Monoacyl lipoteichoic acid from pneumococci stimulates human cells but not mouse cells

We have developed a method for obtaining pneumococcal lipoteichoic acid (LTA) with none, one, or two acyl chains. Anion-exchange chromatography at pH 9.5 yields pneumococcal LTA (labeled LTA-9.5) that has a mass spectrum identical to that of pre-ion-exchange LTA and loses 500 mass units after deacylation by alkali hydrolysis. Anion exchange at pH 10.5 produces LTA (labeled LTA-10.5) with mass peaks that are 264 mass units lower than those of pre-ion-exchange LTA, and deacylation of LTA-10.5 by alkali hydrolysis reduces the mass by only 239 mass units. This result indicates that LTA-10.5 has lost one of the two acyl chains, whereas LTA-9.5 has both acyl chains. When the biological properties of LTA-9.5 and LTA-10.5 are examined with mouse cells, only LTA-9.5 (and not LTA-10.5) is able to stimulate mouse cells to produce tumor necrosis factor alpha, interleukin-1beta, and nitric oxide. In contrast, both LTA-9.5 and LTA-10.5 can stimulate human cells. LTA became inactive when both acyl chains were removed. Thus, acyl chains are critical for LTA function, and small variations in acyl chains can alter biological properties of LTA.     

An analytical model applied to a multicenter pneumococcal enzyme-linked immunosorbent assay study

Pneumococcal conjugate vaccines will eventually be licensed after favorable results from phase III efficacy trials. After licensure of a conjugate vaccine for invasive pneumococcal disease in infants, new conjugate vaccines will likely be licensed primarily on the basis of immunogenicity data rather than clinical efficacy. Analytical methods must therefore be developed, evaluated, and validated to compare immunogenicity results accurately within and between laboratories for different vaccines. At present no analytical technique is uniformly accepted and used in vaccine evaluation studies to determine the acceptable level of agreement between a laboratory result and the assigned value for a given serum sample. This multicenter study describes the magnitude of agreement among 12 laboratories quantifying an identical series of 48 pneumococcal serum specimens from 24 individuals (quality-control sera) by a consensus immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) developed for this study. After provisional or trial antibody concentrations were assigned to the quality-control serum samples for this study, four methods for comparison of a series of laboratory-determined values with the assigned concentrations were evaluated. The percent error between assigned values and laboratory-determined concentrations proved to be the most informative of the four methods. We present guidelines that a laboratory may follow to analyze a series of quality-control sera to determine if it can reproduce the assigned antibody concentrations within an acceptable level of tolerance. While this study focused on a pneumococcal IgG ELISA, the methods that we describe are easily generalizable to other immunological assays.     

C3-containing IgE immune complexes in asthmatic patients.

Higher levels of IgE-containing immune complexes (IC) have been reported in sera from patients with allergic diseases than in sera from controls. To evaluate the possibility of an IC-mediated mechanism in the pathogenesis of bronchial asthma, we measured circulating C3-containing IgE IC (C3-IgE IC) using anti-C3 ELISA from 20 house dust mite (HDM)-sensitive asthmatics, 20 non-atopic asthmatics, and 14 non-atopic controls. C3-IgE IC levels were significantly higher in HDM-sensitive asthmatics (mean +/- S.D.: 12.2 +/- 7.8 AU/ml) than in non-atopic asthmatics (6.5 +/- 7.5 AU/ml) or controls (5.8 +/- 4.4 AU/ml). C3-IgE IC levels were significantly correlated with HDM-specific IgE levels (r = 0.50, p < 0.05), but not with total IgE levels (r = 0.36, p > 0.05) in HDM-sensitive atopic asthmatics. C3-IgE IC levels in sera did not significantly change during HDM-bronchoprovocation test in six HDM-sensitive asthmatics who showed positive reaction. Part of C3-IgE IC could be precipitated by protein G coupled beads. In conclusion, C3-IgE IC levels were elevated in sera from HDM-sensitive asthmatics; moreover IgG antibodies might be a component of C3-IgE IC. Our results suggest that an IgE IC-mediated mechanism could be involved in the pathogenesis of atopic asthma.     

