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In patients with systemic lupus erythematosus (SLE) metabolic alterations are often observed, which may be due to either the disease, the genetic background or the treatment. We studied the serum levels of the adipokines leptin, adiponectin, resistin, visfatin and ghrelin in patients with SLE and controls. Leptin levels were lower and adiponectin, ghrelin and visfatin levels were higher in the patients. No significant differences were encountered for resistin. The values of adipokines were independent of treatment, even after correction for body mass index. Inverse correlations were found among leptin and adiponectin, ghrelin and visfatin. We conclude that adipokines are involved in the metabolic imbalance of patients with SLE.

CD154, a member of the tumor necrosis factor receptor family, is involved in several biological responses. In the sera of systemic lupus erythematosus (SLE) patients, the levels of sCD154 have been shown to be increased, however, few reports have dealt with the biologically active tetramer. Here, we assessed the biological activity of the serum CD154 tetramer using bioassays for BC activation and production nitrite or peroxide. The patients showed a markedly increased total sCD154 serum concentration (12.5 +/- 8.2 vs. 3.9 +/- 1.2 ng/ml; p < 0.001). ba-sCD154 was significantly increased in non-treated patients (7.4 +/- 3.4 ng/ml, n = 22; p < 0.001) and patients with the highest SLE disease activity index (SLEDAI) scores (5.3 +/- 2.9 ng/ml, n = 8), but not in stable patients (1.3 +/- 1.2 ng/ml, n = 30) whose values were similar to normal healthy donors (NHD; 0.8 +/- 0.2 ng/ml). Patients with SLEDAI above 8 that recovered after successful treatment displayed significantly decreased levels of ba-sCD154. We conclude that the bioassay is a useful tool discriminating active and stable SLE, as well as non-treated patients.

The low-density lipoprotein receptor (LDLR) is directly involved in the metabolism of low-density lipoprotein (LDL) and its impairment causes accumulation of plasmatic LDL leading to atherosclerosis, a prevalent disease in patients with systemic lupus erythematosus (SLE). We studied LDLR transcription, expression and function in leukocytes patients with SLE and normal healthy donors (NHD). The ratio LDLR/glyceraldehyde-3-phosphate dehydrogenase (GADPH) mRNAs the expression of LDLR and the uptake of LDL-DiI were significantly lower (p < 0.001) in the patients with SLE. On the contrary, patients with SLE had significantly higher (p < 0.0001) levels of total cholesterol, LDL cholesterol and anti-oxLDL autoantibodies (AAb) as compared to NHD. No correlation between LDLR transcription, expression and function with the SLE disease activity index or with treatment was found. The decreased function of LDLR was independent of treatment. It seems dependent on the sterol regulatory binding protein and may be responsible for the increase of plasma LDL cholesterol and oxLDL AAb further increasing the risk of vascular diseases.

Leishmaniasis is a human parasitic disease caused by infection by the protozoan Leishmania spp. Chemotherapy is currently the only treatment available, but its efficacy is increasingly challenged by the rising incidence of resistance and the frequent severe side effects associated with first-line drugs. Thus the development of leads with distinct mechanisms of action is urgently needed. A strategy often used for this purpose consists of assaying for leishmanicidal activity drugs formerly developed for other applications, such as amphotericin B (antifungal) or miltefosine (antitumor), among others, to profit from previous pharmacological and toxicological studies. Kahalalide F (KF) is a tumoricidal cyclic depsipeptide currently under phase II clinical trials for several types of cancer and psoriasis. Its mechanism of action has not been fully elucidated. Here we report the leishmanicidal activity of KF and its synthetic analogues at a micromolar range of concentrations. Its lethality is strongly linked to the alteration of the plasma membrane (PM) of the parasite based on (i) a rapid depolarization of the PM and uptake of the vital dye SYTOX Green upon its addition; (ii) evidence of severe morphological damage to the membrane of the parasite, as shown by transmission electron microscopy; and (iii) a rapid drop in the intracellular ATP levels, which correlates significantly with the leishmanicidal activity for active analogues, some of them with significant improvement of their therapeutic index with respect to the parental molecule. In addition to the basic knowledge obtained, this class of lethal mechanism is considerably less prone to the induction of resistance than classical drugs. All together, these observations foster further studies for the optimization of KF and its analogues as new anti-Leishmania leads with a new mode of action.

Colistin resistance in Acinetobacter baumannii, a pathogen of clinical concern, was induced in the susceptible strain ATCC 19606 by growth under increasing pressure of the antibiotic, the only drug universally active against multi-resistant clinical strains. In 2-D difference gel electrophoresis (DIGE) experiments, 35 proteins with differences in expression between both phenotypes were identified, most of them appearing as down regulated in the colistin-resistant strain. These include outer membrane (OM) proteins, chaperones, protein biosynthesis factors, and metabolic enzymes, all suggesting substantial loss of biological fitness in the resistant phenotype, as substantiated by complementary experiments in the absence of colistin. Results shed light on the scarcity of widespread clinical outbreaks for resistant phenotypes.

Analogues of the cationic antimicrobial peptide gramicidin S (GS), cyclo(Val-Orn-Leu-D-Phe-Pro)2, with d-Phe residues replaced by different (restricted mobility, mostly) surrogates have been synthesized and used in SAR studies against several pathogenic bacteria. While all D-Phe substitutions are shown by NMR to preserve the overall beta-sheet conformation, they entail subtle structural alterations that lead to significant modifications in biological activity. In particular, the analogue incorporating D-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) shows a modest but significant increase in therapeutic index, mostly due to a sharp decrease in hemolytic effect. The fact that NMR data show a shortened distance between the D-Tic aromatic ring and the Orn delta-amino group may help explain the improved antibiotic profile of this analogue.

