Fabrication of Chitosan/PEGDA Bionanocomposites for Enhanced Drug Encapsulation and Release Efficiency.

Drug delivery systems (DDS) have evolved in the last decades with the development of hydrogels and particles. However, challenges such as high systemic uptake, side effects, low bioavailability, and encapsulation efficiency continue to be significant hurdles faced by such DDSs. Particles and hydrogels can be specifically designed for targeted DDSs to mitigate some of these problems. This study developed chitosan (Cs) particles (Ps) and composite films using poly(ethylene glycol) diacrylate (PEGDA) as a copolymer to encapsulate gentamicin (GtS) for drug delivery. We demonstrated that lysozyme degrades the chitosan ß-1,4 glycosidic bonds to release GtS. PEGDA increased drug encapsulation efficiency by shielding the repelling forces of like charges between Cs and GtS. The data show that PEGDA does not hinder enzymatic degradation while increasing drug encapsulation efficiency and producing more homogeneous particles. Additionally, we utilized Michael's reaction to cross-link Cs, CsPs, and PEGDA to produce a film designed for drug delivery. The film is an anchor for CsPs to prevent premature drug release. The cross-linking of Cs and PEGDA does not affect lysozyme activity, and CsPs could successfully release GtS without affecting GtS activity.

Development and Characterization of Membranes with PVA Containing Silver Particles: A Study of the Addition and Stability.

Developing technologies for the reduction of biofouling and enhancement of membrane functionality and durability are challenging but critical for the advancement of water purification processes. Silver (Ag) is often used in the process of purification due to its anti-fouling properties; however, the leaching of this metal from a filtration membrane significantly reduces its effectiveness. Our study was designed to integrate the positive characteristics of poly vinyl alcohol (PVA) with the controlled incorporation of nano-scale silver ions across the membrane. This approach was designed with three goals in mind: (1) to improve antifouling activity; (2) to prevent leaching of the metal; and (3) to extend the durability of the functionalized membrane. The fabrication method we used was a modified version of manual coating in combination with sufficient pressure to ensure impregnation and proper blending of PVA with cellulose acetate. We then used the spin coater to enhance the cross-linking reaction, which improved membrane durability. Our results indicate that PVA acts as a reducing agent of Ag+ to Ag0 using X-ray photoelectron spectroscopy analysis and demonstrate that the metal retention was increased by more than 90% using PVA in combination with ultraviolet-photo-irradiated Ag+ reduced to Ag0. The Ag+ ions have sp hybrid orbitals, which accept lone pairs of electrons from a hydroxyl oxygen atom, and the covalent binding of silver to the hydroxyl groups of PVA enhanced retention. In fact, membranes with reduced Ag displayed a more effective attachment of Ag and a more efficient eradication of E. coli growth. Compared to pristine membranes, bovine serum albumin (BSA) flux increased by 8% after the initial addition of Ag and by 17% following ultraviolet irradiation and reduction of Ag, whereas BSA rejection increased by 10% and 11%, respectively. The implementation of this hybrid method for modifying commercial membranes could lead to significant savings due to increased metal retention and membrane effectiveness. These enhancements would ultimately increase the membrane's longevity and reduce the cost/benefit ratio.

Passive immunization with Pneumovax® 23 and pneumolysin in combination with vancomycin for pneumococcal endophthalmitis.

