A universal UHPLC-CAD platform for the quantification of polysaccharide antigens.

Several glycoconjugate-based vaccines against bacterial infections have been developed and licensed for human use. Polysaccharide (PS) analysis and characterization is therefore critical to profile the composition of polysaccharide-based vaccines. For PS content quantification, the majority of Ultra High Performance Liquid Chromatography (UHPLC) methods rely on the detection of selective monosaccharides constituting the PS repeating unit, therefore requiring chemical cleavage and tailored development: only a few methods directly quantify the intact PS. The introduction of charged aerosol detector (CAD) technology has improved the response of polysaccharide analytes, offering greater sensitivity than other detector sources (e.g., ELSD). Herein, we report the development of a universal UHPLC-CAD method (UniQS) for the quantification and quality evaluation of polysaccharide antigens (e.g., Streptococcus Pneumoniae, Neisseria meningitidis and Staphylococcus aureus). This work laid the foundation for a universal UHPLC-CAD format that could play an important role in future vaccine research and development helping to reduce time, efforts, and costs.

The Use of Prophylactic Somatostatin Therapy Following Pancreaticoduodenectomy: A Meta-analysis of Randomised Controlled Trials.

BACKGROUND: Prophylactic administration of somatostatin analogues (SA) to reduce the incidence of post-operative pancreatic fistula (POPF) remains contentious. This meta-analysis evaluated its impact on outcomes following pancreaticoduodenectomy (PD). METHODS: The EMBASE, MEDLINE and Cochrane databases were searched for randomised controlled trials (RCTs) investigating prophylactic SA following PD. Comparative effects were summarised as odds ratio and weighted mean difference based on an intention to treat. Quantitative pooling of the effect sizes was derived using the random-effects model. MAIN RESULTS: Twelve RCTs were included involving 1615 patients [SA-treated group (n = 820) and control group (n = 795)]. The SA used included somatostatin-14, pasireotide, vapreotide and octreotide. Pooling of the data showed no significant benefit of its use for the primary outcome measure of all grades of POPF, odds ratio (OR) 0.73 [95% confidence interval (CI), 0.51-1.05, p = 0.09] and clinically relevant POPF, OR 0.48 [95% CI, 0.22-1.06, p = 0.07]. There were no benefits in the secondary outcome measures of delayed gastric emptying, OR 0.98 [95% CI, 0.57-1.69, p = 0.94]; infected abdominal collections, OR 0.80 [95% CI, 0.44-1.43, p = 0.80]; reoperation rates, OR 1.24 [95% CI, 0.73-2.13, p = 0.42]; duration of hospital stay, - 0.23 [95% CI - .59 to 1.13, p = 0.74]; and mortality, 1.78 [95% CI, 0.94-3.39, p = 0.08]. CONCLUSION: SA did not improve the post-operative outcomes following PD, including reducing the incidence of POPF. The routine administration of SA cannot be recommended following PD.

Synthesis and biological evaluation in vitro and in mammalian cells of new heteroaryl carboxyamides as HIV-protease inhibitors.

New heteroaryl HIV-protease inhibitors bearing a carboxyamide spacer were synthesized in few steps and high yield, from commercially available homochiral epoxides. Different substitution patterns were introduced onto a given isopropanoyl-sulfonamide core modifying the type of heteroarene and the central core, with the presence of either H or benzyl group. Their in vitro inhibition activity against recombinant protease showed a general beneficial effect of carboxyamide moiety, the IC50 values ranging between 1 and 15nM. In particular benzofuryl derivatives showed IC50 values among the best for such structurally simple inhibitors. Docking analysis allowed to identify the favorable situation of such benzofuryl derivatives in terms of number of interactions in the active site, supporting the experimental results on activity. The inhibition activity of such molecules has been also evaluated in HEK293 cells expressing the protease fused to green fluorescent protein, by western blotting analysis, fluorescence microscopy and cytofluorimetry.

Spontaneous symmetry breaking on ordered, racemic monolayers of achiral theophylline: formation of unichiral stripes on Au(111).

