Virulence spectra of typed strains of Campylobacter jejuni from different sources: a blinded in vivo study.

Campylobacter jejuni is a major cause of human diarrhoeal disease, but specific virulence mechanisms have not been well defined. The aims of the present blinded study were to measure and compare the in vivo properties of 40 serotyped, biotyped and genotyped C. jejuni isolates from different sources and genetic makeup. An 11-day-old chick embryo lethality assay, which measured embryo deaths and total viable bacteria over 72 h following inoculation of bacteria into the chorioallantoic membrane, revealed a spectrum of activity within the C. jejuni strains. Human and chicken isolates showed similar high virulence values for embryo deaths while the virulence of the bovine isolates was less pronounced. A one-way ANOVA comparison between the capacity of the strains to kill the chick embryos after 24 h with cytotoxicity towards cultured CaCo-2 cells was significant (P=0.025). After inoculation with a Campylobacter strain, mouse ligated ileal loops were examined histologically and revealed degrees of villous atrophy, abnormal mucosa, dilation of the lumen, congestion and blood in lumen, depending on the isolate examined. A 'total pathology score', derived for each C. jejuni strain after grading the pathology features for degree of severity, showed no apparent relationship with the source of isolation. Some relationship was found between amplified fragment length polymorphism groups and total ileal loop pathology scores, and a one-way ANOVA comparison of the mouse pathology scores against total chick embryo deaths after 72 h was significant (P=0.049).

Innovative materials processing strategies: a biomimetic approach.

Many organisms construct structural ceramic (biomineral) composites from seemingly mundane materials; cell-mediated processes control both the nucleation and growth of mineral and the development of composite microarchitecture. Living systems fabricate biocomposites by: (i) confining biomineralization within specific subunit compartments; (ii) producing a specific mineral with defined crystal size and orientation; and (iii) packaging many incremental units together in a moving front process to form fully densified, macroscopic structures. By adapting biological principles, materials scientists are attempting to produce novel materials. To date, neither the elegance of the biomineral assembly mechanisms nor the intricate composite microarchitectures have been duplicated by nonbiological processing. However, substantial progress has been made in the understanding of how biomineralization occurs, and the first steps are now being taken to exploit the basic principles involved.

Comparison of virulence-associated in vitro properties of typed strains of Campylobacter jejuni from different sources.

Campylobacter jejuni is a major cause of human diarrhoeal disease, but specific virulence mechanisms have not been well defined. This blinded study was undertaken with 40 C. jejuni isolates from different sources to determine their haemolytic, cytotoxic and adhesion and invasion activities towards mammalian cells. The results were correlated with source of isolation and genetic makeup by amplified fragment length polymorphism (AFLP) typing. The isolates had variable degrees of haemolytic activity against rabbit erythrocytes and cytotoxicity towards CaCo-2, HeLa and Vero cells. The data indicated that the haemolytic and cytotoxic activities were due to separate factors. A range of cytotoxicity was exhibited, whereby some strains had no activity against the target cells and others had activity against all three cell lines. Certain strains had activity against CaCo-2 cells but little or no activity against the other cells, while others exhibited the opposite phenotype. The data suggested that the cytotoxicity assay with the different cell lines may have detected more than one cytotoxin. A wide variation between isolates was observed for both adherence and invasion with all three cell lines, yet, overall, the strains showed a significantly greater invasion capacity for CaCo-2. There was no clear relationship between source of isolation or disease manifestation and possession of statistically significantly higher levels of particular virulence-associated factors although, in some cases, a correlation between cytotoxicity and cell invasion was evident. Five AFLP clusters, each representing two to eleven isolates with similar profiles, were observed at the 90 % similarity level. Some AFLP groups contained isolates with a common serotype, but each group had C. jejuni isolates from more than one source with the exception of group IV, which contained only human isolates. Isolates with high cytotoxic activity against CaCo-2 cells were confined to groups I, III and IV and a group of unrelated strains (U). Group II isolates had uniformly low cytotoxicity. Isolates in groups I, V and U were more invasive for CaCo-2 cells than isolates in groups II, III and IV. The strain differences in cytotoxicity or invasion did not correlate with source of isolation.

Stimuli-responsive liposomes for drug delivery.