Since 2005, symptoms of grapevine decline have been observed on 4- to 8-month-old grapevines (cvs. Red globe and Crimson) grafted onto 1103 P rootstock in Ica and Pisco valleys in southern Peru. Affected plants exhibited weak growth, interveinal chlorosis, necrosis and wilting of leaves, and death. Dark brown-to-black streaking of the xylem was seen when transverse or longitudinal cuts were made in the trunk and shoots. Symptomatic plants were collected and sections (5 cm long) were cut from the zone between the rootstock and the scion, surface sterilized for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. The sections were split longitudinally, and small pieces of discolored tissues were placed onto potato dextrose agar (PDA) supplemented with oxytetracycline (500 mg liter -1 ). Plates were incubated at 25°C in the dark for 15 days. A Phaeoacremonium sp. was consistently isolated from necrotic tissues. Single conidial isolates were obtained and grown on PDA and malt extract agar (MEA) in the dark at 25°C for 3 weeks until colonies produced spores (3). Colonies were brown on PDA and olive brown on MEA. Conidiophores were branched, 27.5 to 67.5 (42.5) μm long, and often consisting of a single phialide. Conidia were hyaline, oblong ellipsoidal, 2.5 to 4.5 (3.6) μm long, and 1.2 to 1.9 (1.6) μm wide. On the basis of these characteristics, the isolates were identified as Phaeoacremonium parasiticum (Ajello, Georg & C.J.K Wang) W. Gams, Crous & M.J. Wingf. (teleomorph Togninia parasitica L. Mostert, W. Gams & Crous) (2,3). Identity of isolate Ppa-1 was confirmed by PCR-restriction fragment length polymorphism of the internal transcribed spacer region (Phaeoacremonium-specific primers Pm1-Pm2) with the restriction enzymes BssKI, EcoO109I, and HhaI (1). Additionally, the beta-tubulin gene fragment (primers T1 and Bt2b) of this isolate was sequenced (GenBank Accession No. FJ151015). The sequence was identical to the sequence of P. parasiticum (GenBank Accession No. AY328379). Pathogenicity tests were conducted using the isolate Ppa-1. Approximately 20 μl of a suspension containing 10 3 conidia ml -1 was injected into the pith of four nodes on each of 10 dormant, unrooted, 15 cm long cuttings of cv. Red Globe. Four nodes on each of 10 cuttings were used as controls and injected with an equal volume of sterile distilled water. Inoculation points were covered with Parafilm. The cuttings were planted in plastic pots and maintained at 24 ± 3°C in diffuse light, watering as needed. Within 2 months of inoculation, all P. parasicitum-inoculated cuttings exhibited shoots with very poor growth with small leaves and short internodes. In the xylem vessels, black streaks identical to symptoms observed in declining vines in the vineyard were observed. Control plants did not show any of these symptoms. The fungus was reisolated from internal tissues of symptomatic shoots of all inoculated cuttings but not from the control shoots. To our knowledge, this is the first report of P. parasiticum causing young grapevine decline in Peru. References: (1) A. Aroca and R. Raposo. Appl. Environ. Microbiol. 73:2911, 2007. (2) P. W. Crous et al. Mycology 88:786, 2006. (3) L. Mostert et al. Stud. Mycol. 54:1, 2006.

Surfactant protein C (SP-C) consists of a hydrophobic alpha-helix inserted in pulmonary surfactant membranes, and a more polar N-terminal palmitoylated segment exposed to the aqueous phase. Previously, we showed that SP-C inserted in lipid vesicles interacts with bacterial lipopolysaccharide (LPS) and reduces LPS-elicited responses. As the N-terminal segment of SP-C was the most likely region responsible for these effects, a set of synthetic analogs of this stretch (SPC((1-13)) ) were studied. Binding studies showed that SPC((1-13)) binds LPS to the same extent as porcine SP-C under lipid-free conditions. In the absence of serum, both, palmitoylated and non-palmitoylated analogs enhanced the binding of tritiated LPS to macrophages as well as the LPS-induced production of TNF-alpha by these cells. These effects were reversed in the presence of serum; the analogs reduced the production of TNF-alpha in LPS-stimulated macrophages, probably by interfering with the formation of LPS/CD14/LBP complexes as suggested by analysis of the fluorescence emitted by a FITC derivative of Re-LPS. Our data indicate that water-soluble analogs of the N-terminal segment of SP-C can reduce LPS effects in the presence of serum, and thus might help in the design of new derivatives to fight endotoxic shock and pro-inflammatory events.

Two general synthetic methods are described, by which the highly fluorescent and photostable BODIPY group can be inserted in and aligned with the alkyl backbone of linear lipids. These methods have been used to prepare strongly emitting analogues of the leishmanicidal drug miltefosine, in which the antiparasite activity in vitro of the original drug is preserved.

Histatin 5 (Hst5) is a human salivary antimicrobial peptide that targets fungal mitochondria. In the human parasitic protozoa Leishmania, the mitochondrial ATP production is essential, as it lacks the bioenergetic switch between glycolysis and oxidative phosphorylation described in some yeasts. On these premises, Hst5 activity was assayed on both stages of its life cycle, promastigotes and amastigotes (LC(50)=7.3 and 14.4 microM, respectively). In a further step, its lethal mechanism was studied. The main conclusions drawn were as follows: 1) Hst5 causes limited and temporary damage to the plasma membrane of the parasites, as assessed by electron microscopy, depolarization, and entrance of the vital dye SYTOX Green; 2) Hst5 translocates into the cytoplasm of Leishmania in an achiral receptor-independent manner with accumulation into the mitochondrion, as shown by confocal microscopy; and 3) Hst5 produces a bioenergetic collapse of the parasite, caused essentially by the decrease of mitochondrial ATP synthesis through inhibition of F(1)F(0)-ATPase, with subsequent fast ATP exhaustion. By using the Hst5 enantiomer, it was found that the key steps of its lethal mechanism involved no chiral recognition. Hst5 thus constitutes the first leishmanicidal peptide with a defined nonstereospecific intracellular target. The prospects of its development, by its own or as a carrier molecule for other leishmanicidal molecules, into a novel anti-Leishmania drug with a preferential subcellular accumulation are discussed.

Many strains of Shiga toxigenic Escherichia coli (STEC), particularly the serotype O157:H7, are foodborne pathogens causing disease in many countries throughout the world. E. coli O157:H7 is able to attach and survive on various surfaces such as stainless steel (SS) found within the food processing environment. We examined the outer membrane protein (OMP) profiles of four E. coli O157 (three toxigenic O157:H7 and one nontoxigenic O157:HR) and one non-STEC strain (O1:H7), previously reported to have different abilities to attach to SS following growth in planktonic (nutrient broth) and sessile (nutrient agar) culture. The OMPs of the five E. coli strains grown in planktonic and sessile culture were extracted using N-lauroyl sarcosine and the OMP profiles were separated using two-dimensional (2D) gel electrophoresis. Qualitative and quantitative variations in the total number of OMPs expressed between planktonic and sessile cultures were found for all E. coli isolates tested. A number of differentially expressed protein spots were selected from 2D gels and were identified. FlgE was found to be expressed in planktonic culture but not sessile culture. MipA and OmpX had higher expression in sessile culture than planktonic culture, while expression of OmpA did not differ between E. coli strains or between the two modes of growth. Although differential expression of OMPs was found between isolates grown in planktonic and sessile culture, further investigations are required to determine a role of some of these identified proteins during growth of E. coli in planktonic and sessile culture and their influence during the attachment process.

Autotransporter (AT) proteins have been identified in many Gram-negative pathogens and are unique in that their primary sequence is sufficient to direct their transport across the bacterial membrane system. Where characterized they are uniformly associated with virulence. Using conserved AT motifs as a search tool, four putative AT proteins were identified in the Enterohemorrhagic Escherichia coli O157:H7 EDL933 genome. The genes encoding these proteins (z0402/ehaA, z0469/ehaB, z3487/ehaC and z3948/ehaD) were PCR amplified, cloned and expressed in an E. coli K-12 MG1655flu background. Preliminary characterization revealed that ehaA, ehaB and ehaD encode proteins associated with increased biofilm formation. One of these genes (ehaA) resides on a genomic island in E. coli O157:H7 strains EDL933 and Sakai. Over-expression of EhaA in E. coli K-12 demonstrated it is located at the cell surface and resulted in the formation of large cell aggregates, promoted significant biofilm formation and mediated adhesion to primary epithelial cells of the bovine terminal rectum. The expression of ehaA was demonstrated in E. coli EDL933 by RT-PCR. An EhaA-specific antibody revealed the EhaA protein was expressed in 24/50 generic Shiga toxin-producing E. coli (STEC) strains of various serotypes including O157:H7. However, the deletion of ehaA from E. coli EDL933 and a STEC strain from serotype O111:H(-) did not affect biofilm growth. Our results suggest that EhaA may contribute to adhesion, colonization and biofilm formation by E. coli O157:H7 and possibly other STEC serotypes.