We report the observation of spontaneous chiral symmetry breaking within ordered, racemic monolayers of theophylline, manifesting itself as extended, nanoscale unichiral stripes at the interface between molecular domains. Theophylline is a xanthine derivative playing an important role in several biochemical processes. Molecular chirality is induced by adsorption on the Au(111) surface, resulting in extended domains with two different racemic, ordered structures, coexisting with a disordered phase. By combining low-temperature scanning tunneling microscopy (LT-STM) and ab initio density functional theory calculations, we first provide a detailed picture of the interactions within the ordered assemblies, and we uncover the origin of the distinct contrast features in STM images. Secondly, experiments reveal the existence of nanoscale stripes of unichiral molecules separating racemic domains of one of the two ordered phases, giving rise to a local enantiomeric imbalance. Systematic theoretical investigation of their structure and chiral composition confirm their unichirality, with the specific handedness related to the registry between the two ordered domains facing the stripes. These findings can open the way to new insights into the elusive mechanisms leading to local chiral imbalances in racemic systems, possibly at the origin of biomolecular homochirality, as well as suggest novel approaches for stereoselective heterogeneous catalysis.

Preclinical studies on new proteins as carrier for glycoconjugate vaccines.

Glycoconjugate vaccines are made of carbohydrate antigens covalently bound to a carrier protein to enhance their immunogenicity. Among the different carrier proteins tested in preclinical and clinical studies, five have been used so far for licensed vaccines: Diphtheria and Tetanus toxoids, the non-toxic mutant of diphtheria toxin CRM197, the outer membrane protein complex of Neisseria meningitidis serogroup B and the Protein D derived from non-typeable Haemophilus influenzae. Availability of novel carriers might help to overcome immune interference in multi-valent vaccines containing several polysaccharide-conjugate antigens, and also to develop vaccines which target both protein as well saccharide epitopes of the same pathogen. Accordingly we have conducted a study to identify new potential carrier proteins. Twenty-eight proteins, derived from different bacteria, were conjugated to the model polysaccharide Laminarin and tested in mice for their ability in inducing antibodies against the carbohydrate antigen and eight of them were subsequently tested as carrier for serogroup meningococcal C oligosaccharides. Four out of these eight were able to elicit in mice satisfactory anti meningococcal serogroup C titers. Based on immunological evaluation, the Streptococcus pneumoniae protein spr96/2021 was successfully evaluated as carrier for serogroups A, C, W, Y and X meningococcal capsular saccharides.

Carrier priming effect of CRM197 is related to an enhanced B and T cell activation in meningococcal serogroup A conjugate vaccination. Immunological comparison between CRM197 and diphtheria toxoid.

Glycoconjugate vaccines are composed of capsular polysaccharides (CPSs) of a pathogenic bacteria covalently linked to carrier proteins. Pre-exposure to the carrier is known to influence the efficacy of the glycoconjugate, by inducing enhanced or suppressed anti-CPS response. Following our previous work on the immunogenicity of diphtheria toxin mutant CRM197 and formaldehyde-treated diphtheria toxoid (DT) as carriers for meningococcal A (MenA) conjugates in mouse model, we further investigated the role of the carrier on the immunological response to glycoconjugate vaccines. We previously showed that high dosage DT priming could result in carrier-induced epitopic suppression (CIES), an event that did not occur for CRM197 priming, and we observed that anti-DT IgGs could cross-react with DT based conjugates in vitro. Here, we confirmed the cross-reactivity of anti-carrier IgGs with DT conjugates in vivo. Furthermore, we analyzed the splenocytes of animals primed with the carrier and subsequently immunized with the MenA conjugate. Pre-exposure to the carrier protein, both CRM197 and DT, resulted in increased carrier-specific plasma and memory B cell response. However, only for CRM197 priming an enhanced carbohydrate-specific plasma cell response was observed. Analysis of circulating IgGs confirmed these observations. Memory to the CPS resulted to be non-influenced by carrier priming. Analysis of T helper response showed an enhancement effect for CRM197 priming, while DT priming resulted in constrained T cell activation. Stimulation with CRM197, which does not require formaldehyde detoxification, of splenocytes from animal immunized with DT suggested that the formaldehyde treatment used to produce DT might be the cause of limited presentation of the antigen to the T cells. We concluded that the dominant carrier-specific B cell response in case of limited T cell recruitment might explain the previously observed CIES phenomenon in case of DT priming.