The ultimate goal of drug delivery is to increase the bioavailability and reduce the toxic side effects of the active pharmaceutical ingredient (API) by releasing them at a specific site of action. In the case of antitumor therapy, association of the therapeutic agent with a carrier system can minimize damage to healthy, nontarget tissues, while limit systemic release and promoting long circulation to enhance uptake at the cancerous site due to the enhanced permeation and retention effect (EPR). Stimuli-responsive systems have become a promising way to deliver and release payloads in a site-selective manner. Potential carrier systems have been derived from a wide variety of materials, including inorganic nanoparticles, lipids, and polymers that have been imbued with stimuli-sensitive properties to accomplish triggered release based on an environmental cue. The unique features in the tumor microenvironment can serve as an endogenous stimulus (pH, redox potential, or unique enzymatic activity) or the locus of an applied external stimulus (heat or light) to trigger the controlled release of API. In liposomal carrier systems triggered release is generally based on the principle of membrane destabilization from local defects within bilayer membranes to effect release of liposome-entrapped drugs. This review focuses on the literature appearing between November 2008-February 2016 that reports new developments in stimuli-sensitive liposomal drug delivery strategies using pH change, enzyme transformation, redox reactions, and photochemical mechanisms of activation. WIREs Nanomed Nanobiotechnol 2017, 9:e1450. doi: 10.1002/wnan.1450 For further resources related to this article, please visit the WIREs website.

Triggered release of hydrophilic agents from plasmalogen liposomes using visible light or acid.

Triggered release from liposomes composed of semi-synthetic 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphocholine (plasmalogen) lipids has been demonstrated using either aerobic visible illumination or low pH to induce leakage. The photodynamic release system consists of three functional components: (1) small (less than 1000 A) unilamellar plasmalogen vesicles (SUVs) containing encapsulated glucose, (2) oxygen and (3) zinc phthalocyanine (ZnPc) incorporated within the hydrophobic region of the SUV membrane. Irradiation (lambda greater than 640 nm) at 37 degrees C of air-saturated 1-alk-1'-enyl-2-palmitoyl-sn-glycero-3-phosphocholine (PlasPPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (8:1, mol/mol) liposomes at physiologically relevant temperatures results in glucose release rates that are twice those of the corresponding dark control. Photolysis of argon-saturated PlasPPC/DPPC liposomes or of identical vesicles lacking either ZnPc or the plasmalogen vinyl ether bond exhibit glucose release curves which are indistinguishable from the dark control. Irradiation under identical conditions, but in the presence of 100 mM sodium azide, also results in no increased rate of glucose release above that of the dark control. TLC analysis indicates that oxidized lipid species are produced only in air-saturated, irradiated plasmalogen liposomes. The acid lability of the plasmalogen vinyl ether linkage has also been used to trigger release of entrapped calcein. At pH 4.2, the release rate at 37 degrees C is increased 4-fold over rates observed at pH 8. TLC analysis indicates formation of a lysoplasmalogen product. Taken together, these results indicate that both photodynamic and acid triggering can be used to increase plasmalogen liposome permeability and suggest that these liposomes are potentially useful for drug delivery applications.

Acid-catalyzed plasmenylcholine hydrolysis and its effect on bilayer permeability: a quantitative study.