Multilaboratory Evaluation of a Viability Assay for Measurement of Opsonophagocytic Antibodies Specific to the Capsular Polysaccharides of Streptococcus pneumoniae

Opsonophagocytosis is a correlate of protection that measures the functional activity of vaccine-induced antibodies. A standardized opsonophagocytosis assay (OPA) should be used as part of the evaluation of current and future pneumococcal (Pnc) polysaccharide (Ps)-based vaccines. We enrolled five laboratories to evaluate a previously standardized viability OPA. Each laboratory was provided with a detailed OPA protocol, seven target Pnc strains (serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F), two quality control sera and 12 paired sera (blinded) from adult donors who received one dose of the 23-valent Pnc Ps vaccine. Laboratories sent their results to the Centers for Disease Control and Prevention for analysis. Sera were tested in duplicate (single run), and the results were averaged to yield a single OPA titer (≥50% killing) for each serum sample. The percentage of sera within one or two dilutions of the calculated median OPA titer was determined for each laboratory and for each serotype. In general, laboratories were capable of detecting OPA titers within one or two dilutions of the median for at least 75 and 88%, respectively, of the sera tested. The level of agreement with the median OPA titers varied depending on the participating laboratory (overall agreement = 0.8 [99% confidence interval = 0.75 to 0.85]). All OPA median titers reported for quality control sera were within one dilution of the expected titer. We conclude that this OPA can be done in multiple laboratories with a high degree of interlaboratory reproducibility.     

Cytokine production by T helper cell subpopulations during prolonged in vitro stimulation.

In man, CD4+ T cells can be divided into phenotypically distinguishable subsets with different function whereas CD4+ T cells with the opposite pheno-CD45RO and low levels of CD45RA antigen provide help for mitogen-induced immunoglobulin production whereas CD4+ cells with the opposite phenotype suppress immunoglobulin production. However, studies examining cytokine production by phenotypically defined CD4+ T cell subsets have led to different conclusions. Further, very few studies have examined cytokine production by freshly isolated CD4+ T cell subsets during extended culture periods. Thus, we examined the production of several cytokines (at various time points) by CD4+ T cell subsets that were isolated in several ways, and stimulated with PWM, Con A, and PHA in a well-defined serum-free culture system. We found that CD4+, CD45RA- (or CD45RO+) T cells consistently produced the most IL-2, IFN-gamma, and TNF-alpha after mitogen stimulation for 2 days. PWM induced the largest quantities of each cytokine, although a similar pattern of production was observed in response to Con A and PHA. We were unable to detect IL-4 production by mononuclear cells and CD4+ T cell subsets suggesting that, if it is produced at all, IL-4 is produced in extremely small quantities. When the culture period of initially CD45RO- T cells was extended beyond 2 days, the culture supernatant contained increased quantities of each cytokine and the cells in the culture had an increased number of cells expressing CD45RO antigen. Together, these data indicate that CD4, CD45RA- (or CD45RO+) T cells in peripheral blood are the major producers of IL-2, IFN-gamma, and TNF-alpha following short-term mitogen stimulation, and that phenotypically defined peripheral blood T cell subsets do not maintain a distinct pattern of cytokines during extended culture periods.     

Boehmite enhances hair follicle growth via stimulation of dermal papilla cells by upregulating β-catenin signalling

Hair growth, a complex process, has long been the subject of intense research. Recent developments in material technology have revealed boehmite as a new therapeutic modality for use in wound healing and scar reduction, indicating its beneficial effects. Nonetheless, the biological bases of the beneficial effects of boehmite remain unknown. We investigated the hair growth properties of boehmite in vitro and in vivo and observed dose-dependent proliferation of human dermal papilla cells (hDPCs) in vitro and hair regrowth in a mouse model. To investigate the effects of boehmite on the promotion of cell transition to the anagen phase, we evaluated hDPC viability, alkaline phosphatase (ALP) activity, protein expression and vascular endothelial growth factor (VEGF) secretion in vitro and assessed the anagen-promoting effects of boehmite via gross observation and histological analysis in a mouse model. Boehmite increased hDPC viability, ALP activity, AKT/GSK3ß/ß-catenin pathway activity, anagen-related gene expression and VEGF secretion; moreover, it accelerated hair regrowth in a catagen-anagen transition model via upregulation of β-catenin signalling and follicular cell proliferation. Collectively, our results indicate that boehmite accelerates hair growth, partly via its effects on critical events in the active phase of the hair follicle cycle, including the promotion of the proliferation of hDPCs and their immediate progeny to the follicle base.     

