Advances on chemically modified antimicrobial peptides for generating peptide antibiotics.

Antimicrobial peptides (AMPs) are pinpointed as promising molecules against antibiotic-resistant bacterial infections. Nevertheless, there is a discrepancy between the AMP sequences generated and the tangible outcomes in clinical trials. AMPs' limitations include enzymatic degradation, chemical/physical instability and toxicity toward healthy human cells. These factors compromise AMPs' bioavailability, resulting in limited therapeutic potential. To overcome such obstacles, peptidomimetic approaches, including glycosylation, PEGylation, lipidation, cyclization, grafting, D-amino acid insertion, stapling and dendrimers are promising strategies to fine-tune AMPs. Here we focused on chemical modifications applied for AMP optimization and how they have helped these peptide-based antibiotic candidates' design and translational potential.

An acidic model pro-peptide affects the secondary structure, membrane interactions and antimicrobial activity of a crotalicidin fragment.

In order to study how acidic pro-peptides inhibit the antimicrobial activity of antimicrobial peptides, we introduce a simple model system, consisting of a 19 amino-acid long antimicrobial peptide, and an N-terminally attached, 10 amino-acid long acidic model pro-peptide. The antimicrobial peptide is a fragment of the crotalicidin peptide, a member of the cathelidin family, from rattlesnake venom. The model pro-peptide is a deca (glutamic acid). Attachment of the model pro-peptide only leads to a moderately large reduction in the binding to- and induced leakage of model liposomes, while the antimicrobial activity of the crotalicidin fragment is completely inhibited by attaching the model pro-peptide. Attaching the pro-peptide induces a conformational change to a more helical conformation, while there are no signs of intra- or intermolecular peptide complexation. We conclude that inhibition of antimicrobial activity by the model pro-peptide might be related to a conformational change induced by the pro-peptide domain, and that additional effects beyond induced changes in membrane activity must also be involved.

An Immunomodulatory Peptide Confers Protection in an Experimental Candidemia Murine Model.

Fungal Candida species are commensals present in the mammalian skin and mucous membranes. Candida spp. are capable of breaching the epithelial barrier of immunocompromised patients with neutrophil and cell-mediated immune dysfunctions and can also disseminate to multiple organs through the bloodstream. Here we examined the action of innate defense regulator 1018 (IDR-1018), a 12-amino-acid-residue peptide derived from bovine bactenecin (Bac2A): IDR-1018 showed weak antifungal and antibiofilm activity against a Candida albicans laboratory strain (ATCC 10231) and a clinical isolate (CI) (MICs of 32 and 64 µg · ml-1, respectively), while 8-fold lower concentrations led to dissolution of the fungal cells from preformed biofilms. IDR-1018 at 128 µg · ml-1 was not hemolytic when tested against murine red blood cells and also has not shown a cytotoxic effect on murine monocyte RAW 264.7 and primary murine macrophage cells at the tested concentrations. IDR-1018 modulated the cytokine profile during challenge of murine bone marrow-derived macrophages with heat-killed C. albicans (HKCA) antigens by increasing monocyte chemoattractant protein 1 (MCP-1) and interleukin-10 (IL-10) levels, while suppressing tumor necrosis factor alpha (TNF-α), IL-1ß, IL-6, and IL-12 levels. Mice treated with IDR-1018 at 10 mg · kg-1 of body weight had an increased survival rate in the candidemia model compared with phosphate-buffered saline (PBS)-treated mice, together with a diminished kidney fungal burden. Thus, IDR-1018 was able to protect against murine experimental candidemia and has the potential as an adjunctive therapy.

Permeation of substituted silanes and siloxanes through selected gloves and protective clothing.

