments performed with 1 microg/mL of RC-112 and retrocyclin-1 revealed that both glycoproteins were bound to a similar extent. The superior antiviral performance of RC-112 most likely reflected its resistance to degradation by surface-associated or secreted proteases of the JC53-BL target cells. Theta-defensins composed exclusively of D-amino acids merit consideration as starting points for designing microbicides for topical application to the vagina or rectum.

Determinants of Staphylococcus aureus nasal carriage.

Nasal carriage of Staphylococcus aureus has been identified as a risk factor for community-acquired and nosocomial infections. We screened 230 donors of diverse ethnic and socioeconomic backgrounds and identified 62 (27%) whose nasal secretions were colonized by S. aureus. In 18 donors in whom the various regions of the nasal luminal surface were separately sampled, the predominant region of S. aureus colonization was the moist squamous epithelium on the septum adjacent to the nasal ostium. Nasal fluid from carriers was defective in killing endogenous S. aureus and nasal carrier isolates of S. aureus but not a laboratory S. aureus strain. Transmission electron microscopy revealed that S. aureus isolates incubated in nasal fluid from carriers for 2 h at 37 degrees C were less damaged than those incubated in noncarrier fluid and were coated with an electron-dense layer. Compared with that from healthy donors and patients with acute rhinitis, nasal fluid from carriers contained elevated concentrations of the neutrophil-derived defensins human neutrophil peptides 1 to 3 (47- and 4-fold increases, respectively), indicative of a neutrophil-mediated inflammatory host response to S. aureus colonization. The concentration of the inducible epithelial antimicrobial peptide human beta-defensin 2 was also highly elevated compared to that in healthy donors, in whom the level was below the detection limit, or patients with acute rhinitis (sixfold increase). Thus, nasal carriage of S. aureus takes hold in nasal fluid that is permissive for colonization and induces a local inflammatory response that fails to clear the colonizing bacteria.

Calcitermin, a novel antimicrobial peptide isolated from human airway secretions.

The human airways are protected from pathogenic colonization by a blanket of fluid impregnated with innate antimicrobial effector molecules. Among several previously uncharacterized components, we isolated a peptide that had activity primarily targeting Gram-negative bacteria. We named the peptide 'calcitermin' since its amino acid sequence and mass were equivalent to the 15 C-terminal residues of the S100 protein, calgranulin C. The antimicrobial activity of calcitermin was enhanced in acidic buffers (pH 5.4) and in the presence of micromolar concentrations of ZnCl(2). Analysis revealed a putative zinc-binding consensus sequence as well as an alpha-helical conformation in structure-promoting solvents.

Cutting edge: IFN-inducible ELR- CXC chemokines display defensin-like antimicrobial activity.

Recent reports highlighted the chemotactic activities of antimicrobial peptide defensins whose structure, charge, and size resemble chemokines. By assaying representative members of the four known families of chemokines we explored the obverse: whether some chemokines exert antimicrobial activity. In a radial diffusion assay, only recombinant monokine induced by IFN-gamma (MIG/CXCL9), IFN-gamma-inducible protein of 10 kDa (IP-10/CXCL10), and IFN-inducible T cell alpha chemoattractant (I-TAC/CXCL11), members of the IFN-gamma-inducible tripeptide motif Glu-Leu-Arg (ELR)(-) CXC chemokines, were antimicrobial against Escherichia coli and Listeria monocytogenes. Similar to human defensins, antimicrobial activities of the chemokines were inhibited by 50 and 100 mM NaCl. The concentration of MIG/CXCL9 and IP-10/CXCL10 released from IFN-gamma-stimulated PBMC in 24 h were, respectively, 35- and 28-fold higher than from unstimulated cells. Additionally, the amounts of chemokines released per monocyte suggest that, in tissues with mononuclear cell infiltration, IFN-gamma-inducible chemokines may reach concentrations necessary for microbicidal activity. IFN-gamma-inducible chemokines may directly inactivate microbes before attracting other host defense cells to the area of infection.

Inhibition of neutrophil elastase prevents cathelicidin activation and impairs clearance of bacteria from wounds.