Evaluation of the non-toxic mutant of the diphtheria toxin K51E/E148K as carrier protein for meningococcal vaccines.

Diphtheria toxin mutant CRM197 is a common carrier protein for glycoconjugate vaccines, which has been proven an effective protein vector for, among others, meningococcal carbohydrates. The wide-range use of this protein in massive vaccine production requires constant increase of production yields and adaptability to an ever-growing market. Here we compare CRM197 with the alternative diphtheria non-toxic variant DT-K51E/E148K, an inactive mutant that can be produced in the periplasm of Escherichia coli. Biophysical characterization of DT-K51E/E148K suggested high similarity with CRM197, with main differences in their alpha-helical content, and a suitable purity for conjugation and vaccine preparation. Meningococcal serogroup A (MenA) glycoconjugates were synthesized using CRM197 and DT-K51E/E148K as carrier proteins, obtaining the same conjugation yields and comparable biophysical profiles. Mice were then immunized with these CRM197 and DT-K51E/E148K conjugates, and essentially identical immunogenic and protective effects were observed. Overall, our data indicate that DT-K51E/E148K is a readily produced protein that now allows the added flexibility of E. coli production in vaccine development and that can be effectively used as protein carrier for a meningococcal conjugate vaccine.

Carrier priming with CRM 197 or diphtheria toxoid has a different impact on the immunogenicity of the respective glycoconjugates: biophysical and immunochemical interpretation.

Glycoconjugate vaccines play an enormous role in preventing infectious diseases. The main carrier proteins used in commercial conjugate vaccines are the non-toxic mutant of diphtheria toxin (CRM197), diphtheria toxoid (DT) and tetanus toxoid (TT). Modern childhood routine vaccination schedules include the administration of several vaccines simultaneously or in close sequence, increasing the concern that the repeated exposure to conjugates based on these carrier proteins might interfere with the anti-polysaccharide response. Extending previous observations we show here that priming mice with CRM197 or DT does not suppress the response to the carbohydrate moiety of CRM197 meningococcal serogroup A (MenA) conjugates, while priming with DT can suppress the response to DT-MenA conjugates. To explain these findings we made use of biophysical and immunochemical techniques applied mainly to MenA conjugates. Differential scanning calorimetry and circular dichroism data revealed that the CRM197 structure was altered by the chemical conjugation, while DT and the formaldehyde-treated form of CRM197 were less impacted, depending on the degree of glycosylation. Investigating the binding and avidity properties of IgGs induced in mice by non-conjugated carriers, we found that CRM197 induced low levels of anti-carrier antibodies, with decreased avidity for its MenA conjugates and poor binding to DT and respective MenA conjugates. In contrast, DT induced high antibody titers able to bind with comparable avidity both the protein and its conjugates but showing very low avidity for CRM197 and related conjugates. The low intrinsic immunogenicity of CRM197 as compared to DT, the structural modifications induced by glycoconjugation and detoxification processes, resulting in conformational changes in CRM197 and DT epitopes with consequent alteration of the antibody recognition and avidity, might explain the different behavior of CRM197 and DT in a carrier priming context.

Synthesis and biological evaluation of new simple indolic non peptidic HIV Protease inhibitors: the effect of different substitution patterns.

New structurally simple indolic non peptidic HIV Protease inhibitors were synthesized from (S)-glycidol by regioselective methods. Following the concept of targeting the protein backbone, different substitution patterns were introduced onto the common stereodefined isopropanolamine core modifying the type of functional group on the indole, the position of the functional group on the indole and the type of the nitrogen containing group (sulfonamides or perhydroisoquinoline), alternatively. The systematic study on in vitro inhibition activity of such compounds confirmed the general beneficial effect of the 5-indolyl substituents in presence of arylsulfonamide moieties, which furnished activities in the micromolar range. Preliminary docking analysis allowed to identify several key features of the binding mode of such compounds to the protease.