Dimeric cinchona ammonium salts with benzophenone linkers: enantioselective phase transfer catalysts for the synthesis of α-amino acids

Chiral phase transfer catalysts of dimeric cinchona ammonium salts linked with a benzophenone bridge showed high enantioselectivity in the α-alkylation of a glycinate ester under mild industry-applicable conditions: 0.5 mol% PTC and near equivalents of alkyl halide. A dual function of the dimeric quinuclidiniums was proposed for the high efficiency.

Novel cinchona-alkaloid derived dimeric phase transfer catalysts (PTC) with benzophenone linkers and their α-alkylation of glycinate esters has been presented.

Since cinchona alkaloids were transformed to asymmetric quaternary quinuclidinium salts with benzyl halides and were used as a phase-transfer catalyst (PTC) (1) by Dolling¹ and O''Donnell,² cinchona alkaloids have been widely utilized as chiral templates for phase-transfer catalysis.³ (Scheme 1) These organic PTCs can be easily prepared from natural and low cost chiral cinchona alkaloids in a few synthetic steps and they are stable and facile under normal reaction conditions in water. Later N-9-anthracenylmethyl quinuclidinium salt PTC (2) was introduced and showed high enantioselectivity for the alkylation of the protected glycine tert-butyl ester.⁴ Quinuclidinium PTCs with each pseudo-enantiomeric cinchona alkaloids, such as (−)-cinchonidine and (+)-cinchonine, show enantiomeric selectivity each other, and have been successfully applied in various asymmetric organic synthesis including α-alkyl glycine derivatives synthesis.³Open in a separate windowScheme 1Representative cinchona-derived PTCs.Enantioselectivity in the alkylation of glycine esters has been probed by several experimental trials. Crystallographic study of the p-nitrophenoxide salt of PTC 2 showed that N-anthracenylmethy moiety of 2 is located in staggered position between the C^c and the C^d,^4a which provides more hindered spaces around the C^b–C^c–C^d face (F4) around the ammonium.⁵ The C^a–C^b–C^d face (F2) is blocked by O-allyl group and the C^a–C^c–C^d face (F3) are covered by bicyclic ring, but the C^a–C^b–C^c face (F1) is less hindered. Therefore, anionic glycinate derivatives could approach toward the F1. Enolates of glycine esters would form tight ionic complexes with the ammonium nitrogen on the F1 face of PTC 2, and the alkylation with alkyl halides could follow along the direction of less hindered side of the si/re-face of the enolates.^4aNOE correlations study of PTC 2 with borohydride ion⁶ indicated the borohydride occupies the F1 face of PTC 2. Computational simulation⁷ also described the stable transition states where an enolate locates on the F1 face.Dimeric cinchona-derived PTCs linked by either benzene or naphthalene ligand have been introduced by Park et al.^8a–c Among ortho, meta and para-connected PTCs, the meta-disubstituted phenyl PTC 3a or 2,7-disubstituted naphthyl PTC 3b showed highly improved catalytic effects compared to monomeric PTC 2, such as lower dosage of catalyst (1–5 mol%) and high enantioselectivity. Role of additional quinuclidinium was thought to be a steric blocker which could increase the stereoselectivity of the enolate complex on the F1 face.Other dimeric cinchona alkaloid PTCs were also developed with various linkers, such as 9,10-dimethylanthracenyl,⁹ biphenyl, alkenyl,¹⁰ macrocyclic amine and calixarene,¹¹ and their enantioselectivities were equal or lower than those of monomeric PTCs. Some dimeric PTCs were converted to ionic polymers by replacing bromides to a disufonate anion without loss of reactivity and enantioselectivity.¹²Alkylation of tert-butyl glycinate ester was performed with 1–10 mol% of PTC 1–3 and excess 5 equivalents of alkyl halide at 0 to −78 °C. These catalytic conditions are still to be improved for practical application; low mol% of PTC, near equimolar amount of alkyl halides and ambient temperature. Hence we investigated dimeric PTCs with various linkers for facile catalytic condition. Here we introduce new dimeric cinchona PTCs with a benzophenone linker and their application in asymmetric alkylation of glycine derivatives.Monomeric PTC 4p, N-(4-benzoylbenzyl)-O(9)-allylcinchonidium bromide, which has a benzoyl benzyl at N(1), was synthesized from 4-bromomethyl-benzophenone and (−)-cinchonidine. (Scheme 2) Dimeric cinchona-based quarternay ammonium salts (PTC 5–6) were synthesized from meta/para-di(bromomethyl) benzophenone¹³ and two equivalent cinchona alkaloids. Coupling reaction of the di(bromomethyl)benzophenone and (−)-cinchonidine or (+)-cinchonine in EtOH/DMF/CHCl₃ (5 : 6 : 2)⁸ for 5 h at 100 °C and the O(9)-allylation with allyl bromide gave the dimeric quarternary salts, bis(4-(O(9)-allyl-cinchonidium-N-methyl)phenyl) methanone dibromide (5) and bis(4-(O(9)-allyl-cinchonium-N-methyl)phenyl) methanone dibromide (6), respectively, in good yields.^‡Open in a separate windowScheme 2Monomeric and dimeric cinchona-PTC with a benzophenone bridge.Enantioselective PTC 4–6 system was applied in the alkylation of N-(diphenylmethylene)glycine tert-butyl ester (7) to the α-alkylated glycinate (8) under the condition of 0.5–1.0 mol% catalysts and 1.2 equivalent alkyl halides. We also explored the variation of enantioselectivity depending on the various positions of dimeric cinchonidium at benzophenone; 5pp, 5mp and 5mm.Monomeric PTC 4p showed enantioselectivity of 87% ee (S) at 25 °C (2 Geometric difference between PTC 1 and 4p is the extra p-benzoyl substituent on N-benzyl. Apparently the p-benzoyl moiety gives no big enhancement in enantioselectivity of 4p.Catalytic phase-transfer benzylation of 7 with monomeric and dimeric cinchona-based catalysts (4–6)^a