Testing of the permeation resistance of eight glove and suit barriers against commercially available substituted silanes and siloxanes was performed using the ASTM F739-96 standard test method. In addition to barrier performance to the pure organosilanes, the permeation rates of the hydrolysis product (usually ethanol or methanol) were investigated. The silanes and siloxanes used as the challenge agents were N-2-(aminoethyl)-3-aminopropyltrimethoxysilane; 3-aminopropyltriethoxysilane; 3-chloropropyltrimethoxysilane; ethyltriacetoxysilane; 3-glycidoxypropyltrimethoxysilane; 1,1,1,3,3,3-hexamethyldisilazane; hexamethyldisiloxane; 3-methacryloxypropyltrimethoxysilane; methyltriacetoxysilane (50%)/ethyltriacetoxysilane (50%); methyltrimethoxysilane; methyltris(methylethylketoxime)silane; phenyltrimethoxysilane; polydimethyl siloxanes (PS 340); octamethylcyclotetrasiloxane (D4); tetraethoxysilane; tetramethoxysilane; 1,1,3,3-tetramethyl disiloxane; triethoxysilane; trimethoxysilane; vinyltrimethoxysilane; and vinyltris(methylethylketoxime)silane. Protective gloves tested were nitrile rubber, neoprene rubber, butyl rubber, 4H laminate, and polyvinyl chloride. Garments tested included Tyvek/Saranex 23P, CPF 2, and Responder, all made by Kappler Safety Group. In all cases the protective suit materials lasted 8 hours or more. The only glove that lasted 8 hours against all chemicals was the 4H laminate. The polyvinyl chloride glove lasted 10 min to 8 hours or more depending on the chemical. The nitrile, neoprene, and butyl rubber gloves lasted from 53 min to 8 hours or more depending on the chemical. The alcohol permeation was similar to the organosilicon compounds. The suit materials and the butyl glove all lasted more than 8 hours for both methanol and ethanol.

Immunologic interference from sequential administration of live attenuated alphavirus vaccines.

Two different human vaccine trials examined interference arising from sequential administration of vaccines against heterologous alphaviruses. The first trial indicated that persons previously vaccinated against Venezuelan equine encephalitis virus (VEEV) exhibited poor neutralizing antibody responses to a live attenuated chikungunya virus (CHIKV) vaccine (46% response rate). The second trial prospectively examined neutralizing antibody responses to live attenuated VEEV vaccine in persons previously inoculated with either CHIKV vaccine or placebo. Following seroconversion to CHIKV, CHIKV vaccine recipients' geometric mean titers (GMTs) to VEEV by 80% plaque-reduction neutralization titration never exceeded 10, compared with a peak GMT of 95 after VEEV vaccination for alphavirus-naive volunteers who initially received placebo (P < .003). ELISA antibody responses demonstrated cross-reactive IgG to VEEV after primary CHIKV immunization and then an anamnestic response upon subsequent VEEV vaccination. These data indicate that preexisting alphavirus immunity in humans interferes with subsequent neutralizing antibody response to a live attenuated, heterologous vaccine.

Antibiotic treatment of experimental pneumonic plague in mice.

A mouse model was developed to evaluate the efficacy of antibiotic treatment of pneumonic plague; streptomycin was compared to antibiotics with which there is little or no clinical experience. Infection was induced by inhalation of aerosolized Yersinia pestis organisms. Antibiotics were administered by intraperitoneal injection every 6 hours for 5 days, at doses that produced levels of drug in serum comparable to those observed in humans treated for other serious infections. These studies compared in vitro to in vivo activity and evaluated the efficacy of antibiotics started at different times after exposure. Early treatment (started 24 h after challenge, when 0 of 10 mice tested had positive blood cultures) with netilmicin, ciprofloxacin, ofloxacin, ceftriaxone, ceftazidime, aztreonam, ampicillin, and rifampin (but not cefazolin, cefotetan, or ceftizoxime) demonstrated efficacy comparable to streptomycin. Late treatment (started 42 h after exposure, when five of five mice tested had positive blood cultures) with netilmicin, ciprofloxacin, ofloxacin, and a high dose (20 mg/kg of body weight every 6 h) of gentamicin produced survival rates comparable to that with streptomycin, while all of the beta-lactam antibiotics (cefazolin, cefotetan, ceftriaxone, ceftazidime, aztreonam, and ampicillin) and rifampin were significantly inferior to streptomycin. In fact, all groups of mice treated late with beta-lactam antibiotics experienced accelerated mortality rates compared to normal-saline-treated control mice. These studies indicate that netilmicin, gentamicin, ciprofloxacin, and ofloxacin may be alternatives for the treatment of pneumonic plague in humans. However, the beta-lactam antibiotics are not recommended, based upon poor efficacy in this mouse model of pneumonic plague, particularly when pneumonic plague may be associated with bacteremia.