BACKGROUND: Capsule and pneumolysin (PLY) are two major virulence factors of Streptococcus pneumoniae. S. pneumoniae is one of the leading causes of bacterial endophthalmitis. The aim of this study is to determine whether passive immunization with the 23-valent pneumococcal polysaccharide vaccine (Pneumovax® 23; PPSV23) or PLY protects against pneumococcal endophthalmitis. METHODS: New Zealand white rabbits were passively immunized with antiserum to PLY, PPSV23, a mixture of PPSV23/PLY, or PBS (mock). Vitreous was infected with a clinical strain of S. pneumoniae. In a separate group of experiments, vancomycin was injected 4 hours post-infection (PI) for each passively immunized group. Severity of infection, bacterial recovery, myeloperoxidase (MPO) activity and percent loss of retinal function were determined. RESULTS: Passive immunization with each antiserum significantly lowered clinical severity compared to mock immunization (PPSV23 = 9.19, PPSV23/PLY = 10.45, PLY = 8.71, Mock = 16.83; P = 0.0467). A significantly higher bacterial load was recovered from the vitreous of PLY passively immunized rabbits 24 hours PI (7.87 log10 CFU) compared to controls (7.10 log10 CFU; P = 0.0134). Retinas from immunized rabbits were more intact. Vitreous of PLY (2.88 MPO untis/mL) and PPSV23/PLY (2.17) passively immunized rabbits had less MPO activity compared to controls (5.64; P = 0.0480), and both passive immunizations (PLY = 31.34% loss of retinal function, PPSV23/PLY = 27.44%) helped to significantly preserve retinal function compared to controls (64.58%; P = 0.0323). When vancomycin was administered 4 hours PI, all eyes were sterile at 24 hours PI. A significantly lower clinical severity was observed for rabbits administered the combination immunization (5.29) or PPSV23 (5.29) with vancomycin treatment compared to controls (17.68; P = 0.0469). CONCLUSIONS: Passive immunization with antisera to these antigens is effective in reducing clinical severity of pneumococcal endophthalmitis in rabbits. Addition of vancomycin to immunization is effective at eliminating the bacteria.

Active Immunization with Pneumolysin versus 23-Valent Polysaccharide Vaccine for Streptococcus pneumoniae Keratitis.

PURPOSE: The purpose of this study was to determine whether active immunization against pneumolysin (PLY), or polysaccharide capsule, protects against the corneal damage associated with Streptococcus pneumoniae keratitis. METHODS: New Zealand White rabbits were actively immunized with Freund's adjuvant mixed with pneumolysin toxoid (ψPLY), Pneumovax 23 (PPSV23; Merck, Whitehouse Station, NJ), or phosphate-buffered saline (PBS), before corneal infection with 105 colony-forming units (CFU) of S. pneumoniae. Serotype-specific rabbit polyclonal antisera or mock antisera were passively administered to rabbits before either intravenous infection with 10¹¹ CFU S. pneumoniae or corneal infection with 105 CFU of S. pneumoniae. RESULTS: After active immunization, clinical scores of corneas of the rabbits immunized with ψPLY and Freund's adjuvant were significantly lower than scores of the rabbits that were mock immunized with PBS and Freund's adjuvant or with PPSV23 and Freund's adjuvant at 48 hours after infection (P ≤ 0.0010), whereas rabbits immunized with PPSV23 and Freund's adjuvant failed to show differences in clinical scores compared with those in mock-immunized rabbits (P = 1.00) at 24 and 48 hours after infection. Antisera from rabbits actively immunized with PPSV23 and Freund's adjuvant were nonopsonizing. Bacterial loads recovered from infected corneas were higher for the ψPLY- and PPSV23-immunized rabbits after infection with WU2, when compared with the mock-immunized rabbits (P ≤ 0.007). Conversely, after infection with K1443, the ψPLY-immunized rabbits had lower bacterial loads than the control rabbits (P = 0.0008). Quantitation of IgG, IgA, and IgM in the sera of ψPLY-immunized rabbits showed high concentrations of PLY-specific IgG. Furthermore, anti-PLY IgG purified from ψPLY-immunized rabbits neutralized the cytolytic effects of PLY on human corneal epithelial cells. Passive administration of serotype-specific antisera capable of opsonizing and killing S. pneumoniae protected against pneumococcal bacteremia (P ≤ 0.05), but not against keratitis (P ≥ 0.476). CONCLUSIONS: Active immunization with pneumococcal capsular polysaccharide and Freund's adjuvant fails to produce opsonizing antibodies, and passive administration of serotype specific opsonizing antibodies offers no protection against pneumococcal keratitis in the rabbit, whereas active immunization with the conserved protein virulence factor PLY and Freund's adjuvant is able to reduce corneal inflammation associated with pneumococcal keratitis, but has variable effects on bacterial loads in the cornea.