This laboratory has previously shown (Anderson, V.C. and Thompson, D.H. (1992) Biochim. Biophys. Acta 1109, 33-42; Thompson, D.H., Gerasimov, O.V., Wheeler, J.J., Rui, Y. and Anderson, V.C. (1996) Biochim. Biophys. Acta 1279, 25-34), that plasmenylcholine (1-alk-1'-enyl-2-palmitoyl-sn-glycero-3-phosphocholine; PlsPamCho) liposomes release hydrophilic contents upon photooxidation or acid-catalyzed hydrolysis. We now report the kinetics and chemical mechanism of the acid-catalyzed reaction and its effect on calcein leakage rates. Hydrolysis of the plasmenylcholine vinyl ether linkage generates fatty aldehydes and 1-hydroxy-2-palmitoyl-sn-glycero-3-phosphocholine (lysolipid); HPLC and 1H-NMR experiments establish that the former is readily air-oxidized to fatty acids, while the latter undergoes rapid acid-catalyzed rearrangement to 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine. Lysolipid formation obeys first order kinetics, yielding observed pseudo-first order rate constants that are pH-dependent. Bimolecular hydrolysis rate constants, k(bi), have also been determined. Calcein release rates from plasmenylcholine liposomes are strongly dependent on both the dihydrocholesterol (DHC) content and the extent of PlsPamCho hydrolysis within the bilayer. DHC-free plasmenylcholine liposomes (38 degrees C, pH 2.5) require < 5% PlsPamCho hydrolysis to effect > 50% calcein release within 10 min. The presence of > or = 25 mol% DHC, however, greatly reduces the observed calcein release rate; nearly 30% PlsPamCho hydrolysis is required to effect 50% calcein release over a 70-min period in 6:4 PlsPamCho/DHC liposomes. Bacteriochlorophyll a-sensitized photooxidation of plasmenylcholine liposomes also produces fatty aldehyde and another intermediate, tentatively described as 1-formyl-2-palmitoyl-sn-glycero-3-phosphocholine, that hydrolyzes to form the 1-hydroxy lysolipid. These results have important implications for the quantitative description of lysolipid effects on membrane permeability and on the design of triggerable liposomes for drug delivery.

Preparation and electrochemical behavior of gramicidin-bipolar lipid monolayer membranes supported on gold electrodes.

Gramicidin-containing synthetic bolalipid membranes comprised of 2,2'-di-O-decyl-3,3'-O-1,20-eicosanyl-bis-rac-glycero-1,1'-diphosphocholine (C20BAS) have been synthesized and supported on gold electrodes. Supported membranes were prepared by first depositing a partial bolalipid layer on the electrode using a thioctic acid-modified bolalipid (1'-O-omega-thioctamidetetraethylene glycol-2,2'-di-O-decyl-3,3'-di-O-1,20-eicosanyl-bis-rac-glycero-1-phosphate, SSC20BAS) as an anchoring group, followed by a vesicle fusion step using either pure C20BAS or gramicidin-containing C20BAS (C20BAS-GA) vesicles. The latter configuration was designed to immobilize single, continuously-on channels of gramicidin in the C20BAS membrane. Vesicle deposition to form supported bolalipid monolayer membranes was monitored by impedance spectroscopy and cyclic voltammetry. Impedances were observed to increase with vesicle deposition time. Pretreatment of the impedance electrode with SSC20BAS accelerated the supported monolayer membrane deposition rate. Impedances decreased in a gramicidin concentration-dependent manner when gramicidin was incorporated into the C20BAS membrane. These supported bolalipid membranes are also surprisingly inert to organic solvent exposure (CH(3)CH(2)OH;CH(2)Cl(2)), suggesting that they may serve as robust host matrices for integral membrane protein-based sensors.

Triggerable plasmalogen liposomes: improvement of system efficiency.

A photoactivated liposome release system that is generally applicable for triggered release of encapsulated hydrophilic materials is described. This approach to phototriggered release, derived from the known effects of plasmalogen photooxidation on membrane permeability in whole cells and model membrane systems, relies on producing a lamellar phase change or increase in permeability upon cleaving its constitutive lipids to single-chain surfactants using 630-820 nm light to sensitize the photooxidation of the plasmalogen vinyl ether linkage. Semi-synthetic plasmenylcholine liposomes containing encapsulated calcein and a membrane-bound sensitizer, such as zinc phthalocyanine, tin octabutoxyphthalocyanine, or bacteriochlorophyll a, were prepared by extrusion. Irradiation of air-saturated liposome solutions enhanced membrane permeability toward calcein and Mn2+, and promoted membrane fusion processes compared to non-irradiated or anaerobic controls. Bacteriochlorophyll a sensitization produced the fastest observed photoinitiated release rate from these liposomes (100% calcein release in less than 20 min; 800 nm irradiation at 300 mW); the observed release rate was two orders of magnitude slower for egg lecithin liposomes prepared and irradiated under identical experimental conditions. Liposome aggregation, interlipidic particle formation, and membrane fusion between adjoining liposomes was observed by 31P-NMR, freeze-fracture/freeze-etch TEM, and cryo-TEM as a function of irradiation time. The use of near-infrared sensitizers and the capacity of photolyzed plasmenylcholine liposomes to undergo membrane fusion processes make photodynamic therapy with these liposome-borne sensitizers an attractive adjunct to biochemical targeting methods.

