Renovascular disease (RVD) induces renal microvascular (MV) rarefaction that drives progressive kidney injury. In previous studies, we showed that renal vascular endothelial growth factor (VEGF) therapy attenuated MV damage, but did not resolve renal injury at practical clinical doses. To increase the bioavailability of VEGF, we developed a biopolymer-stabilized elastin-like polypeptide (ELP)-VEGF fusion protein and determined its in vivo potential for therapeutic renal angiogenesis in RVD using an established swine model of chronic RVD. We measured single-kidney blood flow (RBF) and GFR and established the degree of renal damage after 6 weeks of RVD. Pigs then received a single stenotic kidney infusion of ELP-VEGF (100 mg/kg), a matching concentration of unconjugated VEGF (18.65 mg/kg), ELP alone (100 mg/kg), or placebo. Analysis of organ distribution showed high renal binding of ELP-VEGF 4 hours after stenotic kidney infusion. Therapeutic efficacy was determined 4 weeks after infusion. ELP-VEGF therapy improved renal protein expression attenuated in RVD, restoring expression levels of VEGF, VEGF receptor Flk-1, and downstream angiogenic mediators, including phosphorylated Akt and angiopoietin-1 and -2. This effect was accompanied by restored MV density, attenuated fibrogenic activity, and improvements in RBF and GFR greater than those observed with placebo, ELP alone, or unconjugated VEGF. In summary, we demonstrated the feasibility of a novel therapy to curtail renal injury. Recovery of the stenotic kidney in RVD after ELP-VEGF therapy may be driven by restoration of renal angiogenic signaling and attenuated fibrogenic activity, which ameliorates MV rarefaction and improves renal function. 27: 174127: -175227: , 201627: . doi: 10.1681 Renal vascular disease (RVD), usually caused by renal artery stenosis, can lead to CKD and ESRD. RVD increases cardiovascular morbidity and mortality, hospitalization, shortens life expectancy, and is increasing at a sustained pace in the United States. 1 Moreover, renal function does not improve or even deteriorates in almost half of the patients with RVD despite treatment. Recent clinical studies suggest that patients undergoing current therapeutic strategies, which include drugs and renal angioplasty, do not show differences in outcomes that could demonstrate distinct benefits of one treatment over the other when compared side by side. 2,3 Moreover, renal function does not improve or even deteriorates in almost half of patients with RVD despite treatment, highlighting a pressing need for novel therapeutic strategies for the growing population of patients suffering from RVD. J Am Soc NephrolUsing a clinically relevant swine model of chronic RVD that mimics several of the pathologic features

Purpose The pneumococcal capsule is required for pathogenesis in systemic infections, yet reports show most conjunctivitis outbreaks are caused by nonencapsulated pneumococci, while keratitis infections are caused by encapsulated strains. This study aims to determine the effect of capsule in pneumococcal keratitis and conjunctivitis in rabbit models of infection. Methods A capsule-deficient isogenic mutant was created using homologous transformation. Parent and mutant strains were injected within the upper bulbar conjunctiva (conjunctivitis) or into the corneal stroma (keratitis) of New Zealand white rabbits. Clinical examinations were performed 24 and 48 hr post-infection at which time corneas or conjunctivae were removed, homogenized, and plated to determine the recovered bacterial load. Whole eyes were removed for histological examination. The neuraminidase activity was determined following in vitro and in vivo growth. Results There were no significant differences in clinical scores between the eyes infected with the parent or mutant for either infection, nor was there a difference in the amount of bacteria recovered from the cornea. In the conjunctivae, however, the mutant strain was cleared by the host faster than the parent strain. Histological examination showed slightly more infiltrating polymorphonuclear leukocytes (PMN) and macrophages in the conjunctivae infected with the parent strain. The neuraminidase activity of both strains was not significantly different when the strains were grown in vitro. However, the neuraminidase activity of the parent was significantly less than that of the mutant at 3 and 12 hr post conjunctival infection. Conclusions Although more outbreaks of pneumococcal conjunctivitis are tied to nonencapsulated S. pneumoniae strains, this study showed that an encapsulated strain was capable of establishing conjunctivitis in a rabbit injection model and survive attack by the host immune system longer than its nonencapsulated isogenic mutant. Nonetheless, the nonencapsulated pneumococci had an increased neuraminidase activity level in vivo when compared to the parent strain.