Comparison of besifloxacin, gatifloxacin, and moxifloxacin against strains of pseudomonas aeruginosa with different quinolone susceptibility patterns in a rabbit model of keratitis.

PURPOSE: Determine the effectiveness of topical besifloxacin, gatifloxacin, and moxifloxacin in treating keratitis caused by 2 strains of Pseudomonas aeruginosa with different quinolone susceptibility profiles. METHODS: Minimal inhibitory concentrations (MICs) were determined for each fluoroquinolone. Sequence analysis was performed on the quinolone resistance determining regions of the ciprofloxacin/levofloxacin-resistant strain. Rabbit corneas were injected with 10 colony-forming units (CFU). After 16 hours, phosphate-buffered saline, besifloxacin (6 mg/mL), gatifloxacin (3 mg/mL), or moxifloxacin (5 mg/mL) was applied topically every 15 minutes for 5 doses, then every 30 minutes for 14 doses. Eyes were examined pre- and posttreatment. Corneas were harvested for bacterial quantitation. RESULTS: MICs against the fully susceptible strain were 0.5, 0.25, and 0.5 µg/mL for besifloxacin, gatifloxacin, and moxifloxacin, respectively. The MICs against the ciprofloxacin/levofloxacin-resistant strain were 2, 16, and 32 µg/mL for besifloxacin, gatifloxacin, and moxifloxacin, respectively. Sequence analysis revealed amino acid mutations in all 4 fluoroquinolone target genes. None of the treatments had an effect on clinical severity of eyes infected with the fully susceptible strain (P > 0.05); however, all were effective at significantly reducing the bacterial CFU in the corneas (P < 0.05). For the ciprofloxacin/levofloxacin-resistant strain, clinical scores of besifloxacin-treated eyes were significantly lower than moxifloxacin-treated eyes (P < 0.037). The quantities of ciprofloxacin/levofloxacin-resistant bacteria recovered from corneas of all treatment groups were significantly lower than those recovered from phosphate-buffered saline-treated corneas (P < 0.05). Besifloxacin-treated eyes had significantly lower CFU recovered as compared with that of gatifloxacin- and moxifloxacin-treated eyes (P < 0.05). CONCLUSIONS: These results support clinical investigation of the effectiveness of besifloxacin in treating Pseudomonas keratitis.

The Streptococcus pneumoniae capsule is required for full virulence in pneumococcal endophthalmitis.

PURPOSE: To determine whether Streptococcus pneumoniae capsule was necessary for pathogenesis of pneumococcal endophthalmitis. METHODS: An isogenic capsule-deficient strain was created using homologous recombination. New Zealand White rabbits were injected intravitreously with 10(2) colony-forming units (CFU) of the parent strain or the capsule mutant. Slit lamp examination (SLE), electroretinography, and myeloperoxidase activity were performed 24 and 48 hours postinfection (PI). Serial dilutions of vitreous were plated to quantitate CFU, eyes were extracted for histology, and host cytokine mRNA expression was determined. RESULTS: Eyes infected with the parent strain had significantly higher SLE scores than eyes infected with the capsule-deficient strain 24 and 48 hours PI (P < 0.001). CFU recovered from eyes infected with the capsule mutant were significantly fewer than CFU recovered from eyes infected with the parent strain 24 and 48 hours PI (P < 0.001). The parent strain caused a significantly greater decrease in retinal function and more retinal destruction than the mutant strain 48 hours PI (P = 0.026). Vitreal IL-1ß, IL-6, and TNF-α were upregulated by both the parent and mutant strain 12 hours PI. By 48 hours PI, there was significantly more neutrophil infiltration in the vitreous infected with the parent strain. CONCLUSIONS: Endophthalmitis caused by the encapsulated strain is more damaging to retinal function and structural integrity. These findings indicate that capsule is an important virulence factor of S. pneumoniae endophthalmitis, in contrast to keratitis, suggesting that the anatomic host site in pneumococcal ocular infections is important.