Potentiation of inhaled staphylococcal enterotoxin B-induced toxicity by lipopolysaccharide in mice.

Nonhuman primates are the established model for evaluating toxic responses to staphylococcal enterotoxins (SEs), as they react similarly to humans. Rodents are generally considered unresponsive to SEs. Binding affinities and T-cell reactivity suggest that SE binds more efficiently to primate major histocompatability complex class II receptors than to mouse receptors. We investigated the potentiation of staphylococcal enterotoxin B (SEB) inhalation toxicity by lipopolysaccharide (LPS) in BALB/c mice. Lethality occurred only when SEB was potentiated by LPS. Neither SEB nor LPS produced lethal effects alone. Temporal responses of interleukin 1 alpha, tumor necrosis factor alpha, interleukin 2, and interferon-gamma evoked by inhaled SEB were enhanced by LPS. By 24 hr after intoxication, serum cytokines decreased to baseline levels, and consistent pulmonary perivascular leukocytic infiltrates were evident histologically. Histologic lesions induced by inhalation exposure to SEB by mice, with or without potentiation by LPS, were similar to those in the rhesus monkey. Predominant pulmonary lesions included severe, diffuse interstitial and alveolar pulmonary edema, leukocytic infiltrates, mild perivascular edema, and alveolar fibrin deposition. Although the mechanism of aerosolized SEB-induced toxicity has not been completely resolved, similarities in histologic lesions, cytokine responses, and acute dose-response suggest the LPS-potentiated mouse model may be a credible alternative to the nonhuman primate model.

Cell-mediated and humoral immune responses after vaccination of human volunteers with the live vaccine strain of Francisella tularensis.

The specific humoral and cell-mediated immune responses of human volunteers vaccinated with the Francisella tularensis live vaccine strain (LVS) were evaluated. In the search for an optimal antigen to measure the immunogenicity of the vaccine in an enzyme-linked immunosorbent assay, we tested irradiation-killed LVS, an aqueous ether extract of the LVS (EEx), lipopolysaccharide (LPS) from LVS, and a virulent strain (SCHU4). Volunteers were immunized with LVS by scarification. Immunoglobulin G (IgG) responses to LVS and LPS gave the highest background titers when tested with sera from unimmunized volunteers, whereas IgA, IgG, and IgM background titers to EEx and SCHU4 were low. Vaccination caused a significant rise (P < 0.01) in IgA, IgG, and IgM titers to all antigens tested, except for the IgG response to LPS. Eighty percent of vaccinated volunteers developed a positive IgG response to EEx 14 days postvaccination, while 50% were positive to LVS. By day 14 after vaccination, 70% of immunized volunteers exhibited a positive response to EEx in an in vitro peripheral blood lymphocyte proliferation assay. EEx, a specific and sensitive antigen for evaluating immune responses of vaccinated volunteers, may be a superior antigen for the diagnosis of tularemia.

Exposure variables in bancroftian filariasis in the Nile Delta.