Objective: To study the effect of autologous serum eyedrop application in aniridic keratopathy., Design: Prospective, consecutive, comparative, interventional case series., Participants: Twenty-six eyes from 13 patients (7 males and 6 females) with aniridic keratopathy treated with autologous serum eyedrops., Methods: All patients underwent a complete ophthalmic examination. The ocular surface examinations included corneal impression cytologic analysis and tear film evaluation. The eyes were divided into 4 groups according to the Mackman classification. Ocular surface photography was used to evaluate the corneal surface and tear film before treatment and every 2 or 3 days until serum drops were stopped. Tear films were evaluated by tear film break-up time (BUT) (normal, 10 seconds or more), Schirmer's test with anesthesia (normal, 10 mm/5 minutes or more), tear meniscus level (normal, 0.5 mm or more), and rose bengal and fluorescein staining pattern of the cornea. Impression cytologic analysis was carried out both before starting the serum eyedrops treatment and a few days after its finalization., Main Outcomes Measures: Tear film production and stability, corneal epithelialization, and corneal epithelium squamous metaplasia., Results: There were no local side effects from autologous serum treatment. Clinical manifestations and slit-lamp findings were in relation to the severity of keratopathy. All patients showed a subjective improvement of keratopathy symptoms after the autologous serum applications. The corneal epithelialization, corneal epithelial cell squamous metaplasia, and tear stability improved significantly with the treatment, but visual acuity, regression of vascular pannus, and subepithelial scarring showed only slight improvement with treatment., Conclusions: Autologous serum eyedrops improved the aniridic keratopathy in all patients, particularly in patients with light or moderate severity. In these patients, use of autologous serum eyedrops was superior to conventional therapy with substitute tears for improving the ocular surface and subjective comfort.

The leishmanicidal mechanism of miltefosine (hexadecylphosphocholine, MT) is not clearly understood. Valuable insights into its mode of action could be obtained by fluorescence techniques, given suitably emitting analogues. In this regard, the synthesis and biological characterization of two fully competent MT fluorescent analogues is reported here: all-(E)-13-phenyltrideca-6,8,10,12-tetraenylphosphocholine (PTE-MT) and all-(E)-13-phenyltrideca-8,10,12-trien-6-ynylphosphocholine (PTRI-MT). Both compounds show large absorption coefficients and a modest, but usable, fluorescence yield. Their activities were very similar to that of MT and were recognized by the MT uptake system of Leishmania. Their localization in living L. donovani promastigotes by confocal microscopy show a homogeneous intracellular distribution of the fluorescence. The concentration of PTRI-MT within the parasites (ca. 1.7 mM) showed a 100-fold enrichment relative to its external concentration. These results are consistent with a multiple target leishmanicidal mechanism for MT and validate the application of these analogues for pharmacokinetic and diagnostic studies concerning the chemotherapy of leishmaniasis.

AM3 (Inmunoferon) is an orally effective immunomodulator that influences the regulatory and effector functions of the immune system whose molecular mechanisms of action are mostly unknown. We hypothesized that the polysaccharide moiety of AM3 (IF-S) might affect immune responses by modulating the lectin-dependent pathogen recognition abilities of human dendritic cells. IF-S inhibited binding of viral, fungal, and parasite pathogens by human monocyte-derived dendritic cells in a dose-dependent manner. IF-S specifically impaired the pathogen recognition capabilities of DC-SIGN, as it reduced the attachment of Candida, Aspergillus, and Leishmania to DC-SIGN transfectants. IF-S also inhibited the interaction of DC-SIGN with both its cellular counterreceptor (intercellular adhesion molecule 3) and the human immunodeficiency virus (HIV) type 1 gp120 protein and blocked the DC-SIGN-dependent capture of HIV virions and the HIV trans-infection capability of DC-SIGN transfectants. IF-S promoted DC-SIGN internalization in DCs without affecting mannose receptor expression, and (1)D saturation transfer difference nuclear magnetic resonance demonstrated that IF-S directly interacts with DC-SIGN on the cell surface. Therefore, the polysaccharide moiety of AM3 directly influences pathogen recognition by dendritic cells by interacting with DC-SIGN. Our results indicate that DC-SIGN is the target for an immunomodulator and imply that the adjuvant and immunomodulatory actions of AM3 are mediated, at least in part, by alteration of the DC-SIGN functional activities.

Background: The objective of this study was to compare the utility of plain radiographs to multislice computed tomography (MCT) for cervical spine (c-spine) evaluation. We hypothesized that plain radiographs add no clinically relevant diagnostic information to MCT in the screening evaluation of the c-spine of trauma patients., Methods: This was a prospective, unblinded, consecutive series of injured patients requiring c-spine evaluation that were imaged with three-view plain films and MCT (occiput to T1 with 3-dimensional reconstruction). The final discharge diagnosis based on all prospectively collected clinical data, MCT, and plain films was utilized as the gold standard for the sensitivity calculation., Results: From October 2004 to February 2005, 667 trauma patients requiring c-spine evaluation were enrolled. Average age was 35.4 years and 70% were male. The mechanism of injury was blunt in 99% and 48.7% occurred as a result of motor vehicle collision. Sixty of 667 (9%) sustained acute c-spine injuries. MCT had a sensitivity of 100% and specificity of 99.5%. Plain films had a sensitivity of 45% and specificity of 97.4%. Plain radiography missed 15 of 27 (55.5%) clinically significant c-spine injuries., Conclusion: MCT outperformed plain radiography as a screening modality for the identification of acute c-spine injury in trauma patients. All clinically significant injuries were detected by MCT. Plain films failed to identify 55.5% of clinically significant fractures identified by MCT and added no clinically relevant information.

Purpose: To study the effect of several morphometric characteristics of the deep scleral flap on intraocular pressure (IOP) control after deep sclerectomy., Methods: The authors conducted a nonrandomized prospective study of 56 eyes of 51 patients who underwent deep sclerectomy alone or combined with phacoemulsification. A reticulated hyaluronic acid implant was used in all cases. The surface, volume, and weight of the dissected deep scleral flap were measured and recorded. The correlation between these parameters and the postoperative IOP was evaluated at 6 and 12 months postoperatively., Results: Forty-nine eyes (87.50%) achieved an IOP of 21 mmHg or less without treatment, and the need for medication was decreased significantly (p<0.001) at 12 months postoperatively. The authors found a significant inverse correlation between the mean postoperative IOP at 6 months and the surface, volume, and weight of the deep scleral flap, but not at 12 months postoperatively., Conclusions: Greater weight, surface area, and volume of the deep scleral flap dissected during deep sclerectomy achieve significantly lower postoperative IOPs in the early postoperative period.

Miltefosine (hexadecylphosphocholine), the first oral drug against visceral leishmaniasis, triggered pneumococcal autolysis at concentrations higher than 2.5 microM. Bactericidal activity was also observed in cultures of other streptococci, although these failed to undergo lysis. The autolysis elicited by miltefosine can be attributed to triggering of the pneumococcal autolysin LytA.