Comparison of CRM197, diphtheria toxoid and tetanus toxoid as protein carriers for meningococcal glycoconjugate vaccines.

Glycoconjugate vaccines are among the most effective and safest vaccines ever developed. Diphtheria toxoid (DT), tetanus toxoid (TT) and CRM197 have been mostly used as protein carriers in licensed vaccines. We evaluated the immunogenicity of serogroup A, C, W-135 and Y meningococcal oligosaccharides conjugated to CRM197, DT and TT in naïve mice. The three carriers were equally efficient in inducing an immune response against the carbohydrate moiety in immunologically naïve mice. The effect of previous exposure to different dosages of the carrier protein on the anti-carbohydrate response was studied using serogroup A meningococcal (MenA) saccharide conjugates as a model. CRM197 showed a strong propensity to positively prime the anti-carbohydrate response elicited by its conjugates or those with the antigenically related carrier DT. Conversely in any of the tested conditions TT priming did not result in enhancement of the anti-carbohydrate response elicited by the corresponding conjugates. Repeated exposure of mice to TT or to CRM197 before immunization with the respective MenA conjugates resulted in a drastic suppression of the anti-carbohydrate response in the case of TT conjugate and only in a slight reduction in the case of CRM197. The effect of carrier priming on the anti-MenA response of DT-based conjugates varied depending on their carbohydrate to protein ratio. These data may have implications for human vaccination since conjugate vaccines are widely used in individuals previously immunized with DT and TT carrier proteins.

Meningococcal X polysaccharide quantification by high-performance anion-exchange chromatography using synthetic N-acetylglucosamine-4-phosphate as standard.

A method for meningococcal X (MenX) polysaccharide quantification by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) is described. The polysaccharide is hydrolyzed by strong acidic treatment, and the peak of glucosamine-4-phosphate (4P-GlcN) is detected and measured after chromatography. In the selected conditions of hydrolysis, 4P-GlcN is the prevalent species formed, with GlcN detected for less than 5% in moles. As standard for the analysis, the monomeric unit of MenX polysaccharide, N-acetylglucosamine-4-phosphate (4P-GlcNAc), was used. This method for MenX quantification is highly selective and sensitive, and it constitutes an important analytical tool for the development of a conjugate vaccine against MenX.

Sandwich ELISA assay for the quantitation of palytoxin and its analogs in natural samples.

Palytoxins are potent marine biotoxins that have recently become endemic to the Mediterranean Sea, and are becoming more frequently associated with seafood. Due to their high toxicity, suitable methods to quantify palytoxins are needed. Thus, we developed an indirect sandwich ELISA for palytoxin and 42-hydroxy-palytoxin. An intralaboratory study demonstrated sensitivity (limit of detection, LOD = 1.1 ng/mL; limit of quantitation, LOQ = 2.2 ng/mL), accuracy (bias of 2.1%), repeatability (RSDr = 6% and 9% for intra- and interassay variability, respectively) and specificity: other common marine toxins (okadaic acid, domoic acid, saxitoxin, brevetoxin-3, and yessotoxin) do not cross-react in this assay. It performed well in three different matrices: observed LOQs were 11.0, 9.6, and 2.4 ng/mL for mussel extracts, algal net samples and seawater, respectively, with good accuracy and precision. The LOQ in seafood is 11 µg palytoxin/kg mussel meat, lower than that of the most common detection technique, LC-MS/MS.

A scalable method for O-antigen purification applied to various Salmonella serovars.