To demonstrate focality of filariasis within endemic rural areas and to define exposure variables which may influence this phenomenon, the population of an agrarian endemic village, of 12,500 individuals, in the Nile Delta of Egypt was censused. A sequential sample of individuals residing in every fifth house was tested for microfilaremia (239 households with 8.6 +/- 3.5 individuals per household (HHD). Three areas of the village were tested simultaneously and a questionnaire was filled out for each sampled HHD with special emphasis given to the entomological and environmental factors that might affect filarial infection. One area (area A) had a higher intensity of larvae and biting adults of the main filarial vector, Culex pipiens, than the other two areas (areas B and C). Of the 1488 persons who agreed to be tested in the three areas 181 (12.2%) were microfilaremic. Microfilaremia prevalences were the same in males and females and microfilariae were present in all age groups. Filarial infection was most prevalent in area "A" (1.16 +/- 0.14 infected people per HHD) than in area "B" (0.44 +/- 0.11) or "C" (0.72 +/- 0.10) (ANOVA; p = 0.0003). several possible predictor variables were analyzed by logistic regression with the presence of infection as the response variable. Among individuals residing around the main Cx. pipiens development sites, those living in houses facing vacant land are exposed to more mosquito bites and had a greater chance of having filarial infection (relative risk [RR] = 1.5; logistic regression, P = 0.0089). People residing in large households had a reduced chance of having filarial infection (RR = 0.87; logistic regression, p = 0.0015). These data show that the distribution of microfilaremic individuals is uneven within the study village and suggest that small HHD and houses that bordered open areas containing mosquito development sites are potential risk factors for acquiring filarial infection.

Efficacy of a standard human anthrax vaccine against Bacillus anthracis spore challenge in guinea-pigs.

The efficacy of an anthrax vaccine licensed for human use, MDPH-PA, was tested in guinea-pigs intramuscularly challenged with 10, 100 or 1000 LD50 of spores from two virulent strains of Bacillus anthracis, Vollum 1B and Ames. As demonstrated in other investigations, immunization with MDPH-PA provided better protection against challenge with the Vollum 1B strain than with the Ames strain, although vaccine efficacy against the Ames strain was better than previously reported. Enzyme-linked immunosorbent assay of serum antibody titres to B. anthracis protective antigen showed that there was no significant correlation between survival and antibody titres.

A longitudinal study of Junin virus activity in the rodent reservoir of Argentine hemorrhagic fever.

We monitored Junin virus (JV) activity in rodent populations for 30 months at seven mark-recapture grids located in agricultural fields and adjacent roadsides and fence lines in endemic and nonendemic areas of Argentine hemorrhagic fever. Blood and oral swabs taken from rodents captured at five-week intervals were analyzed by enzyme-linked immunosorbent assay for JV antigen (Ag). Calomys laucha and C. musculinus were the most frequently captured rodents, making up 47% and 22% of captures, respectively. Of 41 Ag-positive captures, 37 were C. musculinus and four were C. laucha; 34 were from two trapping grids in the same locality. Antigen-positive Calomys were more frequently male (76%), and were found significantly more frequently among the oldest animals and the largest body mass classes. These patterns, combined with the greater mobility and higher frequencies of wounds among males than females, implicated horizontal transmission as the primary route of JV transmission between rodents. Seasonal maximum levels in JV prevalence (up to 25% of captured Ag-positive C. musculinus) occurred during periods of maximal population densities of Calomys. Spatial distribution of Ag-positive rodents reflected habitat preferences; most Ag-positive C. musculinus were captured from border habitats (roadsides and fence lines), and all Ag-positive C. laucha were captured in crop fields. These distinct, but previously undocumented, habitat preferences suggest that the disease in humans may be related to exposures to the primary reservoir species, C. musculinus, in border habitats rather than in crop fields.

Cell-mediated and humoral immune responses induced by scarification vaccination of human volunteers with a new lot of the live vaccine strain of Francisella tularensis.