The host defense roles of neutrophil elastase in a porcine skin wound chamber model were explored. Analysis of wound fluid by acid-urea polyacrylamide gel electrophoresis, Western blot, and bacterial overlay confirmed that the neutrophil-derived protegrins constituted the major stable antimicrobial polypeptide in the wound fluid. The application to the wound of 0.10 and 0.25 mM N-methoxysuccinyl-alanine-alanine-proline-valine (AAPV) chloromethyl ketone, a specific neutrophil elastase inhibitor (NEI), blocked the proteolytic activation of protegrins and diminished the associated antimicrobial activity as detected by radial diffusion assay against Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans or by bacterial gel overlay against S epidermidis and E coli. The application of the related cathepsin G inhibitor (CGI), benzyloxycarbonyl-glycine-leucine-phenylalanine (ZGLF) chloromethyl ketone, had no effect. In wound chambers that received 10(6) colony-forming unit (CFU)/mL of S epidermidis, the presence of NEI significantly decreased the 24-hour clearance of bacteria from the wound compared to wounds treated with CGI or solvent only. Neither inhibitor, at 0.10 or 0.25 mM concentration, affected leukocyte accumulation or degranulation in the wound chambers. The in vitro microbicidal decrement due to NEI was restored by an amount of the specific protegrin (PG-1), which was equivalent to the measured difference of protegrin between control and inhibited chambers. Administration of 1 microg/mL exogenous PG-1 4 hours after chamber preparation was sufficient to normalize in vivo antimicrobial activity. Although pharmacologic NEIs are promising candidates as anti-inflammatory drugs, they may impair host defense in part by inhibiting the activation of cathelicidins by neutrophil elastase.

Therapeutic effect of a pig-derived peptide antibiotic on porcine wound infections.

PURPOSE: We investigated the therapeutic potential of the pig-derived antimicrobial peptide protegrin-1 (PG-1) against porcine skin wounds infected with Pseudomonas aeruginosa. MATERIALS AND METHODS: Using a porcine skin wound model, PG-1 was added to the wound fluid either at the time of P. aeruginosa inoculation, four hours after inoculation or 24 hours after inoculation. Wound fluids were analyzed 20-24 hours later by use of colony-forming unit (CFU) assays, semiquantitative immunoblot analysis for PG-1, and radial diffusion assays (RDA) for residual in vitro activity. RESULTS: Results of the CFU assays indicated a 10,000-fold decrease in the number of bacteria when PG-1 was added at the time of inoculation, a 120-fold decrease when added 4 hours after inoculation and a 10-fold decrease when added 24 hours after inoculation. Results of immunoblot analysis and RDA indicated that PG-1 concentrations for each of the three conditions remained increased in wound fluid 20 to 24 hours after treatment, and correlated with increased residual in vitro antimicrobial activity. CONCLUSIONS: These results document that the endogenous antibiotic PG-1 significantly prevented the colonization of P. aeruginosa in wounds and reduced the in vivo bacterial concentration in established wound infections. Therapeutics used in the same animal species from which they were derived are a promising means for preventing and treating localized infections.

Human antimicrobial peptides: analysis and application.

Antimicrobial peptides are innate host defense molecules that have a direct effect on bacteria, fungi and enveloped viruses. They are found in evolutionarily diverse species ranging from prokaryotes and plants to invertebrate and vertebrate animals. Humans express several families of antimicrobial peptides in myeloid cells and on various epithelial surfaces where they are poised to defend against pathogens. Recently, antimicrobial peptides from animals and plants have served as templates for the design of new therapeutic antibiotics. This review provides an introduction to the biology of human antimicrobial peptides, followed by a more detailed discussion of their isolation from tissues and biological fluids, their purification by gel electrophoresis and chromatography and assays of their antimicrobial activities.

Characterization of a fish antimicrobial peptide: gene expression, subcellular localization, and spectrum of activity.