Percutaneous transluminal renal angioplasty/stenting (PTRAS) is frequently used to treat renal artery stenosis and renovascular disease (RVD); however, renal function is restored in less than one half of the cases. This study was designed to test a novel intervention that could refine PTRAS and enhance renal recovery in RVD. Renal function was quantified in pigs after 6 weeks of chronic RVD (induced by unilateral renal artery stenosis), established renal damage, and hypertension. Pigs with RVD then underwent PTRAS and were randomized into three groups: placebo (RVD+PTRAS), chronic endothelin-A receptor (ET-A) blockade (RVD+PTRAS+ET-A), and chronic dual ET-A/B blockade (RVD+PTRAS+ET-A/B) for 4 weeks. Renal function was again evaluated after treatments, and then, ex vivo studies were performed on the stented kidney. PTRAS resolved renal stenosis, attenuated hypertension, and improved renal function but did not resolve renal microvascular rarefaction, remodeling, or renal fibrosis. ET-A blocker therapy after PTRAS significantly improved hypertension, microvascular rarefaction, and renal injury and led to greater recovery of renal function. Conversely, combined ET-A/B blockade therapy blunted the therapeutic effects of PTRAS alone or PTRAS followed by ET-A blockade. These data suggest that ET-A receptor blockade therapy could serve as a coadjuvant intervention to enhance the outcomes of PTRAS in RVD. These results also suggest that ET-B receptors are important for renal function in RVD and may contribute to recovery after PTRAS. Using clinically available compounds and techniques, our results could contribute to both refinement and design of new therapeutic strategies in chronic RVD. Chronic renovascular disease (RVD) increases the risk of cardiovascular morbidity and mortality and may progressively induce renal injury, leading to ESRD. 1 Percutaneous transluminal renal angioplasty/stenting (PTRAS) is a frequently used therapeutic strategy to treat patients with chronic RVD. Targeting the renal stenosis is a logical choice for treating RVD, because the resolution of the vascular obstruction followed by restoration of blood flow to the site of injured tissues should play an important role to initiate successful repairing responses. The use of PTRAS in RVD grew significantly during the past 20 years, 2 with tremendous progress in successfully resolving renal stenosis and restoring blood flow (.95% of the cases). 3 However, despite the high technical success of PTRAS, improvement in renal function is still observed in a relatively small portion of the cases. 4 The reasons for the persistent poor outcomes after PTRAS in RVD are still unclear. Furthermore, the dissociation between the technical success of PTRAS and renal outcomes underscores a pressing need to identify more effective therapeutic strategies in RVD.Endothelin-1 (ET-1) is a powerful renal vasoconstrictor and mitogenic peptide that plays important roles in controlling BP and renal function.

Streptococcus pneumoniae (pneumococcus) is an opportunistic bacterial pathogen responsible for causing several human diseases including pneumonia, meningitis, and otitis media. Pneumococcus is also a major cause of human ocular infections and is commonly isolated in cases of bacterial keratitis, an infection of the cornea. The ocular pathology that occurs during pneumococcal keratitis is partly due to the actions of pneumolysin (Ply), a cholesterol-dependent cytolysin produced by pneumococcus. The lytic mechanism of Ply is a three step process beginning with surface binding to cholesterol. Multiple Ply monomers then oligomerize to form a prepore. The prepore then undergoes a conformational change that creates a large pore in the host cell membrane, resulting in cell lysis. We engineered a collection of single amino acid substitution mutants at residues (A370, A406, W433, and L460) that are crucial to the progression of the lytic mechanism and determined the effects that these mutations had on lytic function. Both PlyWT and the mutant Ply molecules (PlyA370G, PlyA370E, PlyA406G, PlyA406E, PlyW433G, PlyW433E, PlyW433F, PlyL460G, and PlyL460E) were able to bind to the surface of human corneal epithelial cells (HCECs) with similar efficiency. Additionally, PlyWT localized to cholesterol-rich microdomains on the HCEC surface, however, only one mutant (PlyA370G) was able to duplicate this behavior. Four of the 9 mutant Ply molecules (PlyA370E, PlyW433G, PlyW433E, and PlyL460E) were deficient in oligomer formation. Lastly, all of the mutant Ply molecules, except PlyA370G, exhibited significantly impaired lytic activity on HCECs. The other 8 mutants all experienced a reduction in lytic activity, but 4 of the 8 retained the ability to oligomerize. A thorough understanding of the molecular interactions that occur between Ply and the target cell, could lead to targeted treatments aimed to reduce the pathology observed during pneumococcal keratitis.

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.

BackgroundCapsule 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.MethodsNew 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.ResultsPassive 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).ConclusionsPassive 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.

Possibly by inducing a pro-survival renal microenvironment, macitentan increased renal microvascular density, promoted cell survival and decreased injury, which in turn improved stenotic kidney hemodynamics in our model. Our results further support the safety of using macitentan in patients with concomitant chronic renal disease and supported the feasibility of a new strategy that may preserve the stenotic kidney in RVD.

TLR2 and TLR4 are involved in the response to pneumococcal keratitis and TLR2 may aid in bacterial clearance by recruitment of neutrophils to the cornea, whereas TLR4 may be necessary to modulate the immune response to limit cellular damage.