Tularemia is a disease caused by the facultative intracellular bacterium Francisella tularensis. We evaluated a new lot of live F. tularensis vaccine for its immunogenicity in human volunteers. Scarification vaccination induced humoral and cell-mediated immune responses. Indications of a positive immune response after vaccination included an increase in specific antibody levels, which were measured by enzyme-linked immunosorbent and immunoblot assays, and the ability of peripheral blood lymphocytes to respond to whole F. tularensis bacteria as recall antigens. Vaccination caused a significant rise (P less than 0.05) in immunoglobulin A (IgA), IgG, and IgM titers. Lymphocyte stimulation indices were significantly increased (P less than 0.01) in vaccinees 14 days after vaccination. These data verify that this new lot of live F. tularensis vaccine is immunogenic.

Immunization against anthrax with Bacillus anthracis protective antigen combined with adjuvants.

The protective efficacy of immunization against anthrax with Bacillus anthracis protective antigen (PA) combined with different adjuvants was tested in Hartley guinea pigs and CBA/J and A/J mice. Adjuvant components derived from microbial products that were tested included threonyl-muramyl dipeptide (threonyl-MDP); monophosphoryl lipid A (MPL); trehalose dimycolate (TDM); and the delipidated, deproteinized, cell wall skeleton (CWS) from either Mycobacterium phlei or the BCG strain of Mycobacterium bovis. Non-microbially derived adjuvants tested included aluminum hydroxide and the lipid amine CP-20,961. In guinea pigs, all adjuvants and adjuvant mixtures enhanced antibody titers to PA as well as survival after a parenteral challenge of virulent B. anthracis Ames spores. In contrast, PA alone or combined with either aluminum hydroxide or CP-20,961 failed to protect mice. Vaccines containing PA combined with threonyl-MDP or MPL-TDM-CWS protected a majority of female CBA/J mice. Statistical analysis of survival data in the guinea pigs indicated that PA-MPL-CWS and PA-MPL-TDM-CWS were more efficacious than the currently licensed human anthrax vaccine.

Egg hatching of Aedes mosquitoes during successive floodings in a Rift Valley fever endemic area in Kenya.

Floodwater Aedes breeding habitats in central Kenya were sequentially flooded to determine the numbers of mosquito eggs hatching during each flooding. Approximately 90% of the larvae sampled during 4 floodings emerged during the initial flooding. The number of Aedes eggs hatching during the second flooding was lowest of all 4 floodings, and no significant differences in the amount of egg hatching during floodings 3 and 4 were seen. Unhatched Aedes eggs were present in soil samples collected after the final flooding. The possible implications of these findings with regard to Rift Valley fever virus control are discussed.

Influence of body weight on response of Fischer 344 rats to anthrax lethal toxin.

Groups of Fischer 344 rats were injected intravenously with Bacillus anthracis culture supernatant containing crude anthrax toxin. Times to death of rats given identical toxin preparations varied directly with the weights of the rats (P = 0.0001). In contrast to previous reports, the data indicate that rat weight must be taken into account during in vivo assays of anthrax lethal toxin activity.

Resistance to the Sterne strain of B. anthracis: phagocytic cell responses of resistant and susceptible mice.

Inflammatory responses were compared in vivo, and host phagocytic cell functions compared in vitro, of mice resistant (CBA/J) and susceptible (A/J) to lethal infection with the Sterne strain of Bacillus anthracis. Polymorphonuclear leukocyte (PMN) and macrophage responses at the initial site of infection were slower in A/J mice than in CBA/J mice. Whereas in A/J mice, the number of PMN ultimately responding to infection was equal to, or greater than, that in CBA/J mice, fewer macrophages accumulated. A/J mice failed to clear relatively low doses of the organisms and died. In vitro, chemotactic responses to both serum- and bacteria-derived attractants were similar for macrophages from A/J and CBA/J mice but were reduced for PMN from A/J mice. PMN and macrophages from the two mouse strains phagocytosed and killed spores in vitro to a similar extent, although killing by A/J PMN could be blocked by prior uptake of large numbers of killed spores. Thus susceptibility to lethal infection with Sterne strain correlated with the delayed influx (PMN) and reduced accumulation (macrophages) of phagocytes at the initial site of infection, but not with defective in vitro uptake or killing of spores.