Relationship between Angiographic Late Loss and 5-Year Clinical Outcome after Drug-Eluting Stent Implantation

Purpose

Currently, insufficient data exist to evaluate the relationship between angiographic late loss (LL) and long-term clinical outcome after drug-eluting stent (DES) implantation. In this study, we hypothesized that angiographic LL between 0.3 and 0.6 mm correlate with favorable long-term clinical outcomes.

Materials and Methods

Patients were enrolled in the present study if they had undergone both DES implantation in single coronary vessel and a subsequent follow-up angiogram (n=634). These individuals were then subdivided into three groups based on their relative angiographic LL: group I (angiographic LL <0.3 mm, n=378), group II (angiographic LL between 0.3 and 0.6 mm, n=124), and group III (angiographic LL >0.6 mm, n=134). During a 5-year follow-up period, all subjects were tracked for critical events, defined as any cause of death or myocardial infarction, which were then compared among the three groups.

Results

Mean follow-up duration was 63.0±10.0 months. Critical events occurred in 25 subjects in group I (6.6%), 5 in group II (4.0%), and 17 in group III (12.7%), (p=0.020; group I vs. group II, p=0.293; group II vs. group III, p=0.013). In a subsequent multivariate logistic regression analysis, chronic renal failure [odds ratio (OR)=3.29, 95% confidence interval (CI): 1.48-7.31, p=0.003] and long lesion length, defined as lesion length >28 mm (OR=1.88, 95% CI: 1.02-3.46, p=0.042) were independent predictors of long-term critical events.

Conclusion

This retrospective analysis fails to demonstrate that post-DES implantation angiographic LL between 0.3 and 0.6 mm is protective against future critical events.