Antimicrobial peptides are proposed to act as the first line of mucosal host defense by exerting broad-spectrum microbicidal activity against pathogenic microbes. Pleurocidin, a new 25-residue linear antimicrobial peptide, was recently isolated from the skin secretions of winter flounder (Pleuronectes americanus). The present study identifies the cDNA and gene encoding pleurocidin. The pleurocidin gene comprises four exons. Its upstream region demonstrates consensus binding sequences for transcription factors found in host defense genes in mammals, including sequences identical to the NF-IL6 and alpha and gamma interferon response elements. Pleurocidin is predicted to exist as a 68-residue prepropeptide that undergoes proteolytic cleavage of its amino-terminal signal and carboxy-terminal anionic propiece to form the active, mature peptide. Transmission electron microscopy localized pleurocidin to the mucin granules of skin and intestinal goblet cells. Significant synergy was shown to occur between pleurocidin and D-cycloserine targeting Mycobacterium smegmatis. Pleurocidin was functionally active at physiologic concentrations of magnesium and calcium; however, high concentrations of these divalent cations ablated pleurocidin's activity against a standard test strain, Escherichia coli D31. Pleurocidin was tested against bacterial and fungal clinical isolates and showed broad-spectrum antimicrobial activity. Together, these data support the hypothesis that pleurocidin participates in innate mucosal immunity, and it may prove to be a beneficial therapeutic agent.

Microanalysis of antimicrobial properties of human fluids.

Host defense responses of animals and plants to pathogenic microbes are mediated in part by the release of antimicrobial substances into tissue fluids. Exploration of the antimicrobial properties of tissue fluids is often limited by their small quantity. We have developed assays of antimicrobial activity that require only 1 microl of fluid. Using normal nasal secretions as a model mucosal fluid we demonstrated that the kinetics of the 1 microl colony-forming unit (CFU) assays were equivalent to the larger CFU assays. The handling of viscous mucin-containing fluids was facilitated by pretreatment with N-acetylcysteine (NAC), a treatment that did not alter the performance of the assay. This low-volume assay will facilitate studies of the antimicrobial properties of scarce biological fluids.

Innate antimicrobial activity of nasal secretions.

Minimally manipulated nasal secretions, an accessible form of airway surface fluid, were tested against indigenous and added bacteria by using CFU assays. Antimicrobial activity was found to vary between donors and with different target bacteria and was markedly diminished by dilution of the airway secretions. Donor-to-donor differences in electrophoresis patterns of nasal secretions in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and acid urea-PAGE analyses were readily observed, suggesting that polymorphic genes encode the secreted proteins. Three donors (of twenty-four total), whose nasal fluid yielded similar protein band patterns and did not kill indigenous bacteria, were determined to be heavy nasal carriers of Staphylococcus aureus. Their fluid was deficient in microbicidal activity toward a colonizing strain of S. aureus but the defect was corrected in vitro by a 1:1 addition of nasal fluid from noncarriers. The microbicidal activity of normal fluid was inactivated by heating it for 10 min to 100 degrees C and could not be restored solely by the addition of two major nasal antimicrobial proteins, lysozyme and lactoferrin. Several other known antimicrobial proteins and peptides, including statherin, secretory phospholipase A2, and defensins, were identified in nasal secretions and likely contribute to their total antimicrobial properties. Nasal fluid may serve as a useful model for the analysis of lower-airway secretions and their role in host defense against airway colonization and pulmonary infections.

Isolation and characterization of pleurocidin, an antimicrobial peptide in the skin secretions of winter flounder.

Antimicrobial peptides are found in both myeloid cells and mucosal tissues of many vertebrates and invertebrates. These peptides are predicted to operate as a first-line host defense mechanism exerting broad-spectrum activity against pathogenic bacteria, fungi, parasites, and enveloped viruses. We report the characterization of a novel 25-residue linear antimicrobial peptide found in the skin mucous secretions of the winter flounder (Pleuronectes americanus). This peptide was purified through multiple chromatographic methods to obtain a single peak by reversed-phase high performance liquid chromatography. This purified peptide, which we named pleurocidin, exhibited antimicrobial activity against Escherichia coli in a bacterial cell lysis plate assay. Mass spectrometry and amino acid sequence analysis indicated that it is 25 amino acids in length. Pleurocidin is predicted to assume an amphipathic alpha-helical conformation similar to many other linear antimicrobial peptides. There is a high degree of homology between pleurocidin and two antimicrobial peptides, ceratotoxin from the Mediterranean fruit fly and dermaseptin from the skin of a hylid frog. The minimal inhibitory concentration and minimal bactericidal concentration of pleurocidin were determined against 11 different Gram-negative and Gram-positive bacteria. Immunohistochemistry locates pleurocidin in the epithelial mucous cells of flounder skin. Pleurocidin represents a novel antimicrobial peptide found in fish and may play a role in innate host defense.