Shiga toxigenic Escherichia coli (STEC) are important foodborne pathogens causing gastrointestinal disease worldwide. Bacterial attachment to food surfaces, such as stainless steel may lead to cross contamination of foods and subsequent foodborne disease. A variety of STEC isolates, including E. coli O157:H7/H- strains, were grown in planktonic (broth) and sessile (agar) culture, following which initial attachment to stainless steel was determined using epifluorescence microscopy. Experiments were performed to determine whether the number of bacteria attached to stainless steel differed between STEC strains and between the two modes of growth. No relationship was found between STEC strains and the number of bacteria attached to stainless steel. Five STEC strains, including one non-toxigenic O157 isolate, attached in significantly greater (p<0.05) numbers to stainless steel following growth in planktonic culture compared to sessile culture. In contrast, two clinical strains of O157:H7 attached in significantly greater (p<0.05) numbers following growth in sessile culture compared to planktonic culture. Thirteen out of twenty E. coli strains showed no significant difference (p>0.05) in attachment when grown in planktonic or sessile culture. The change of interfacial free energy between the bacterial strains and stainless steel was calculated and the influence of free energy in attachment was determined. Although a significant variation (p<0.05) in free energy values was found between STEC strains, no correlation was found between free energy values and bacterial counts on stainless steel. In addition, no correlation was also found between bacterial hydrophobicity and surface charge values or production of surface structures (type I fimbriae or flagella) (previously determined) with the number of bacteria attached to stainless steel. The results of this study suggest that different growth conditions (planktonic and sessile) can influence the attachment of STEC to stainless steel. Factors other than bacterial physicochemical properties and these surface structures may also influence STEC attachment to stainless steel.

Attachment of Shiga toxigenic Escherichia coli (STEC) to surfaces and the formation of biofilms may enhance persistence in a food processing environment and present a risk of contaminating products. Seven strains of STEC and three non-STEC strains were selected to compare two biofilm quantification methods; epifluorescence microscopy on stainless steel (SS) and a microtitre plate assay. The influence of prior growth in planktonic (nutrient broth) and sessile (nutrient agar) culture on biofilm production, as well as expression of surface structures and the possession of antigen 43 (encoded by agn43) on biofilm formation were also investigated. Biofilms were produced in diluted nutrient broth at 25 degrees C for 24 and 48 h. Curli expression was determined using congo red indicator agar, while the presence of agn43 was determined using polymerase chain reaction. No correlation was found between counts for epifluorescence microscopy on SS and the absorbance values obtained with the microtitre plate method for planktonic and sessile grown cultures. Different abilities of individual STEC strains to attach to SS and microtitre plates were found with some strains attaching better to each surface following growth in either planktonic or sessile culture. All O157 STEC strains had low biofilm counts on SS for planktonic and sessile grown cultures; however, one STEC O157:H- strain (EC516) had significantly greater (p<0.05) biofilm production on microtitre plates compared to the other O157 STEC strains. EC516 and other STEC (O174:H21 and O91:H21) strains expressing curli fimbriae were found to produce significantly greater (p<0.05) biofilms on microtitre plates compared to the non-curli expressing strains. No relationship was found between the production of type-I fimbriae, motility, agn43 and bacterial physicochemical properties (previously determined) and biofilm formation on SS or microtitre plates. Variations between the two biofilm determination methods may suggest that the biofilm production on microtitre plates may not be appropriate to represent other surfaces such as SS and that caution should be taken when selecting a method to quantify biofilm production on a surface.

Miltefosine (hexadecylphosphocholine [HePC]) is currently on trial as a first-choice, orally active drug for the treatment of visceral leishmaniasis when resistance to organic pentavalent antimonials becomes epidemic. However, data on the targets involved in its leishmanicidal mechanism have, until now, been only fragmentary. We have carried out a systematic study of the alterations induced on the bioenergetic metabolism of Leishmania donovani promastigotes by HePC. Overnight incubation with HePC caused a significant decline in the intracellular ATP levels of the parasites, together with a reduction in the oxygen consumption rate and mitochondrial depolarization, while the integrity of the plasma membrane remained undamaged. In a further step, the effects of HePC on the respiratory chain were addressed in digitonized parasites. The inhibition of the oxygen consumption rate caused by HePC was not reverted either with the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone or with tetramethyl-p-phenylenediamine plus ascorbate, which feeds the electron transport chain at the level of cytochrome c. These results suggest that cytochrome c oxidase is a likely target in the complex leishmanicidal mechanism of HePC. This was further confirmed from the finding that this enzyme was specifically inhibited in a dose-dependent manner by HePC, but not the cytochrome c reductase, ruling out an unspecific effect of HePC on the respiratory chain.

Purpose: To describe the different cellular adaptive patterns found in the conjunctival epithelium from patients with aqueous-deficient and mucous-deficient dry eyes., Methods: The authors studied different conjunctival areas, by impression cytology and by biopsy, 50 eyes with facial nerve paralysis (FNP), 50 eyes with ocular cicatricial pemphigoid (OCP), and 50 eyes from patients with primarily Sjögren syndrome (1SS)., Results: Eyes with FNP from the first clinical grade showed a progressive alteration of the nonsecretory cells, with a significant decrease in density goblet cells, generally with a PAS-positive staining. Eyes with OCP, during clinical grades 1 and 2, showed a slow deterioration of the nonsecretory cells; but from clinical grade 3, there was a significant increase of the cellular size and the thickness of the conjunctiva. Goblet cells showed a significant decrease in density from clinical grade 1, generally with a PAS-negative staining. Eyes with 1SS during clinical grades 1 and 2 showed a progressive alteration of the nonsecretory cells, with a significant decrease in density goblet cells, and a PAS-positive staining. From clinical grade 3 appeared a significant increase of nonsecretory cellular size and thickness of conjunctiva, with a significant decrease in goblet cell counts, and a PAS-negative staining., Conclusions: Patients with FNP (a primarily aqueous-deficient alteration) follow completely the squamous metaplasia process. Patients with OCP (a primarily mucous-deficient syndrome) have a hypertrophy and hyperplasia process along the ocular surface. Patients with 1SS (a primarily aqueous-deficient and mucin-deficient alteration) have a squamous metaplasia process, but from clinical grade 3 also appears a hypertrophy and hyperplasia process.

Objective: To establish a protocol for the use of autologous serum in the ophthalmic practice., Methods: Personal experience and a literature review., Results: The use of autologous serum, in eye drop form, has been reported as a new treatment for several ocular surfaces diseases. These products have biomechanical and biochemical properties similar to normal tears. They contain components such as fibronectin, vitamin A and growth factors that have an epitheliotrophic effect on the ocular surface epithelial cells., Conclusions: The clinical studies performed showed a variable efficacy, with the preparative process and use of the autologous serum eye drops varying considerably between different studies.