Phototriggering of liposomal drug delivery systems.

Over the past several years, photodynamic therapy (PDT) has been approved for the treatment of various cancers. Additional applications of photochemical processes for triggering site-specific drug delivery are in early stages of development at this time. This review focuses on the literature appearing between January 1996-June 2001 that describe new and ongoing studies of phototriggering mechanisms that may ultimately find utility in drug delivery applications.

Efficient synthesis of 40- and 48-membered tetraether macrocyclic bisphosphocholines.

An efficient route toward the synthesis of unsaturated (bis-diacetylenic) and saturated 40- and 48-membered macrocyclic biphosphocholines has been developed using 2-phenyl-5-hydroxy-1,3-dioxane as a common glycerol synthon. Ring closure was accomplished using either high-dilution Glaser oxidation of [(Cy3P)2RU==CHPh]Cl2-catalyzed olefin metathesis conditions. Deprotection of benzyl ethers using trimethylsilyl iodide (TMS-I) in the presence of diacetylenic moieties has also been demonstrated for the first time.

Synthesis of acid-labile diplasmenyl lipids for drug and gene delivery applications.

The low pH environments characteristic of endosomal compartments and ischemic tissues provide an intrinsic pathway for triggering site-specific contents release from appropriately designed delivery vehicles. Accordingly, research in this group has focused on the design, synthesis and application of novel acid-sensitive lipids that will undergo facile lamellar (L alpha) to hexagonal (HII) phase transitions within these acidic sites. Previously, it has been demonstrated that plasmenylcholine-type lipids have excellent acid hydrolysis and contents release kinetics (Gerasimov et al., Biochim. Biophys. Acta. 1324 (1997) 200-214; Rui et al., J. Am. Chem. Soc. 120 (1998) 11213-11218). This paper describes the synthesis of three new acid sensitive lipids, based on a chiral 1,2-di-O-(1Z',9Z'-octadecadienyl)-sn-glycerol (6) platform, displaying phosphocholine (7), poly(ethyleneoxide) (8), and O-carbamoyl-N-diethylen-etriamine (10) headgroups. Intermediate 6 was obtained in 28% overall yield via a six step synthesis from (S)-(+)-2,2-dimethyl-1,2-dioxolane-4-methanol. Subsequent conversion to the final products was acheived in moderate (7 and 10) to excellent yields (8).

Aerobic and anaerobic microbiology of peritonsillar abscess.

Thirty-four aspirates of pus from peritonsillar abscesses that were studied for aerobic and anaerobic bacteria showed bacterial growth. A total 107 bacterial isolates (58 anaerobic and 49 aerobic and facultative) were recovered, accounting for 3.1 isolates per specimen (1.7 anaerobic and 1.4 aerobic and facultatives). Anaerobic bacteria only were present in 6 (18%) patients, aerobic and facultatives in 2 (6%), and mixed aerobic and anaerobic flora in 26 (76%). Single bacterial isolates were recovered in 4 infections, 2 of which were Streptococcus pyogenes and 2 were anaerobic bacteria. The predominant bacterial isolates were Staphylococcus aureus (6 isolates), Bacteroides sp (21 isolates, including 15 Bacteroides melaninogenicus group), and Peptostreptococcus sp (16) and S. pyogenes (10). beta-Lactamase-producing organisms were recovered from 13 (52%) of 25 specimens tested. This retrospective study highlights the polymicrobial nature and importance of anaerobic bacteria in peritonsillar abscess.

Aerobic and anaerobic microbiology of acute suppurative parotitis.

Aspirates of pus from acute suppurative parotitis were studied for aerobic and anaerobic bacteria. Bacterial growth was present in 23 specimens. A total of 36 bacterial isolates (20 anaerobic and 16 aerobic and facultative) were recovered, accounting for 1.6 isolates per specimen (0.9 anaerobic and 0.7 aerobic and facultative). Anaerobic bacteria only were present in 10 (43%) patients, aerobic and facultatives in 10 (43%), and mixed aerobic and anaerobic flora in 3 (13%). Single bacterial isolates were recovered in 9 infections, 6 of which were Staphylococcus aureus and 3 were anaerobic bacteria. The predominant bacterial isolates were S. aureus (8 isolates), Bacteroides sp. (6 isolates, including 4 Bacteroides melaninogenicus group), and Peptostreptococcus sp. (5). beta-Lactamase-producing organisms were recovered from 11 (73%) of the 15 specimens tested. This study highlights the polymicrobial nature and importance of anaerobic bacteria in acute suppurative parotitis.