Production of active bovine tracheal antimicrobial peptide in milk of transgenic mice.

Tracheal antimicrobial peptide (TAP) is a member of the beta-defensin family of antibiotic peptides found in the tracheal mucosa of the cow. TAP gene expression in the bovine airway is inducible by lipopolysaccharide and inflammatory mediators, suggesting that it functions to protect the upper airway from infection. Limited availability of bovine TAP (bTAP) has precluded investigation of its potential utility in agriculture and medicine. To overcome this problem, transgenic mice expressing bTAP using an expression vector driven by control sequences from the murine whey acidic protein (WAP) gene have been generated. The WAP/bTAP transcript was detected in RNA isolated from mammary tissue of transgenic females. bTAP was purified to homogeneity from milk via acid precipitation, reverse-phase HPLC, and ion-exchange chromatography. This milk-derived bTAP had antimicrobial activity against Escherichia coli. Amino-terminal peptide sequencing confirmed the identity of this material as a bTAP isoform. bTAP available from a mammary gland bioreactor will allow evaluation of bTAP for use as an antibiotic in agriculture and medicine.

A comparison of caregivers for elderly stroke and dementia victims.

OBJECTIVE: To compare elderly co-resident caregivers of stroke and dementia patients on measures of burden and psychological morbidity. DESIGN: Cohort study. SETTING: Caregivers interviewed at home. SUBJECTS: Convenience sample, 99 co-resident caregivers of dementia and stroke patients registered with a community rehabilitation and geriatric service and who were 60 years or over. MAIN OUTCOME MEASURES: Caregiver burden as measured by self-administered questionnaire and subsequent interview, using the Relatives Stress Scale (RSS) and psychological morbidity as measured on the General Health Questionnaire (GHQ). RESULTS: Forty-six percent of caregivers had significant psychological morbidity (GHQ greater than 4). Mean RSS score was 12.2 (SD 5.4). No significant differences were found between stroke and dementia caregivers on these measures. Caregiver burden was significantly correlated with psychological morbidity in both caregiver groups. Behavior and mood disturbance in the patient was significantly correlated with burden (dementia caregivers r = 0.66; stroke caregivers r = 0.49, P less than 0.0001) and psychological morbidity (dementia caregivers r = 0.44, P less than 0.01; stroke caregivers r = 0.30, P less than 0.05). Caregiver's dissatisfaction with participation in life activities was correlated with burden (dementia caregivers r = 0.58; stroke caregivers r = 0.63, P less than 0.0001) and psychological morbidity (dementia caregivers r = 0.67, stroke caregivers r = 0.56, P, 0.0001). CONCLUSION: Elderly co-resident caregivers for stroke and dementia patients experience similar degrees of burden and high levels of psychological morbidity. Psychiatric aspects of chronic disability, rather than physical aspects, were found to be more stressful to caregivers. All assessments of the disabled elderly should include measures of caregiver burden and psychological distress.

Objective assessment of spasticity, strength, and function with early exhibition of dantrolene sodium after cerebrovascular accident: a randomized double-blind study.

A double-blind, placebo-controlled trial was conducted to determine whether early exhibition of Dantrium (Dantrolene Sodium) in patients with cerebrovascular accidents, before the onset of significant spasticity, would enhance the functional outcome of rehabilitation. Thirty-eight patients were enrolled in the trial and 31 satisfactorily completed the study. A modified Cybex isokinetic dynamometer was used to gather information on strength and muscle tone. Clinical, functional, and biochemical data were also collected. It was found that Dantrium reduced strength in the unaffected limbs but did not alter strength in the paretic limbs. Dantrium produced no alteration in clinical tone, functional outcome, or biochemical tests at the dosage (200 mg per day) used in this study.