The alkylphosphocholine miltefosine (n-hexadecylphosphocholine, MT) has been introduced recently as a very effective drug for the oral treatment of human leishmaniasis. However, the parasiticidal mechanism of MT at a molecular level is far from being understood. Here we report the synthesis and biological characterization of 16-mercaptohexadecylphosphocholine, a thiol analog of MT which was designed to facilitate the search of MT interacting targets within the parasite by a variety of analytical methods. This analog presents the same leishmanicidal effect as the parent drug against Leishmania donovani promastigotes and Leishmania pifanoi axenic amastigotes, and has been used to develop an affinity chromatography method to attempt the isolation of putative Leishmania proteins that bind to the phosphocholine part of the molecule.

Purpose: To compare corneal surface evolution after moderate alkaline burns by impression cytology in patients treated with medical therapy or with amniotic membrane transplantation (AMT)., Methods: A prospective study of 24 eyes from 18 patients (13 men and 5 women) with moderate alkaline burns was performed. All patients were divided according to the clinical ocular severity and the therapy used. Twelve eyes were treated surgically with AMT and the other 12 eyes received only medical therapy. Corneal cytology was obtained immediately after the burns, and 1, 2, 5, and 9 months later. We differentiated between samples obtained from affected areas and areas not affected by the burns. Cellular size, nuclear size, and nuclear-cytoplasmic (N:C) ratio were examined in corneal epithelial cells, as was the presence of goblet cells in corneal epithelium., Results: Nuclear size, cellular size, and N:C ratio in non-burn-affected corneal areas had no significant alterations in comparison with normal eyes. In contrast, in burn-affected corneal areas, these parameters were significantly worse, and the presence of goblet cells in corneal epithelium was frequent 1 month after severe burns. Cellular size, nuclear size, N:C ratio, and corneal conjunctivalization improved during the study in all patients, but corneal reepithelialization occurred earlier in patients treated with AMT than in patients with only medical therapy., Conclusion: Morphologic and morphometric analysis of corneal cells by impression cytology after ocular burns permits the establishment of cellular reepithelialization patterns in relation with limbal deficiency level and with clinical ocular severity. AMT improves corneal reepithelialization earlier than medical therapy in moderate alkaline burns.

Objective: To attempt to review the aniridia keratopathy pathogenesis and establish a treatment protocol according to the severity of the symptoms., Methods: Personal experience in aniridic keratopathy management and a bibliography review., Results: The ocular manifestations of this anomaly include defects of the cornea, glaucoma, lens subluxation, cataracts, hypoplasia of the iris, fovea and optic nerve, amblyopia and nystagmus. The keratopathy occurs in a 20% of patients with aniridia. The correct PAX6 expression is necessary for normal corneal development, limbal stem cell activity and correct corneal epithelial cell migration and adhesion., Conclusions: The management of ocular surface diseases due to limbal stem cell deficiency in aniridia is complex but has changed in recent years, as an understanding of the limbal stem cells and their microenvironment has modified the therapeutic approach. The use of autologous serum eye drops, amniotic membrane transplantation, limbal transplantation or cultivated limbal cell transplantation have all been reported as a treatment for several ocular surface diseases.

Background: The optimal management strategy for patients sustaining penetrating neck injury without an urgent indication for operative exploration remains controversial. The objective of this study was to prospectively assess multislice helical computed tomography angiography (MCTA) as a stand alone screening modality for the initial evaluation of hemodynamically stable patients with penetrating neck injuries. Our hypothesis was that MCTA is a sensitive diagnostic screening test that could noninvasively evaluate the vascular and aerodigestive structures of the neck., Methods: After Institutional Review Board approval, all penetrating neck injuries assessed during a 16-month period were prospectively evaluated at a Level I trauma center. Patients without an indication for urgent neck exploration underwent MCTA screening. MCTA accuracy was tested against an aggregate gold standard of final diagnosis encompassing all imaging, surgical procedures and clinical follow-up obtained., Results: In all, 106 injuries penetrated the platysma; 15 required urgent exploration and 91 underwent MCTA (34 gunshot wounds/57 stab wounds). Nineteen external wounds were in zone 1, 39 were in zone 2, 10 in zone 3, and 23 traversed multiple zones. MCTA was nondiagnostic in 2.2% secondary to artifact from retained missile fragments. Follow-up was achieved in 84.5% of patients for a mean of 33.3 days (range: 2-150). MCTA achieved 100% sensitivity and 93.5% specificity in detecting all vascular and aerodigestive injuries sustained. MCTA correctly identified two tracheal and two carotid artery injuries requiring operative or endovascular repair in asymptomatic patients. No injuries requiring intervention were missed by MCTA., Conclusion: In the initial evaluation of stable penetrating neck injuries, MCTA appears to be a sensitive and safe screening modality. Further investigation is warranted.

Objectives: Acinetobacter baumannii has successfully developed resistance against all common antibiotics, including colistin, one of the last active drugs against this pathogen. We have tested whether the differences in lethal mechanism between polymyxin B and the cecropin A-melittin hybrid peptide CA(1-8)M(1-18), shown previously with a colistin-susceptible strain, can be exploited as a new chemotherapeutic alternative against colistin-resistant clinical isolates. Furthermore, the effect of capsule on the bactericidal activity of cecropin A-melittin analogues (CAMs) was tested., Methods: MICs and MBCs of the four CAMs were determined for 13 clinical isolates. The bactericidal activity of the antimicrobial peptides was measured using time-kill curves. The presence or absence of capsule was determined using Indian ink stain., Results: The MIC ranges of CA(1-8)M(1-18) and three of its shortened analogues, namely CA(1-7)M(2-9), its Nalpha-terminal octanoylated analogue and CA(1-7)M(5-9), for A. baumannii strains were 2-8, 2-4, 2-8 and 4-4 mg/L, respectively. MBCs differed by a factor of two at the most. All of the cecropin A-melittin peptides showed bactericidal activity in time-kill curves against four A. baumannii strains. The bactericidal activity of CAMs was not affected by the presence of capsule., Conclusions: These results indicate that this class of peptides has a fast microbicidal effect on the colistin-resistant A. baumannii isolates, regardless of considerable structural variation among the four peptides and varying colistin MIC for the strains included in the study. Overall, the cecropin A-melittin peptides appear to be a promising alternative to overcome polymyxin resistance in A. baumannii.

Background: The purpose of this study was to develop a computed tomography (CT) scan screening test to predict the need for intervention in patients with splenic injury., Methods: CT scans of 20 patients with blunt injury to the spleen were reviewed to identify findings that correlated with the need for intervention (surgery or embolization). A screening test was created and then validated in CT scans from 56 consecutive patients., Results: Three findings correlated with the need for intervention: 1) devascularization or laceration involving 50% or more of the splenic parenchyma, 2) contrast blush greater than one centimeter in diameter (from active extravasation of intravenous contrast material or pseudoaneurysm formation), and 3) a large hemoperitoneum. The sensitivity of the screening test was 100%, specificity was 88%, and overall accuracy was 93%., Conclusions: These CT scan grading criteria appears to reliably predict the need for invasive management in patients with blunt injury to the spleen.