Pathogenesis of A Clinical Ocular Strain of Streptococcus pneumoniae and the Interaction of Pneumolysin with Corneal Cells.

Streptococcus pneumoniae is an important cause of bacterial keratitis, an infectious disease of the cornea. This study aimed to determine the importance of pneumolysin (PLY), a pneumococcal virulence factor, in keratitis using a clinical keratitis isolate (K1263) and its isogenic mutant deficient in PLY (K1263ΔPLY) and determine the effect of these strains on primary rabbit corneal epithelial (RCE) cells. Each strain was injected into the corneal stromas of rabbits, clinical examinations were performed, and the recovered bacterial loads were determined. Bacterial extracts were exposed to RCE cells, and morphology and viability were assessed. The mutant strain deficient in PLY, K1263ΔPLY, caused significantly lower ocular disease scores than the parent strain (K1263), although a higher bacterial load was recovered from corneas infected with the mutant strain. Histological examination showed increased inflammatory cells in the anterior chamber and increased edema in eyes infected with the parent strain. RCE cells exposed to the parent strain had significantly decreased cell viability and showed increased evidence of cellular damage. This study confirms that in a strain that can cause clinical keratitis, PLY is a significant cause of the damage associated with pneumococcal keratitis. It also shows for the first time that the results from an in vitro model using RCE cells correlates with in vivo results thereby establishing a less invasive way to study the mechanisms of pneumococcal keratitis.

Immunization with pneumolysin protects against both retinal and global damage caused by Streptococcus pneumoniae endophthalmitis.

PURPOSE: To determine whether immunization with pneumolysin (PLY) protects against pneumococcal endophthalmitis. METHODS: New Zealand white rabbits were immunized with a mutant form of PLY that retains only 1% of its cytolytic activity until serum IgG titers were ≥51,200. For a negative control, rabbits were immunized with phosphate-buffered saline (mock). Each vitreous was injected with 10(2) colony-forming units of a clinical endophthalmitis isolate of Streptococcus pneumoniae. Severity of endophthalmitis was graded by slit lamp examination at 24 and 48 h postinfection (PI). Serial dilutions of vitreous were plated for bacterial colony-forming units quantitation, eyes were extracted for histology, and a whole blood survival assay was performed. RESULTS: Immunized rabbits had a significantly lower mean slit lamp examination score at 24 and 48 h PI when compared to mock immunized rabbits (P ≤ 0.002). There was not a significant difference in bacterial load in the vitreous at 24 or 48 h PI. Histological sections showed that retinas of mock immunized rabbits appeared to be destroyed, whereas those of PLY immunized rabbits remained largely intact. Damage spread to the aqueous humor, stroma, and conjunctiva of mock immunized rabbits by 48 h PI. Minimal damage was observed in the vitreous of PLY immunized rabbits and did not spread to other parts of the eye. Whole blood from immunized rabbits inhibited the growth of bacteria better than whole blood from mock immunized rabbits. CONCLUSION: Immunization with PLY helps protect the eye from damage caused by pneumococcal endophthalmitis.

Efficacy of besifloxacin in an early treatment model of methicillin-resistant Staphylococcus aureus keratitis.