The surface lipopolysaccharide of gram-negative bacteria is both a virulence factor and a B cell antigen. Antibodies against O-antigen of lipopolysaccharide may confer protection against infection, and O-antigen conjugates have been designed against multiple pathogens. Here, we describe a simplified methodology for extraction and purification of the O-antigen core portion of Salmonella lipopolysaccharide, suitable for large-scale production. Lipopolysaccharide extraction and delipidation are performed by acetic acid hydrolysis of whole bacterial culture and can take place directly in a bioreactor, without previous isolation and inactivation of bacteria. Further O-antigen core purification consists of rapid filtration and precipitation steps, without using enzymes or hazardous chemicals. The process was successfully applied to various Salmonella enterica serovars (Paratyphi A, Typhimurium, and Enteritidis), obtaining good yields of high-quality material, suitable for conjugate vaccine preparations.

Relative stability of meningococcal serogroup A and X polysaccharides.

Prior to the introduction of the MenAfriVac™ serogroup A glycoconjugate vaccine in September 2010, serogroup A was the major epidemic disease-causing meningococcal serogroup in the African meningitis belt. However, recently serogroup X meningococcal (MenX) disease has received increased attention because of outbreaks recorded in this region, with increased endemic levels of MenX disease over the past 2 years. Whereas polysaccharide-protein conjugate vaccines against meningococcal serogroups A, C, W and Y (MenA, MenC, MenW, MenY) are on the market, a vaccine able to protect against MenX has never been achieved. The structure of serogroup A, C, W and Y meningococcal polysaccharides has been already fully elucidated by NMR. MenX capsular polysaccharide (MenX CPS) structure is also documented but fewer characterization data have been published. We have applied here (1)H NMR, (31)P NMR and HPLC to evaluate the stability of MenX CPS in aqueous solution as compared to MenA capsular polysaccharide (MenA CPS). The stability study demonstrated that MenA CPS is more susceptible to hydrolytic degradation than MenX CPS. The different stereochemistry of the N-acetyl group at position C(2) of mannosamine (MenA CPS) and glucosamine (MenX CPS) respectively might play a fundamental role in this susceptibility to polysaccharide chain degradation. The satisfactory stability of MenX CPS predicts the possibility that a stable fully-liquid MenX polysaccharide or glycoconjugate vaccine could be developed.

Vi-CRM 197 as a new conjugate vaccine against Salmonella Typhi.

An efficacious, low cost vaccine against typhoid fever, especially for young children, would make a major impact on disease burden in developing countries. The virulence capsular polysaccharide of Salmonella Typhi (Vi) coupled to recombinant mutant Pseudomonas aeruginosa exoprotein A (Vi-rEPA) has been shown to be highly efficacious. We investigated the use of carrier proteins included in infant vaccines, standardized the conjugation process and developed key assays required for routine lot release at production scale. Vi from a BSL1 organism, Citrobacter freundii, strain WR7011, was used as an alternative to Vi from S. Typhi. We showed that Vi conjugated to CRM(197), a non-toxic mutant of diphtheria toxin, widely used in commercial vaccines, was produced at high yield. Vi-CRM(197) proved immunogenic in animal studies, even without adjuvant. Thus, Vi-CRM(197) appears to be a suitable candidate for the development of a commercially viable, effective typhoid vaccine for developing countries.

Bronchodilator activity of (3R)-3-[[[(3-fluorophenyl)[(3,4,5-trifluorophenyl)methyl]amino] carbonyl]oxy]-1-[2-oxo-2-(2-thienyl)ethyl]-1-azoniabicyclo[2.2.2]octane bromide (CHF5407), a potent, long-acting, and selective muscarinic M3 receptor antagonist.