Objectives: An altered production of cytokines underlies inflammatory bowel disease (IBD) susceptibility. Various polymorphisms at the IL-10 and TNFalpha gene promoters control cytokine production levels. The influence of these polymorphisms on susceptibility to ulcerative colitis (UC) and Crohn's disease (CD) and their association with clinical features were analyzed., Subjects and Methods: Genetic polymorphisms of TNFalpha (-308 G/A) and IL-10 (-1082 G/A, -812 C/T, and -592 C/A) were determined using the LightCycler system with hybridization probes matched with one sequence variant. The study population included 99 UC patients, 146 CD patients, and 343 matched controls., Results: We did not find association between TNFalpha or IL-10 gene polymorphisms and UC or CD susceptibility, though a slight influence of -1082*G allele in UC appearance was observed. In a stratified analysis, a highly significant association between the -1082 AA IL-10 genotype and the steroid dependency was observed in IBD (p < 0.0001), contributing both UC (p = 0.004) and CD (p = 0.003) to this association. In contrast, TNFalpha genotypes did not influence steroid dependency in IBD. Further, the contribution of cytokine genotypes and of clinical features to the appearance of steroid-dependent status (dependent variable) was studied by multivariate analysis. The steroid-dependent phenotype correlated in UC with extensive disease (p = 0.010) and with the low producer -1082 AA IL-10 genotype (p = 0.002) and in CD with penetrating disease (p = 0.010), arthritis (p = 0.011), and the -1082 AA IL-10 genotype (p = 0.006)., Conclusions: The main conclusion is that carriage of the -1082 AA IL-10 genotype (low producer) is a relevant risk factor for developing steroid-dependent IBD.

Shiga toxigenic Escherichia coli (STEC) serotypes are important foodborne pathogens that cause gastrointestinal disease worldwide. An understanding of how STEC strains attach to surfaces may provide insight into the potential persistence of and contamination with STEC in food environments. The initial attachment of a selection of STEC serotypes to beef muscle and adipose tissue was evaluated for isolates grown in planktonic and sessile culture. Initial experiments were performed to determine whether attachment differed among STEC strains and between the two modes of growth. Viable counts were obtained for loosely and strongly attached cells, and the strength of attachment (Sr) was calculated. All bacterial isolates grown in sessile culture attached in higher numbers to muscle and adipose tissue than did bacteria in planktonic cultures. For all attachment assays performed, mean concentrations for loosely attached cells were consistently higher than concentrations for strongly attached cells. The mean concentrations for strongly attached bacteria for planktonic and sessile cultures were significantly higher (P < 0.05) on adipose than on muscle tissue. However, some strains of STEC, particularly those from sessile culture, did not differ in their attachment to muscle or adipose tissue. Sr values were not significantly different (P > 0.05) among STEC isolates for all assays. No correlation was found between bacterial hydrophobicity and surface charge values (previously determined) and production of surface structures, viable counts, and Sr values. STEC grown in planktonic and sessile culture seems to behave differently with respect to attachment to muscle and adipose tissue. Cells in sessile culture may have a greater potential to strongly attach to meat surfaces.

Purpose: To determine the correlation between the squamous metaplasia grade of the corneal surface and the clinical severity of the limbal deficiency in patients with this disorder., Methods: We studied 98 eyes of patients with limbal deficiency by impression cytology. These patients were divided into four groups in relation to their clinical severity: 14 eyes had no symptoms, 34 eyes showed a mild grade of limbal deficiency, 28 eyes had a moderate grade and 22 eyes had severe limbal deficiency. Corneal cytology was performed in each patient. Cellular size, nuclear size, nuclear changes and the nuclear-cytoplasmic (N:C) ratio were defined in corneal epithelial cells, in addition to evaluation for the presence of goblet cells in the corneal epithelium., Results: In patients with limbal deficiency without symptoms, we found that the cellular size was 477 (SD140) microm2 and the N:C ratio was 1:5.25 (SD1.5). In patients with mild limbal deficiency, the cellular size was 764 (SD122.6) microm2 and the N:C ratio was 1:8.2 (SD1.4). These patients did not show corneal conjunctivalization. In patients with moderate limbal deficiency the cellular size was 1,162 (SD340.2) microm2 and the N:C ratio was 1:13.2 (SD3.6). Some patients showed corneal conjunctivalization that was defined by the presence of goblet cells in the corneal epithelium. In patients with severe limbal deficiency, the cellular size was 2,036 (SD382) microm2 and the N:C ratio was 1:23.6 (SD4.0). Corneal conjunctivalization was present in all of these patients., Conclusions: There was a correlation between limbal deficiency severity and squamous metaplasia grade defined by impression cytology. Impression cytology was very useful for the diagnosis of cases of mild or subclinical limbal deficiency.

Antimicrobial peptides (AMPs) are evolutionarily old components of innate immunity found in all living pluricellular organisms. Interestingly, some organisms express families of AMPs with only a slight variation among their members, possibly to increase their spectrum of activity. Despite the growing body of knowledge about their biological activity and mode of action on bacteria, only a few of them have been tested on Leishmania, a worldwide spread protozoan pathogen, and the parameters contributing to this activity are yet to be determined. We report on the anti-Leishmania activity and mode of action of bombinins H2 and H4 isolated from the skin secretion of the frog Bombina variegata. H4, the most active, is the first natural AMP of animal origin with a single L- to D-amino acid isomerization. Membrane depolarization and membrane permeation assays, as well as electron microscopy, suggest that the lethal mechanism involves plasma membrane permeation and/or disruption. To better understand the enhanced activity of H4, we determined the peptide's structure in membranes mimicking those of mammals, bacteria, and Leishmania by using ATR-FTIR and CD spectroscopies and assessed their membrane binding by using surface plasmon resonance. The data reveal that (i) H2 but not H4 partially aggregates in membranes mimicking those of Leishmania, (ii) H2 is slightly more helical than H4 in all membranes, and (iii) H4 binds the Leishmania model membrane approximately 5-fold better than H2. This study highlights the importance of a single alpha-amino acid epimerization as a tool used by nature to modulate the activity of AMPs. In addition, our findings suggest bombinins H as potential templates for the development of new drugs with a new mode of action against Leishmania.

Acinetobacter baumannii has successfully developed resistance against all common antibiotics, including colistin (polymyxin E), the last universally active drug against this pathogen. The possible widespread distribution of colistin-resistant A. baumannii strains may create an alarming clinical situation. In a previous work, we reported differences in lethal mechanisms between polymyxin B (PXB) and the cecropin A-melittin (CA-M) hybrid peptide CA(1-8)M(1-18) (KWKLFKKIGIGAVLKVLTTGLPALIS-NH2) on colistin-susceptible strains (J. M. Saugar, T. Alarcón, S. López-Hernández, M. López-Brea, D. Andreu, and L. Rivas, Antimicrob. Agents Chemother. 46:875-878, 2002). We now demonstrate that CA(1-8)M(1-18) and three short analogues, namely CA(1-7)M(2-9) (KWKLFKKIGAVLKVL-NH2), its Nalpha-octanoyl derivative (Oct-KWKLFKKIGAVLKVL-NH2), and CA(1-7)M(5-9) (KWKLLKKIGAVLKVL-NH2) are active against two colistin-resistant clinical strains. In vitro, resistance to colistin sulfate was targeted to the outer membrane, as spheroplasts were equally lysed by a given peptide, regardless of their respective level of colistin resistance. The CA-M hybrids were more efficient than colistin in displacing lipopolysaccharide-bound dansyl-polymyxin B from colistin-resistant but not from colistin-susceptible strains. Similar improved performance of the CA-M hybrids in permeation of the inner membrane was observed, regardless of the resistance pattern of the strain. These results argue in favor of a possible use of CA-M peptides, and by extension other antimicrobial peptides with similar features, as alternative chemotherapy in colistin-resistant Acinetobacter infections.