PURPOSE: To determine the effectiveness of topically applied besifloxacin, gatifloxacin, and moxifloxacin for the early treatment of experimental methicillin-resistant Staphylococcus aureus (MRSA) keratitis. METHODS: Ten hours post-MRSA infection, rabbit eyes were treated topically with 19 doses of phosphate-buffered saline (PBS), besifloxacin, gatifloxacin, or moxifloxacin. Slit-lamp examinations were performed before and after the inoculation. Corneas were harvested for bacterial quantitation and minimal inhibitory concentrations (MICs) were determined. RESULTS: All 3 fluoroquinolones significantly lowered the clinical severity of the infection as compared to treatment with PBS (P < 0.05). However, the mean log(10) colony-forming unit (CFU) recovered from besifloxacin-treated corneas was significantly lower than all other treatment groups (P < 0.01). CFU recovered from corneas treated with moxifloxacin and PBS showed no significant difference (P = 0.12). Corneas treated with gatifloxacin had a significantly lower log(10) CFU recovered as compared to PBS-treated corneas (P < 0.01). The MICs for gatifloxacin and moxifloxacin were 8 microg/mL, whereas the MIC for besifloxacin was 1 microg/mL. CONCLUSIONS: All 3 fluoroquinolones significantly lowered the clinical severity of the infection. Besifloxacin had an 8-fold lower MIC for MRSA than gatifloxacin and moxifloxacin, and was significantly more effective than gatifloxacin and moxifloxacin in reducing the number of MRSA in the rabbit cornea.

Efficacy of besifloxacin in a rabbit model of methicillin-resistant Staphylococcus aureus keratitis.

PURPOSE: The purpose of this study was to determine the effectiveness of topically applied besifloxacin (0.6%), gatifloxacin (0.3%), and moxifloxacin (0.5%) for the late treatment of experimental methicillin-resistant Staphylococcus aureus (MRSA) keratitis. METHODS: One hundred colony-forming units (CFUs) of bacteria were injected intrastromally into rabbit corneas. Sixteen hours after infection, one topical drop of phosphate-buffered saline, besifloxacin, gatifloxacin, or moxifloxacin was applied to each eye every 15 minutes for 5 doses and then every 30 minutes for 14 doses. Eyes were examined before and after treatment by slit lamp biomicroscopy. Corneas were harvested from treated and untreated rabbits for the quantitation of bacteria. Minimal inhibitory concentrations (MICs) were determined in vitro for each fluoroquinolone. RESULTS: None of the treatments had an effect on clinical severity (P > 0.05). Although there were no differences in clinical severity between any groups after treatment, the mean log10 CFU of MRSA recovered from besifloxacin-treated corneas (5.111 +/- 0.251) was significantly lower than the CFU recovered from corneas treated with phosphate-buffered saline (7.006 +/- 0.144), gatifloxacin (7.108 +/- 0.346), and moxifloxacin (7.473 +/- 0.144; P < 0.001). CFU recovered from gatifloxacin- and moxifloxacin-treated corneas were not significantly different from phosphate-buffered saline-treated corneas (P = 1.000). The MICs against the MRSA strain were 8 microg/mL for both gatifloxacin and moxifloxacin, whereas the MIC for besifloxacin was 1 microg/mL. CONCLUSION: Besifloxacin had an 8-fold lower MIC for MRSA than gatifloxacin and moxifloxacin and was significantly more effective than gatifloxacin and moxifloxacin in reducing the number of MRSA in the rabbit cornea 16 hours after infection.

Development of a Streptococcus pneumoniae keratitis model in mice.

BACKGROUND: Streptococcus pneumoniae is a common cause of bacterial keratitis, and models to examine the ocular pathogenesis of this bacterium would aid in efforts to treat pneumococcal keratitis. The aim of this study was to establish a murine model of pneumococcal keratitis. METHODS: The corneas of A/J, BALB/c or C57BL/6 mice were scratched and topically infected with a clinical strain of S. pneumoniae. Slitlamp examination (SLE), enumeration of bacteria in the corneas and histology were performed. RESULTS: Bacteria were recovered from the eyes of A/J mice on postinfection (PI) days 1 [1.96 +/- 0.61 log(10) colony-forming units (CFU)] and 3 (1.41 +/- 0.71 log(10) CFU). SLE scores were significantly higher in the infected A/J mice as compared to the BALB/c or C57BL/6 mice on PI day 3 (p < 0.0001) and steadily increased over time, reaching a maximal value of 3.00 +/- 0.35 on PI day 10. Histopathology revealed stromal edema and the influx of polymorphonuclear leukocytes on PI days 7 and 10, and corneal disruption on PI day 7. CONCLUSIONS: S. pneumoniae keratitis was established in A/J mice, but not BALB/c or C57BL/6 mice.