Computerized axial tomography versus complex motion as a predictor of surgical findings in middle ear and mastoid cholesteatoma.

In order to evaluate the efficacy of polytomography and computerized tomography in the prediction of surgical findings in cases of suspected cholesteatoma of the middle ear and mastoid, we evaluated preoperatively 52 ears by both radiographic methods. We utilized a check list of 14 radiological signs or surgical findings to generate measures of sensitivity and specificity. The strengths of each radiographic method are outlined and the considerable weaknesses in predictive value are discussed.

Primary small cell (oat cell) carcinoma of the larynx associated with an IgD multiple myeloma.

Primary small cell (oat cell) carcinoma of the larynx is a rare condition. We report a case of primary oat cell carcinoma of the subglottic larynx associated with a synchronous IgD multiple myeloma (an unreported association). An increased incidence of carcinoma associated with plasma cell disorders has been reported, and the theories of this association are discussed. In a review of the reported cases, the most successful management of oat cell carcinoma of the larynx appears to incorporate a combination of radiotherapy and chemotherapy. Our case was treated with a combination of protocols used for oat cell carcinoma of the larynx and multiple myeloma. At 24 months after diagnosis, the patient is free of oat cell carcinoma, and the multiple myeloma is under control.

Microbiology and management of chronic maxillary sinusitis.

OBJECTIVE: Assessment of the microbiology and management of patients who suffered from chronic maxillary sinusitis was studied retrospectively. DESIGN: Retrospective analysis of microbiology and antimicrobial therapy of 68 patients who underwent the Caldwell-Luc procedure for chronic sinusitis had not received antimicrobials before surgery and whose cultures showed bacterial growth. SETTING: This study was performed at the Naval Hospital in Bethesda, Md. INTERVENTION: Amoxicillin-clavulanic acid was given to 18 patients, amoxicillin or ampicillin to 25, cefaclor to 17, and erythromycin to eight. RESULTS: A total of 183 isolates (123 anaerobic and 60 aerobic) were recovered. Anaerobic organisms only were recovered from 35 (51%), specimens, and aerobic or facultative bacteria only in 12 (18%), and mixed aerobic and anaerobic flora in 21 (31%). Thirty-four aerobic and anaerobic beta-lactamase-producing bacteria were isolated from 28 patients. The 18 patients who received amoxicillin-clavulanic acid had the most rapid and complete response to therapy, none required a change in therapy, and surgical drainage was required in one case. Of 25 patients who received amoxicillin or ampicillin, eight required a change of therapy due to clinical failure (32%), including three who also had surgical drainage. Of 17 that received cefaclor, five had an antibiotic change (29%), one with surgical drainage. Of the eight who were treated with erythromycin, three needed antibiotic change (38%), two with surgical drainage. Resistant organisms were recovered from most of the patients that required therapeutic change. CONCLUSIONS: These findings indicate the major role of aerobic and anaerobic beta-lactamase-producing bacteria organisms in the polymicrobial etiology of chronic maxillary sinusitis and illustrate the superiority of therapy effective against these bacteria.

Control of early postoperative pain with bupivacaine in adult local tonsillectomy.

Early postoperative pain following tonsillectomy remains a significant obstacle to speedy recovery and smooth convalescence. Inadequate analgesia causes poor oral intake and occasionally requires overnight hospitalization in same-day surgery practices. Although several otolaryngologists anecdotally support intraoperative infiltration with long-acting anesthetic agents for postoperative pain control, to our knowledge, no previous study confirms this claim. In a prior investigation, we found no difference between bupivacaine hydrochloride and saline placebo in pediatric patients undergoing tonsillectomy. In this trial, we performed a similar study in an adult population. Fifty-one patients undergoing tonsillectomy with local anesthesia were randomized into bupivacaine or saline placebo groups. Patients provided the following data: (1) pain level; (2) oral intake; (3) number of pain medication doses; and (4) level of pain on jaw opening, all at 10 hours postoperatively. Bupivacaine administration resulted in no adverse effects. No difference was noted in pain level, amount of oral intake, or pain on full jaw opening. Bupivacaine group patients received fewer though not statistically significant doses of pain medication than placebo group patients. We conclude that bupivacaine is a safe medication but offers no advantage in the control of early postoperative pain in adult patients undergoing local tonsillectomy.