Background: Accurate screening of the thoracolumbar spine (TLS) remains problematic in the care of trauma patients. The current standard of care for TLS screening is not clearly defined. In trauma patients undergoing computed tomography (CT) of the chest and or abdomen, compelling supportive evidence for reformatting and reusing this CT data to clear the thoracolumbar spine has accumulated over the last 3 years. The objective of this review was to identify and review all published studies comparing reformatted CT to traditional plain radiography for TLS clearance., Methods: A Medline search for all English language articles published on this subject since 1980 identified seven studies. Each was classified according to the levels of evidence classification of the Agency for Health Care Policy and Research. The methodology of each study was reviewed for the CT protocol, utilization of radiologist blinding and whether historical dictated reports or de novo image readings were used for comparison. The sensitivity and specificity of each study and the gold standard utilized in its calculation was noted. Where available, detailed information regarding the missed injuries and their clinical relevance was abstracted for each study., Results: All evaluated studies demonstrated superior sensitivity and interobserver variability for reformatted CT compared with plain radiographic screening. CT was also more accurate in localizing, classifying, and delineating the age, bony intrusion, and soft-tissue damage associated with the fracture. For studies with time-motion components, a protocol utilizing CT clearance was not only more accurate but faster and more economical. Screening with reformatted visceral CT data required no additional scan time or radiation exposure., Conclusion: The evidence to date demonstrates the superior sensitivity of reformatted visceral CT for detecting thoracolumbar spine injury. With no further patient movement, radiation exposure, cost, or time, trauma patients undergoing visceral CT can have their thoracolumbar spine promptly evaluated. Further prospective evaluation of the CT protocols to optimize visualization of both the viscera and the bone is warranted.

Background: With the evolution of multi-slice helical computed tomographic angiography (MCTA), the optimal imaging modality for arterial injury in the traumatized lower extremity has been challenged. The objective of this study was to evaluate the ability of MCTA to detect arterial injury in the traumatized lower extremity. Our hypothesis was that MCTA is a sensitive and specific test for the non-invasive evaluation of lower extremity arterial injury., Methods: After Institutional Review Board approval, we reviewed all patients at our Level I trauma center who sustained lower extremity trauma and underwent initial evaluation by MCTA over a 3-year period ending in February 2005. MCTA accuracy was tested against a gold standard of operative intervention, duplex ultrasonography, catheter-based angiography, or clinical follow-up., Results: Sixty-three MCTAs were performed in 59 patients. MCTA was diagnostic in 62 of the 63 scans (98.4%). The mechanism was penetrating in 45.8%. Lower extremity fractures were present in 38.7% of patients studied. There were 22 positive studies. Out of this group, 19 were confirmed at operation and 3 were managed non-operatively. In the 19 injuries confirmed in the operating theater, there were 5 superficial femoral, 2 profunda, 10 popliteal, 1 posterior tibial, and 1 injury to all 3 mid-calf arteries. There were two injuries below the trifurcation managed non-operatively and there was one popliteal occlusion with distal reconstitution that was confirmed by duplex and managed non-operatively because of patient refusal of surgery. Forty studies were negative for arterial injury, with clinical follow up available in 89.5%, for a mean of 48.2 days (range, 5-287 days). No missed injuries were identified during the follow-up period. MCTA was non-diagnostic in 1 patient (1.6%), secondary to artifact from retained missile fragments. MCTA achieved 100% sensitivity and 100% specificity in detecting clinically significant arterial injury., Conclusion: MCTA is a sensitive and specific non-invasive imaging modality for arterial evaluation in the injured lower extremity that may replace catheter-based angiography in most patients.

Aim: Eradication therapy with proton pump inhibitor, clarithromycin and amoxicillin fails in a considerable number of cases. A rescue therapy still fails in more than 20% of the cases. Our aim was to evaluate the efficacy and tolerability of a third-line levofloxacin-based regimen in patients with two consecutive Helicobacter pylori eradication failures., Design: Prospective multicenter study., Patients: In whom a first treatment with omeprazole-clarithromycin-amoxicillin and a second with omeprazole-bismuth-tetracycline-metronidazole (or ranitidine bismuth citrate with these antibiotics) had failed., Intervention: A third eradication regimen with levofloxacin (500 mg b.i.d.), amoxicillin (1 g b.i.d.), and omeprazole (20 mg b.i.d.) was prescribed for 10 days., Outcome: Eradication was confirmed with 13C-urea breath test 4-8 wk after therapy., Results: One-hundred patients were initially included, and nine were lost for follow-up. All patients but five took all the medications correctly. Per-protocol and intention-to-treat eradication rates were 66% (95% CI = 56-75%) and 60% (50-70%). Adverse effects were reported in 25% of the patients, mainly including metallic taste (8%), nausea (8%), myalgia/arthralgia (5%), and diarrhea (4%); none of them were severe., Conclusion: Levofloxacin-based rescue therapy constitutes an encouraging empirical third-line strategy after multiple previous H. pylori eradication failures with key antibiotics such as amoxicillin, clarithromycin, metronidazole, and tetracycline.

Objective: The objective of this study was to review the outcome of nonoperative treatment for penetrating internal jugular vein (IJ) injuries in a continuous series of prospectively identified, hemodynamically stable patients., Methods: All penetrating neck injuries assessed from February 1, 2004, to August 31, 2004, were prospectively identified. Patients without an indication for urgent neck exploration underwent diagnostic assessment with multislice helical computed tomographic angiography with or without vascular ultrasonography. All IJ injuries with no other indication for surgical exploration were treated nonoperatively. All patients were discharged home and followed up for a minimum of 1 week to document outcomes., Results: From 51 neck injuries penetrating the platysma, 7 required urgent neck exploration, during which 2 IJ injuries were ligated. Forty-four patients underwent multislice helical computed tomographic angiography. Eight IJ injuries (two gunshot wounds and six stab wounds) with no other indication for neck exploration were identified and managed nonoperatively. One external wound was in zone 1, five were in zone 2, one was in zone 3, and one traversed all three zones. The average length of stay was 4.5 days. At follow-up, ranging from 1 week to 5 months, all patients were asymptomatic, and no patient required delayed operation for IJ injury., Conclusions: In hemodynamically stable patients with no other indication for exploration, the nonoperative management of penetrating jugular vein injuries should be considered as a safe alternative.

Hepcidin is a liver-expressed antimicrobial and iron regulatory peptide. A number of studies have indicated that hepcidin is important for the correct regulation of body iron homeostasis. The aims of this study were to analyse the expression, trafficking and regulation of human hepcidin in an in vitro cell culture system. Human hepcidin was transfected into human embryonic kidney cells. Immunofluorescence and confocal microscopy analysis revealed that recombinant hepcidin localised to the Golgi complex. Recombinant hepcidin is secreted from the cell within 1 h of its synthesis. Recombinant hepcidin was purified from the cell culture medium using ion-exchange and metal-affinity chromatography and was active in antimicrobial assays. Amino-terminal sequence analysis of the secreted peptide revealed that it was the mature 25 amino acid form of hepcidin. Our results show that recombinant myc-His tagged human hepcidin was expressed, processed and secreted correctly and biologically active in antimicrobial assays.