Efficacy of a commercial bacterin in protecting strain 13 guineapigs against Bordetella bronchiseptica pneumonia.

Bordetella bronchiseptica is known to be endemic in many guineapig (Cavia porcellus) colonies, and periodically is the aetiological agent of fatal epizootics of bronchopneumonia. A commercial, non-adjuvant B. bronchiseptica bacterin, which is approved for use in canines, was evaluated for induction of a protective immune response in Strain 13/N guineapigs against an airborne challenge of virulent B. bronchispeptica in small-particle aerosol. Seronegative animals were vaccinated on days 0 and 21 with intramuscular injections of 0.2 ml of bacterin. Humoral antibody titres of the vaccinated animals, as determined by ELISA, ranged from 128-1024 on day 49. On day 30 following the second dose of bacterin (study day 51), 12 vaccinated and 12 PBS sham-vaccinated animals were exposed to an inhaled dose of 4.3 x 10(5) CFU of B. bronchiseptica (325 LD50). Vaccinated, challenged animals remained clinically normal, although each guineapig did develop a localized upper respiratory infection. The rate of weight gain as well as rectal temperature of these animals were analogous to those exhibited by the control groups. Examination of 4 of the vaccinated, challenged animals on day 7 after exposure showed bacteria present in moderate to high numbers in the larynx and trachea but only minimally detectable in the lungs; by 30 days after exposure, the numbers of bacteria in the larynx and trachea were diminished, with none being detected in the lungs. Pathological alterations induced by B. bronchiseptica were not detected at either day 7 or day 30 after challenge in any of the vaccinated, challenged animals. Protection induced in Strain 13/N guineapigs by the commercial canine bacterin was sufficient to preclude the development of pulmonary disease, even in animals presented with a massive challenge of virulent bacteria in a small-particle aerosol.

An automated flow-temperature-humidity control system.

An automated system that controls air flow, temperature, and humidity has been developed from a commercially available temperature=humidity indicator and a specially built flow-temperature-humidity control module. Parameters are set using direct-reading dials on the control module. The air flow is maintained using a mass-flow controller while process controllers connected to the indicator regulate humidity and temperature. The system will run indefinitely without need for operator intervention. If the module and indicator are calibrated properly, accurate air flows (+/- 2% of full scale), temperatures (+/- 0.3 degrees C), and humidities (+/- 2% RH) can be achieved.

Enhancement of air filtration using electric fields.

Although polarized electrostatic air filters are efficient air filtrating devices, their main disadvantages are difficulty in collecting conductive particles or in operating at relative humidities above 70%. We describe here a new filter design that eliminates these problems. A nonconductive media, normally a glass fiber mat, is placed between two insulated conductive screens. As the voltage across the screens is increased, the penetration of particles decreases exponentially. Increasing the electric field from 0 to 10 kV/cm will decrease the mass penetration from 60% to less than 10% of a polydispersed 0.8 micrometer ammd(sigma g = 2.0) sodium chloride aerosol. The experimental effects of face velocity, particle charge and size, packing density, fiber size, and screen insulation mirror the theoretical effects of these variables on particle penetration.

Respiratory cartridge efficiency studies: VIII. summary and conclusions.

The theory of solvent vapor adsorption of activated carbon is reviewed. Calculated and experimental cartridge service life values are compared using various breathing rates, relative humidities, concentrations and solvent vapors. Cartridge service life (the 10% breakthrough time) can be estimated from the emperical expression: t 10% = 2.4 X 10(6) WC (A + BT)/C 2/3 MQ Carbon weight (wc), relative solvent volatility (a, b and t) concentration (C), molecular weight (M) and breathing rate (Q) all play a vital role in cartridge performance predictions.