Protection from Streptococcus pneumoniae keratitis by passive immunization with pneumolysin antiserum.

PURPOSE: To determine whether passive immunization with pneumolysin antiserum can reduce corneal damage associated with pneumococcal keratitis. METHODS: New Zealand White rabbits were intrastromally injected with Streptococcus pneumoniae and then passively immunized with control serum, antiserum against heat-inactivated pneumolysin (HI-PLY), or antiserum against cytotoxin-negative pneumolysin (psiPLY). Slit lamp examinations (SLEs) were performed at 24, 36, and 48 hours after infection. An additional four corneas from rabbits passively immunized with antiserum against psiPLY were examined up to 14 days after infection. Colony forming units (CFUs) were quantitated from corneas extracted at 20 and 48 hours after infection. Histopathology of rabbit eyes was performed at 48 hours after infection. RESULTS: SLE scores at 36 and 48 hours after infection were significantly lower in rabbits passively immunized with HI-PLY antiserum than in control rabbits (P < or = 0.043). SLE scores at 24, 36, and 48 hours after infection were significantly lower in rabbits passively immunized with psiPLY antiserum than in control rabbits (P < or = 0.010). The corneas of passively immunized rabbits that were examined up to 14 days after infection exhibited a sequential decrease in keratitis, with an SLE score average of 2.000 +/- 1.586 at 14 days. CFUs recovered from infected corneas were not significantly different between each experimental group and the respective control group at 20 or 48 hours after infection (P > or = 0.335). Histologic sections showed more corneal edema and polymorphonuclear leukocyte (PMN) infiltration in control rabbits compared with passively immunized rabbits. CONCLUSIONS: HI-PLY and psiPLY both elicit antibodies that provide passive protection against S. pneumoniae keratitis.

A comparison of pneumolysin activity and concentration in vitro and in vivo in a rabbit endophthalmitis model.

The purpose of this study was to determine whether the in vitro activity and concentration of Streptococcus pneumoniae pneumolysin correlated to the pathogenesis of S. pneumoniae endophthalmitis. Five S. pneumoniae clinical endophthalmitis strains were grown in media to similar optical densities (OD), and extracellular milieu was tested for pneumolysin activity by hemolysis of rabbit red blood cells. Pneumolysin concentration was determined using a sandwich ELISA. Rabbit vitreous was injected with 10(2) colony-forming units (CFU) of 1 of 2 different strains with low hemolytic activity (n = 10 and 12 for strains 4 and 5, respectively) or 1 of 3 different strains with high hemolytic activity (n = 12 per strain). Pathogenesis of endophthalmitis infection was graded by slit lamp examination (SLE) at 24 hours post-infection. Bacteria were recovered from infected vitreous and quantitated. The SLE scores of eyes infected with strains having high hemolytic activity were significantly higher than the scores of those infected with strains having low hemolytic activity (P < 0.05). Pneumolysin concentration in vitro, however, did not correlate with hemolysis or severity of endophthalmitis. Bacterial concentrations from the vitreous infected with 4 of the strains were not significantly different (P > 0.05). These data suggest that pneumolysin hemolytic activity in vitro directly correlates to the pathogenesis of S. pneumoniae endophthalmitis. The protein concentration of pneumolysin, however, is not a reliable indicator of pneumolysin activity.

Peritoneal challenge modulates expression of pneumococcal surface protein C during bacteremia in mice.