There is an increasing awareness of immune cell modulation by antimicrobial peptides. While this process often requires specific receptors for the peptides involved, several reports point out to a receptor-independent process. The cecropin A-melittin hybrid peptide CA(1-8)M(1-18) (KWKLFKKIGIGAVLKVLTTGLPALIS-amide) modifies gene expression in the macrophage line RAW 264.7 in the absence of any previous macrophage priming, suggesting a membrane permeation process. To further analyze the initial steps of this mechanism, we have studied the interaction of the peptide with these cells. Below 2 microM, CA(1-8)M(1-18) causes a concentration-dependent membrane depolarization partially reversible with time. At 2 microM, the accumulation of the SYTOX green vital dye is one half of that achieved with 0.05% Triton X-100. The binding level, as assessed by fluorescein-labeled CA(1-8)M(1-18), varies from 7.7+/-1.2 to 37.4+/-3.9 x 10(6) molecules/cell over a 0.5-4.0 microM concentration range. Electrophysiological experiments with 0.5 microM CA(1-8)M(1-18), a concentration that triggers maximal NOS2 expression and minimal toxicity, show a reversible current induction in the RAW 264.7 plasma membrane that is maintained as far as peptide is present. This activation of the macrophage involves the production of nitric oxide, a metabolite lethal for many pathogens that results from unspecific membrane permeation by antimicrobial peptides, and represents a new mode of action that may open new therapeutic possibilities for these compounds against intracellular pathogens.

Purpose: To study the ocular surface evolution by impression cytology and limbal biopsy in patients with congenital aniridia treated with amniotic membrane transplantation (AMT)., Methods: We studied 14 eyes of patients with congenital aniridia and moderate limbal deficiency that were treated with AMT. The morphology of the limbocorneal epithelium and stroma was examined by impression cytology and limbal biopsy. Impression cytology was performed prior to surgery and 3, 6, 9, 12 and 24 months after treatment. Limbal biopsy was performed before surgery and 9 months after AMT., Results: Visual acuity showed a mean improvement of 0.3 at 24 months of follow-up. Corneal reepithelialization was completed by 2 months in all patients, however, after 9 months some patients showed persistent epithelial defects and chronic ulceration. Corneal clarity and peripheral neovascularization was markedly improved after 2 months. Impression cytology showed an improvement in squamous metaplasia at 3 and 6 months, however after nine months, until as late as 24 months, a progressive worsening was seen in epithelial cell metaplasia then being similar in extent to that before surgery. Limbal biopsy nine months after surgery showed a nearly normal epithelium and limbocorneal stroma., Conclusions: AMT is a very effective procedure, although its effect is transitory, for restoring the ocular surface integrity in patients with congenital aniridia and moderate limbal deficiency.

Purpose: To study the ocular surface evolution in patients with severe limbal deficiency treated with limbal allograft transplantation (LT) combined with amniotic membrane transplantation (AMT)., Methods: We studied 14 eyes with severe limbal deficiency, treated with LT combined with AMT (LT + AMT). We studied the morphology of the limbocorneal epithelium and stroma by impression cytology and limbal biopsy., Results: Visual acuity showed a mean improvement of 0.4 at 24 months of follow-up. Corneal clarity and loss of neovascularization were markedly improved after 3 and 6 months, respectively. We did not find corneal conjuntivalization on cytology performed 6 months after surgery, except in a patient with chemical burn. Cytology and limbal biopsy performed nine months after surgery showed an epithelium and limbocorneal stroma near to normality. Squamous metaplasia was worse in patients with Stevens-Johnson syndrome and ocular cicatricial pemphigoid after 12 months, but was similar in patients with chemical burns and improved in patients with aniridia., Conclusions: LT + AMT is a very effective procedure for restoring the ocular surface integrity in patients with severe limbal deficiency. This combination improves the outcomes obtained with LT alone.

The tetraheme protein cytochrome c(3) (Cyt-c(3)) from Desulfovibrio gigas, immobilized on a self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid, is studied by theoretical and spectroscopic methods. Molecular dynamics simulations indicate that the protein docks to the negatively charged SAM via its lysine-rich domain around the exposed heme IV. Complex formation is associated with only little protein structural perturbations. This finding is in line with the resonance Raman and surface-enhanced resonance Raman (SERR) spectroscopic results that indicate essentially the same heme pocket structures for the protein in solution and adsorbed on SAM-coated Ag electrodes. Electron- and proton-binding equilibrium calculations reveal substantial negative shifts of the redox potentials compared to the protein in solution. The magnitude of these shifts decreases in the order heme IV (-161 mV) > heme III (-73 mV) > heme II (-57 mV) > heme I (-26 mV), resulting in a change of the order of reduction. These shifts originate from the distance-dependent electrostatic interactions between the SAM headgroups and the individual hemes, leading to a stabilization of the oxidized forms. The results of the potential-dependent SERR spectroscopic analyses are consistent with the theoretical predictions and afford redox potential shifts of -160 mV (heme IV), -90 mV (heme III), -70 mV (heme II), and +20 mV (heme I) relative to the experimental redox potentials for Cyt-c(3) in solution. SERR spectroscopic experiments reveal electric-field-induced changes of the redox potentials also for the structurally very similar Cyt-c(3) from Desulfovibrio vulgaris, although the shifts are somewhat smaller compared to Cyt-c(3) from D. gigas. This study suggests that electric-field-induced redox potential shifts may also occur upon binding to biomembranes or partner proteins and thus may affect biological electron transfer processes.

Leishmaniasis encompasses a wide range of infections caused by the human parasitic protozoan species belonging to the Leishmania genus. It appears frequently as an opportunistic disease, especially in virus-infected immunodepressed people. Similarly to other pathogens, parasites became resistant to most of the first-line drugs. Therefore, there is an urgent need to develop antiparasitic agents with new modes of action. Gene-encoded antimicrobial peptides are promising candidates, but so far only a few of them have shown anti-protozoa activities. Here we found that temporins A and B, 13-amino acid antimicrobial peptides secreted from the skin of the European red frog Rana temporaria, display anti-Leishmania activity at micromolar concentrations, with no cytolytic activity against human erythrocytes. To the best of our knowledge, temporins represent the shortest natural peptides having the highest leishmanicidal activity and the lowest number of positively charged amino acids (a single lysine/arginine) and maintain biological function in serum. Their lethal mechanism involves plasma membrane permeation based on the following data. (i) They induce a rapid collapse of the plasma membrane potential. (ii) They induce the influx of the vital dye SYTOX Green. (iii) They reduce intracellular ATP levels. (iv) They severely damage the membrane of the parasite, as shown by transmission electron microscopy. Besides giving us basic important information, the unique properties of temporins, as well as their membranolytic effect, which should make it difficult for the pathogen to develop resistance, suggest them as potential candidates for the future design of antiparasitic drugs with a new mode of action.