Controlled nerve growth factor release from multi-ply alginate/chitosan-based nerve conduits.

The delivery kinetics of growth factors has been suggested to play an important role in the regeneration of peripheral nerves following axotomy. In this context, we designed a nerve conduit (NC) with adjustable release kinetics of nerve growth factor (NGF). A multi-ply system was designed where NC consisting of a polyelectrolyte alginate/chitosan complex was coated with layers of poly(lactide-co-glycolide) (PLGA) to control the release of embedded NGF. Prior to assessing the in vitro NGF release from NC, various release test media, with and without stabilizers for NGF, were evaluated to ensure adequate quantification of NGF by ELISA. Citrate (pH 5.0) and acetate (pH 5.5) buffered saline solutions containing 0.05% Tween 20 yielded the most reliable results for ELISA active NGF. The in vitro release experiments revealed that the best results in terms of reproducibility and release control were achieved when the NGF was embedded between two PLGA layers and the ends of the NC tightly sealed by the PLGA coatings. The release kinetics could be efficiently adjusted by accommodating NGF at different radial locations within the NC. A sustained release of bioactive NGF in the low nanogram per day range was obtained for at least 15days. In conclusion, the developed multi-ply NGF loaded NC is considered a suitable candidate for future implantation studies to gain insight into the relationship between local growth factor availability and nerve regeneration.

Elevated inflammatory markers combined with positive pneumococcal urinary antigen are a good predictor of pneumococcal community-acquired pneumonia in children.

BACKGROUND: Our objective was to evaluate procalcitonin (PCT) and C-reactive protein (CRP) as predictors of a pneumococcal etiology in community-acquired pneumonia (CAP) in hospitalized children. METHODS: Children requiring hospitalization for CAP were prospectively enrolled. The following indices were determined: antibodies against pneumococcal surface proteins (anti-PLY, pneumococcal histidine triad D, pneumococcal histidine triad E, LytB and pneumococcal choline-binding protein A), viral serology, nasopharyngeal cultures and polymerase chain reaction for 13 respiratory viruses, blood pneumococcal polymerase chain reaction, pneumococcal urinary antigen, PCT and CRP. Presumed pneumococcal CAP (P-CAP) was defined as a positive blood culture or polymerase chain reaction for Streptococcus pneumoniae or as a pneumococcal surface protein seroresponse (≥2-fold increase). RESULTS: Seventy-five patients were included from which 37 (49%) met the criteria of P-CAP. Elevated PCT and CRP values were strongly associated with P-CAP with odds ratios of 23 (95% confidence interval: 5-117) for PCT and 19 (95% confidence interval: 5-75) for CRP in multivariate analysis. The sensitivity was 94.4% for PCT (cutoff: 1.5 ng/mL) and 91.9% for CRP (cutoff: 100 mg/L). A value≤0.5 ng/mL of PCT ruled out P-CAP in >90% of cases (negative likelihood ratio: 0.08). Conversely, a PCT value≥1.5 ng/mL associated with a positive pneumococcal urinary antigen had a diagnostic probability for P-CAP of almost 80% (positive likelihood ratio: 4.59). CONCLUSIONS: PCT and CRP are reliable predictors of P-CAP. Low cutoff values of PCT allow identification of children at low risk of P-CAP. The association of elevated PCT or CRP with a positive pneumococcal urinary antigen is a strong predictor of P-CAP.

Immunity to pneumococcal surface proteins in children with community-acquired pneumonia: a distinct pattern of responses to pneumococcal choline-binding protein A.

Clin Microbiol Infect ABSTRACT: The aetiological diagnosis of community-acquired pneumonia (CAP) is challenging in children, and serological markers would be useful surrogates for epidemiological studies of pneumococcal CAP. We compared the use of anti-pneumolysin (Ply) antibody alone or with four additional pneumococcal surface proteins (PSPs) (pneumococcal histidine triad D (PhtD), pneumococcal histidine triad E (PhtE), LytB, and pneumococcal choline-binding protein A (PcpA)) as serological probes in children hospitalized with CAP. Recent pneumococcal exposure (positive blood culture for Streptococcus pneumoniae, Ply(+) blood PCR finding, and PSP seroresponse) was predefined as supporting the diagnosis of presumed pneumococcal CAP (P-CAP). Twenty-three of 75 (31%) children with CAP (mean age 33.7 months) had a Ply(+) PCR finding and/or a ≥2-fold increase of antibodies. Adding seroresponses to four PSPs identified 12 additional patients (35/75, 45%), increasing the sensitivity of the diagnosis of P-CAP from 0.44 (Ply alone) to 0.94. Convalescent anti-Ply and anti-PhtD antibody titres were significantly higher in P-CAP than in non P-CAP patients (446 vs. 169 ELISA Units (EU)/mL, p 0.031, and 189 vs. 66 EU/mL, p 0.044), confirming recent exposure. Acute anti-PcpA titres were three-fold lower (71 vs. 286 EU/mL, p <0.001) in P-CAP children. Regression analyses confirmed a low level of acute PcpA antibodies as the only independent predictor (p 0.002) of P-CAP. Novel PSPs facilitate the demonstration of recent pneumococcal exposure in CAP children. Low anti-PcpA antibody titres at admission distinguished children with P-CAP from those with CAP with a non-pneumococcal origin.

Pneumolysin Activates the NLRP3 Inflammasome and Promotes Proinflammatory Cytokines Independently of TLR4.

Pneumolysin (PLY) is a key Streptococcus pneumoniae virulence factor and potential candidate for inclusion in pneumococcal subunit vaccines. Dendritic cells (DC) play a key role in the initiation and instruction of adaptive immunity, but the effects of PLY on DC have not been widely investigated. Endotoxin-free PLY enhanced costimulatory molecule expression on DC but did not induce cytokine secretion. These effects have functional significance as adoptive transfer of DC exposed to PLY and antigen resulted in stronger antigen-specific T cell proliferation than transfer of DC exposed to antigen alone. PLY synergized with TLR agonists to enhance secretion of the proinflammatory cytokines IL-12, IL-23, IL-6, IL-1β, IL-1α and TNF-α by DC and enhanced cytokines including IL-17A and IFN-γ by splenocytes. PLY-induced DC maturation and cytokine secretion by DC and splenocytes was TLR4-independent. Both IL-17A and IFN-γ are required for protective immunity to pneumococcal infection and intranasal infection of mice with PLY-deficient pneumococci induced significantly less IFN-γ and IL-17A in the lungs compared to infection with wild-type bacteria. IL-1β plays a key role in promoting IL-17A and was previously shown to mediate protection against pneumococcal infection. The enhancement of IL-1β secretion by whole live S. pneumoniae and by PLY in DC required NLRP3, identifying PLY as a novel NLRP3 inflammasome activator. Furthermore, NLRP3 was required for protective immunity against respiratory infection with S. pneumoniae. These results add significantly to our understanding of the interactions between PLY and the immune system.