Differential expression of pneumococcal virulence proteins has been demonstrated. We previously demonstrated challenge route-dependent differences in pneumococcal surface protein C (PspC) expression during bacteremia. In this study, we investigated differences in PspC expression during the transition of pneumococci from the peritoneum to the blood. Time course analysis of PspC expression using flow cytometry demonstrated that Streptococcus pneumoniae D39 collected from blood expressed significantly more PspC than did D39 collected from the peritoneum of intraperitoneally (i.p.)-infected mice. Various challenge models were then used to determine whether host responses originating from the peritoneum can influence PspC expressed by pneumococci in the blood. Using heat-inactivated D39 (HI-D39) and sterile peritoneal dialysis fluid (PDF), we investigated whether stimulation of peritoneal responses can influence PspC expression. Injection of mice i.p. with HI-D39 or PDF immediately prior to intravenous (i.v.) infection with D39 caused a significant increase in PspC expressed by D39 in the blood. Finally, we used cytokine array analysis to investigate specific inflammatory mediators that may result in differential PspC expression. Of the 96 inflammatory cytokines assayed, D39 i.p. challenge led to increased expression of 33 cytokines in serum; whereas D39 i.v. challenge led to increased expression of 15 and decreased expression of 11 cytokines relative to serum of the uninfected control. These results indicate that PspC is differentially regulated during growth in vivo and that the level of expression varies depending on the host environment.

Factor H binding to PspC of Streptococcus pneumoniae increases adherence to human cell lines in vitro and enhances invasion of mouse lungs in vivo.

Pneumococcal surface protein C (PspC) binds to both human secretory immunoglobulin A (sIgA) and complement factor H (FH). FH, a regulator of the alternative pathway of complement, can also mediate adherence of different host cells. Since PspC contributes to adherence and invasion of host cells, we hypothesized that the interaction of PspC with FH may also mediate adherence of pneumococci to human cells. In this study, we investigated FH- and sIgA-mediated pneumococcal adherence to human cell lines in vitro. Adherence assays demonstrated that preincubation of Streptococcus pneumoniae D39 with FH increased adherence to human umbilical vein endothelial cells (HUVEC) 5-fold and to lung epithelial cells (SK-MES-1) 18-fold, relative to that of D39 without FH on the surface. The presence of sIgA enhanced adherence to SK-MES-1 6-fold and to pharyngeal epithelial cells (Detroit 562) 14-fold. Furthermore, sIgA had an additive effect on adherence to HUVEC; specifically, preincubation of D39 with both FH and sIgA led to a 21-fold increase in adherence. Finally, using a mouse model, we examined the significance of the FH-PspC interaction in pneumococcal nasal colonization and lung invasion. Mice intranasally infected with D39 preincubated with FH had increased bacteremia and lung invasion, but they had similar levels of nasopharyngeal colonization compared to that of mice challenged with D39 without FH.

Immunization with PspA incorporated into a poly(ethylene oxide) matrix elicits protective immunity against Streptococcus pneumoniae.

CBA/N mice were immunized with PspA in a poly(ethylene oxide) matrix to examine its ability to deliver the antigen and modulate the immune response. All mice receiving PspA in the matrix survived a lethal pneumococcal challenge and had serum anti-PspA antibody levels statistically higher than mice receiving PspA alone (P < 0.009).

Pneumolysin, PspA, and PspC contribute to pneumococcal evasion of early innate immune responses during bacteremia in mice.

The pneumococcal virulence factors include capsule, PspA, PspC, and Ply. Cytometric analysis demonstrated that the greatest levels of C3 deposition were on a Deltaply PspA(-) PspC(-) mutant. Also, Ply, PspA, and PspC expression resulted in C3 degradation in vitro and in vivo. Finally, blood clearance assays demonstrated that there was enhanced clearance of Deltaply PspA(-) PspC(-) pneumococci compared to the clearance of nonencapsulated pneumococci.

Enhanced protective immunity against pneumococcal infection with PspA DNA and protein.

The effect of priming and boosting with pspA/EF5668 and purified recombinant PspA/EF5668 was examined. With this strategy CBA/N mice were protected against fatal challenge with Streptococcus pneumoniae EF5668. Anti-PspA antibody titers were elevated, and Western analysis with the immune serum demonstrated cross-reactivity with PspA from several different pneumococcal isolates, representing different PspA clades. Immune serum localized cross-reactive epitopes to the alpha-helical domain of PspA/Rx1 and PspA/EF5668. We demonstrated that DNA/protein prime-boost immunizations can enhance protective immunity against pneumococcal challenge.