The novel quaternary ammonium salt (3R)-3-[[[(3-fluorophenyl)[(3,4,5-trifluorophenyl)methyl]amino]carbonyl]oxy]-1-[2-oxo-2-(2-thienyl)ethyl]-1-azoniabicyclo[2.2.2]octane bromide (CHF5407) showed subnanomolar affinities for human muscarinic M1 (hM1), M2 (hM2), and M3 (hM3) receptors and dissociated very slowly from hM3 receptors (t(½) = 166 min) with a large part of the receptorial complex (54%) remaining undissociated at 32 h from radioligand washout. In contrast, [(3)H]CHF5407 dissociated quickly from hM2 receptors (t(½) = 31 min), whereas [(3)H]tiotropium dissociated slowly from both hM3 (t(½) = 163 min) and hM2 receptor (t(½) = 297 min). In the guinea pig isolated trachea and human isolated bronchus, CHF5407 produced a potent (pIC(50) = 9.0-9.6) and long-lasting (up to 24 h) inhibition of M3 receptor-mediated contractile responses to carbachol. In the guinea pig electrically driven left atrium, the M2 receptor-mediated inhibitory response to carbachol was recovered more quickly in CHF5407-pretreated than in tiotropium-pretreated preparations. CHF5407, administered intratracheally to anesthetized guinea pigs, potently inhibited acetylcholine (Ach)-induced bronchoconstriction with an ED(50) value of 0.15 nmol/kg. The effect was sustained over a period of 24 h, with a residual 57% inhibition 48 h after antagonist administration at 1 nmol/kg. In conscious guinea pigs, inhaled CHF5407 inhibited Ach-induced bronchoconstriction for at least 24 h as did tiotropium at similar dosages. Cardiovascular parameters in anesthetized guinea pigs were not significantly changed by CHF5407, up to 100 nmol/kg i.v. and up to 1000 nmol/kg i.t. In conclusion, CHF5407 shows a prolonged antibronchospastic activity both in vitro and in vivo, caused by a very slow dissociation from M3 receptors. In contrast, CHF5407 is markedly short-acting at M2 receptors, a behavior not shared by tiotropium.

Internal auditory canal metastasis.

Metastasis of the inner auditory canal is a really rare event. Clinically, it usually presents with rapid worsening cranial nerve palsy. Authors present a review of the literature reporting clinical features, radiological findings, intraoperative aspects of an illustrative case. A 56-year-old female patient presented with a peripheral facial nerve palsy. MRI showed two left p-fossa tumors whose one into the inner canal. Rapid worsening of facial damage despite corticosteroid treatment and the possibility to remove both tumors in the same surgical step suggested authors to operated on the patient. Intraoperatively, inner canal tumor looked totally involving the VII-VIII nerve complex so surgical extirpation was only partially feasible. Posterior wall drilling of the meatus was performed which improved facial palsy. Leptomeningeal spinal seeding occurred and spinal irradiation was performed. The case highlights the importance of maintaining a high degree of awareness of the auditory canal metastasis in patients with a previous history of malignancy who develop a rapid progressive peripheral VII nerve palsy. Furthermore, our case and literature data suggest that inner canal metastasis is a distinct entity from temporal bone and ponto-cerebellar angle metastasis on the base of the peculiarity of clinical features, prognosis, therapeutic strategies. In fact, inner canal metastases usually arise in patients apparently cured, and they imply a better prognosis even if with an higher risk of leptomeningeal seeding. Moreover, surgery rarely allows the removal of the lesion, also if symptoms relief may be achieved, as in our case.

Endothelium-mediated modulation of ergoreflex and improvement in exercise ventilation by acute sildenafil in heart failure patients.

Reflex neural oversignaling sensitive to muscle by-products (ergoreflex) causes exercise hyperventilation in heart failure (HF). We probed whether an improved endothelial function with sildenafil intake may prevent this effect. In 16 chronic heart failure patients and 16 normal subjects, before and after sildenafil intake (50 mg) or placebo, we measured ergoreflex, flow-mediated brachial artery dilation (FMD, an index of endothelial function), and, during maximal exercise, the slope of ventilation to carbon dioxide production (VE/VCO2, an index of ventilatory efficiency), the ratio of changes in O2 uptake (VO2) versus work rate (WR) (deltaVO2/deltaWR, an index of aerobic efficiency). After sildenafil intake, patients, unlike controls, showed a significant decrease in ergoreflex and VE/VCO2 slope and an increase in FMD and deltaVO2/deltaWR. Ergoreflex changes with sildenafil intake correlated with those in FMD and VE/VCO2. Phosphodiesterase-5 inhibition, by improving endothelial activity and muscle perfusion, modulates signaling and improves ventilatory and aerobic efficiencies, potentially indicating a novel pathway in the HF therapeutic management.