Microbiology of otitis externa.

Microbiologic and clinical data from 26 patients with otitis externa were prospectively evaluated. Specimens were processed for aerobic and anaerobic bacteria. Bacterial growth was noted in 23 specimens. A total of 33 aerobic and 2 anaerobic bacteria were recovered. Aerobic bacteria only were isolated in 21 (91%) patients, anaerobic bacteria only in 1 (4%), and mixed aerobic and anaerobic bacteria in 1 (4%). The most common isolates were Pseudomonas aeruginosa (14 instances), Staphylococcus aureus (7), Acinetobacter calcoaceticus (2), Proteus mirabilis (2), Enterococcus faecalis (2), Bacteroides fragilis (1), and Peptostreptococcus magnus (1). One isolate was recovered in 13 (57%) patients, 2 isolates in 8 (35%), and 3 isolates in 2 (9%). These data illustrate the polymicrobial nature of otitis externa in about half of the patients and the role of anaerobic bacteria in 8% of them. Further studies are warranted to evaluate the therapeutic implications of these findings.

Effect of anemia on survival of myocutaneous flaps in the pig.

The decision to transfuse patients after major head and neck reconstructive surgery has been influenced by the dictum that a hematocrit level of 30% or more is necessary for the survival of surgical flaps. Pedicled myocutaneous flaps are among the most frequently used methods of reconstruction after major head and neck oncologic surgery. No studies have addressed the survival of myocutaneous pedicled flaps in anemic animals. In this study survival of latissimus dorsi myocutaneous flaps in pigs was evaluated in anemic and control groups. A total of 26 pigs were randomly divided into two groups. The pigs in the anemic group were exsanguinated to normovolemic anemia (average hematocrit, 19%), followed by elevation of a latissimus dorsi myocutaneous flap. In the control group the same operation was performed without exsanguination. All other variables were kept constant. The flap survival was judged on postoperative days 3, 7, and 14 by two evaluators. Ten pigs from each group were found to have 100% flap survival on post-operative day 14. There was no significant difference in mean flap survival rates between two groups for postoperative days 3, 7, and 14. It is concluded that normovolemic anemia does not adversely affect the survival of the myocutaneous flaps. This finding may save unnecessary transfusions in postoperative patients.

Quantitative bacteriology of tonsils removed from children with tonsillitis hypertrophy and recurrent tonsillitis with and without hypertrophy.

The aerobic and anaerobic bacterial species and their numbers were studied in tonsillar specimens from children who had undergone elective tonsillectomy: 6 patients with recurrent tonsillitis (RT), 9 with recurrent tonsillitis with hypertrophy (RTH), and 8 with obstructive tonsillar hypertrophy (OTH). Mixed flora were present in all tonsils, yielding an average of 6.7 isolates (5.6 aerobic or facultative and 1.1 anaerobic bacteria). The highest recovery rate of organisms per tonsil was in patients with OTH (7.7 per tonsil), compared to 6.3 per tonsil in RT and 5.9 per tonsil in RTH. The predominant aerobic and facultative organisms were Haemophilus influenzae (22 isolates), Neisseria sp (16), Staphylococcus aureus (14), and Eikenella corrodens (14), and the predominant anaerobic bacteria were Fusobacterium sp (8), Bacteroides sp (7), and Prevotella melaninogenica (5). The number of bacteria per gram of tonsillar tissue varied between 10(4) and 10(8). A higher concentration of S aureus and H influenzae was found in hypertrophic tonsils (RTH and OTH) as compared to RT. These findings suggest the presence of an increased bacterial load and supports an etiologic role for H influenzae and S aureus in hypertrophic tonsils with and without inflammation (RTH and OTH). Further studies to elucidate the effect of selective antimicrobial therapy directed at these organisms may offer an alternative management of hypertrophic tonsils.